Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Science and Technology 682
_____________________________
_____________________________
Direct Observation of Biomolecule
Adsorption and Spatial Distribution
of Functional Groups in
Chromatographic Adsorbent Particles
BY
ANDERS LJUNGLÖF
ACTA UNIVERSITATIS UPSALIENSIS
UPPSALA 2002
Dissertation for the Degree of Doctor of Philosophy in Surface Biotechnology presented at
Uppsala University in 2002
ABSTRACT
Ljunglöf, A. 2002. Direct observation of biomolecule adsorption and spatial distribution of
functional groups in chromatographic adsorbent particles. ACTA Universitatis Upsaliensis.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and
Technology 682. 66 pp. ISBN 91-554-5212-4.
Confocal microscopy has been used as a tool for studying adsorption of biomolecules to
individual chromatographic adsorbent particles. By coupling a fluorescent dye to protein
molecules, their penetration into single adsorbent particles could be observed visually at
different times during batch uptake. By relating the relative fluorescence intensity obtained at
different times to the value at equilibrium, the degree of saturation versus time could be
constructed. The use of two different fluorescent dyes for protein labeling and two
independent detectors, allowed direct observation of a two-component adsorption process.
The confocal technique was also applied for visualization of nucleic acids. Plasmid DNA and
RNA were visualized with fluorescent probes that binds to double stranded DNA and RNA
respectively. Confocal measurements following single component adsorption to ion exchange
particles, revealed an interesting phenomenon. Under certain experimental conditions,
development of “inner radial concentration rings” (i.e. adsorbed phase concentrations that are
higher at certain radial positions within the particle) were observed. Some examples are given
that show how such concentration rings are formed within a particle.
Methods were also developed for measurement of the spatial distribution of immobilized
functional groups. Confocal microscopy was used to investigate the immobilization of trypsin
on porous glycidyl methacrylate beads. Artefacts relating to optical length differences could
be reduced by use of “contrast matching”. Confocal microscopy and confocal micro-Raman
spectroscopy, were used to analyze the spatial distribution of IgG antibodies immobilized on
BrCN-activated agarose beads. Both these measurement methods indicate an even ligand
distribution. Finally, confocal Raman and fluorescence spectroscopy was applied for
measurement of the spatial distribution of iminodiacetic- and sulphopropyl groups, using Nd3+
ions as fluorescent probes. Comparison of different microscope objectives showed that an
immersion objective should be used for measurement of wet adsorbent particles.
Direct experimental information from the interior of individual adsorbent particles will
increase the scientific understanding of intraparticle mass transport and adsorption
mechanisms, and is an essential step towards the ultimate understanding of the behaviour of
chromatographic adsorbents.
Key words: Confocal microscopy, confocal Raman spectroscopy, protein adsorption, ligand
distribution, visualization, imaging.
Anders Ljunglöf, Center for Surface Biotechnology, Box 577, BMC, SE-751 23 Uppsala,
Sweden, and Amersham Biosciences, Björkgatan 30, SE-751 84 Uppsala, Sweden.
e-mail: [email protected]
© Anders Ljunglöf 2002
ISSN 1104-232X
ISBN 91-554-5212-4
Printed in Sweden by Uppsala University, Tryck & Medier, Uppsala 2002
Contents
1. List of publications
1.1 Papers discussed in the thesis
1.2 Other publications
2. Introduction and aim of the thesis
3. Confocal microscopy
3.1 Introduction
3.2 Resolution in confocal microscopy
3.2.1 Optical resolution
3.2.2 Image format
3.3 Lasers
3.4 Fluorophores
4. Confocal Raman Spectroscopy
5. Chromatographic adsorbents and techniques
5.1 The stationary phase in chromatography
5.2 Chromatographic techniques
5.2.1 Ion exchange chromatography
5.2.2 Affinity chromatography
6. Present investigation
6.1 Measurement of adsorption processes
6.1.1 Visualization of intraparticle protein adsorption
6.1.2 Fractional approach to equilibrium
6.1.3 Dual scanning mode for simultaneous detection of two proteins
6.1.4 Visualization of nucleic acids
6.1.5 Observation of inner radial concentration rings
6.2 Measurement of spatial distribution of immobilized functional groups
6.2.1 Studies of trypsin immobilization on porous glycidyl methacrylate beads
6.2.2 Measurement of ligand distribution in affinity- and ion exchange media
7. Future work
8. Abbreviations and nomenclature
9. Acknowledgements
10. References
Appendixes
A Molecular structure of fluorescent dyes used for protein labeling
B Excitation and emission spectra of fluorescent dyes
C Influence of fluorescent dyes on mass transport kinetics
D Experimental
Papers I-VII
3
1. List of publications
1.1 Papers discussed in the thesis
This thesis is based on the following papers, which will be referred to by their Roman
numerals.
I.
Ljunglöf, A., and Hjorth, R. (1996)
"Confocal microscopy as a tool for studying protein adsorption to
chromatographic matrices" J. Chromatogr. A 743, 75-83
II.
Ljunglöf, A. and Thömmes, J. (1998)
"Visualizing intraparticle protein transport in porous adsorbents by
confocal microscopy" J. Chromatogr. A 813, 387-395
III.
Linden, T., Ljunglöf, A., Kula, M-R. and Thömmes, J. (1999)
”Visualizing two-component protein diffusion in porous adsorbents by confocal
scanning laser microscopy” Biotechnol. Bioeng. 65 (6), 622-630
IV.
Ljunglöf, A., Bergvall, P., Bhikhabhai, R. and Hjorth, R. (1999)
"Direct visualization of plasmid DNA in individual chromatography adsorbent
particles by confocal scanning laser microscopy" J.Chromatogr. A 844, 129-135
V.
Malmsten, M., Xing, K., and Ljunglöf, A. (1999)
”Confocal microscopy studies of trypsin immobilization on porous glycidyl
methacrylate beads” J. Colloid Interf. Sci. 220 (2) 436-442
VI.
Ljunglöf, A., Larsson, M., Knuuttila, K-G., and Lindgren, J. (2000)
”Measurement of ligand distribution in individual adsorbent particles using
confocal scanning laser microscopy and confocal micro-Raman spectroscopy”
J. Chromatogr. A 893, 235-244
VII.
Larsson, M., Lindgren, J., Ljunglöf, A., and Knuuttila, K-G. ”Confocal
Raman and fluorescence spectroscopy applied to polymeric chromatographic
adsorbent particles.” Submitted 2001
Reprints of the publications were made with the kind permission of the publishers.
4
1.2. Other publications
In addition the following papers, have been published or submitted:
VIII. Linden, T., Ljunglöf, A., Hagel, L., Kula, M-R. and Thömmes, J. (2001)
”Visualizing patterns of protein uptake to porous media using confocal scanning
laser microscopy” Separation Sci. Technol. In press.
IX. Danielsson, Å., Ljunglöf, A., and Lindblom. H. (1988) ”One-step purification of
monoclonal IgG antibodies from mouse ascites” J. Immunol. Meth. 115, 79-88
X. Karlstam, B., and Ljunglöf. A. (1991) ”Purification and partial characterization
of a novel hyaluronic acid-degrading enzyme from antarctic krill (Euphausia
superba)” Polar Biology 11, 501-507
5
2. Introduction and aim of the thesis
The adsorption of proteins to different types of chromatographic matrices has been
studied extensively. An increased scientific understanding of the adsorption process is
essential for development of more effective separation methods and chromatography
media, and for optimization of chromatography processes.
Most studies of protein adsorption have been carried out as batch or column
experiments in which the adsorbent is considered as a bulk. A frequently used method
for the determination of the kinetics of protein adsorption and approach to equilibrium
is a batch uptake experiment >Arve, 1987; Skidmore, 1990; Chang, 1998@. Here the
solid phase concentration is calculated indirectly from the decrease in protein
concentration in the fluid phase, assuming that each molecule, which disappears from
the solution, is bound to the adsorbent. Various methods have been used to estimate
effective diffusivities from experimental data. Such estimates are model dependent in
that a specific intraparticle diffusion model must be assumed, while the concentration
profile within the particle is generally unknown. Furthermore, the distribution of
functional groups, like ion exchange- or affinity ligands, is normally assumed to be
homogeneous. For more precise comparison of theoretical predictions and
experimental results, a method for direct measurement of protein uptake in individual
particles is desirable.
One way to obtain images with sufficient resolution for individual particles is to use
mechanical sectioning with a microtome. Adsorbates, e.g. fluorescently labeled
proteins can then be visualized by fluorescence microscopy. This technique has been
used for the visualization of immobilized affinity ligands >Subramanian, 1994@.
Another way is to use autoradiography to record the spatial distribution of
radioisotopes within the specimen. Autoradiographs can be obtained by exposing
photosensitive films to the J radiation from adsorbed 125I-labelled proteins >Liu, 1997@.
However, both mechanical sectioning and autoradiography are quite laborious and
time-consuming.
Holographic laser interferometry makes it possible to study and follow adsorption
processes within a sample as they actually occur >Mattisson, 1999@. This is achieved by
exactly superimposing a stored holographic image and the image of the sample. Any
changes of concentration in sample after the hologram recording can be seen as light
and dark interference fringes. With this technique it is possible to obtain experimental
concentration profiles in both the liquid and the gel phase during a diffusion process.
These profiles can then be used for determination of diffusion coefficients and to
verify intra-particle transport models. However, the method is restricted to porous gel
cast into a diffusion cell, and can not be used to study diffusion in individual adsorbent
particles.
The aim of the work presented in this thesis is to develop new methodology for
measurement of adsorption processes, and of the spatial distribution of immobilized
functional groups, directly in individual chromatographic adsorbent particles.
6
Confocal scanning laser microscopy
3.1 Introduction
Confocal microscopy has become an increasingly important tool in biomedical and
biotechnology research. The technique is based on an idea by Marvin Minsky from
1957 >Minsky, 1961 and 1988@. The advantage of confocal microscopy compared to
conventional microscopy is that distinct optical sections through the sample are
obtained without out-of-focus blur. For many years the development of the technique
was rather slow. However, due to developments in both computer science and laser
technology, it became possible to realize the potential of confocal microscopy. Among
other applications, the technique has been used:
- for analysis of paper structures >Béland, 1995@
- in polymer science >Ribbe, 1997; Thill, 1988@
- in material science >Tata, 1998@
- in neuroscience >Ulfhake,1992@ and biology >Go, 1997; Poliz, 1998, Sing, 1998@
- in biotechnology >Brewer, 1995; Cutts, 1995; Banchel, 1996; Kim, 1996;
de Beer,1997; DeLeo, 1997; Egner, 1997; Amir, 1998; Lundqvist, 1998;
Laca, 1999; Dziennik 1999 and 2000, Spiess, 2001@.
The technique has been extensively described >Wilson, 1990; Pawley, 1995; Masters,
1996; Sheppard and Shotton, 1997@.
In a conventional microscope, the entire field of view of a specimen is
simultaneously illuminated. However in a confocal microscope, laser light is focused
on one spot in the specimen (single-point illumination, fig 3.1b). Reflected light, or
emitted fluorescent light from this spot is focused again, and is allowed to pass
through a pinhole aperture positioned in front of the detector (fig 3.2). The pinhole
aperture effectively blocks light from out-of-focus planes. This depth discriminating
property makes it possible to optically slice the specimen into thin sections >Carlsson,
1985 and 1987; Wilson, 1989@. Thus, different depth layers can be studied much more
clearly, since virtually no out-of-focus light is superimposed on the image. By
moving the focal point throughout the sample, the entire plane of interest can be
scanned. Scanning in two dimensions at a given depth provides a two-dimensional
optical section of the sample at that depth. By gradually moving the acquisition plane
deeper into the sample, a stack of confocal images can be obtained that describes a
three-dimensional volume.
7
Fig 3.1 Illumination of a specimen in conventional and confocal microscopy.
In conventional microscopy (a) the entire depth of specimen is illuminated
continuously which results in the detection of out-of-focus and in-focus signals
together, causing loss of resolution. In confocal microscopy, the specimen is
illuminated sequentially covering specific points at a time. These images of points
which are devoid of out-of-focus signals are then added to form a complete in-focus
image of the specimen. (C: cover slip; S: specimen; S1: slide) >adopted from Sing,
1998@.
8
Fig 3.2 Simplified ray paths in a confocal microscope
Excitatory laser light is reflected by the dichroic mirror and is focused by the objective
lens to a limited spot at the focal plane within the sample. Fluorescence emissions,
excited within the illuminated cones above and below the focus plane (see fig 3.1b)
are collected again by the objective lens, pass through the dichroic mirror and is
focused towards the detector. However, fluorescent light originating from planes
above and below the focal plane is blocked by the detector aperture, and will not reach
the detector.
Detector
Detector aperture
Dichroic mirror
Laser source
Objective lens
Sample
Focal plane
9
3.2 Resolution in confocal microcopy
Resolution is a measure of the ability to distinguish closely spaced points or lines as
separate objects. Both lateral resolution (in the xy-plane) and the axial resolution (in
the direction of the optical axis, i.e. z-direction) are used to characterize a single focal
plane. The optical resolution can be calculated theoretically from the wavelength of
light and the numerical aperture of the objective used. In practice, many other factors
(i.e. coherence properties of the light, depth of the focal plane within the object, image
format and size of the detector aperture) are involved.
3.2.1 Optical resolution
The theoretical lateral resolution (Rf) for an ordinary microscope and incoherent
imaging can be calculated from
Rf = (0.61 u O)/N.A.
(1)
where N.A. is the numerical aperture of the microscope objective in use and Ois the
wavelength of the detected light >Wallen, 1992@. The image brightness is related to the
numerical aperture and the magnification >Majlof, 1993@ according to
Brightness # N.A.4 / magnification2
(2)
Equations 1 and 2 show that even a small increase in numerical aperture has a large
impact on light collection and makes it possible to resolve smaller details in the
specimen. Thus a practical rule for conventional microscopy is to select the objective
lens with the highest numerical aperture.
An advantage of confocal compared to conventional microscopy is due to the single
point illumination of the excitation light (fig 3.1). As fluorescence emission generally
occurs at longer wavelengths than that of the excitation light (Stokes shift), the spatial
resolution will be determined both by the excitation wavelength, which determines the
size of the illuminated spot, and the emission wavelength. Thus, a choice of
fluorophores with shorter excitation wavelength and smaller Stokes shift will
maximize the resolution. The resolution criteria in confocal microscopy are more
critically dependent on the numerical aperture. The theoretical lateral resolution is
given by
Rf = (0.46 u O)/N.A.
(3)
which is a 30% improvement over the conventional case >Wallen, 1992; Majlof, 1993@
10
For registration of an infinitely thin fluorescent layer, the axial resolution, Rd (fullwidth-half-maximum), is given by
Rd = FWHM =
8.5 O
8S n sin2 D / 2
(4)
>Ulfhake, 1992@ where n is the refractive index of the medium between objective and
object, and Dis the half angle of the light cone probing the object >Driscoll, 1978@
obtained from
1$. = n sin(D)
(5)
However, for small numerical apertures the axial resolution is inversely proportional
to the square of the numerical aperture >Carlsson, 1987@, and eq. 4 can be
approximated by
Rd = (1.4 n u O(N.A)2
(6)
From equations 3 and 6 it follows that the numerical aperture is much more important
for axial resolution than for lateral resolution. The higher the numerical aperture of the
objective, the thinner the optical section. It is important to realize that these
expressions apply close to the coverslip (and close to the optical axis), and that the
performance can be expected to decrease further away from the coverslip >Pawley,
1995; Majlof, 1993; Booth, 1998@. Nevertheless, calculation of the theoretical
resolution gives an understanding of the limitations of the technique. Furthermore, a
large aperture is used at the expense of the working distance of the objective. As a
consequence, a large aperture is unsuitable for scanning thick samples. Theoretical
values for some common lenses are listed in Table 3.1.
The resolution is also dependent on the size and shape of the detector aperture. When
the size of the aperture is increased (and thus more out-of-focus light reaches the
detector) the signal intensity increases while the depth resolution decreases. On the
other hand, a smaller aperture reduces the amount of light from out-of-focus-planes
but also reduces the signal originating from the focal plane itself. A good compromise
between intensity and resolution is an aperture diameter equal to that of the Airy disc
(fig 3.3) >Sheppard, 1997@.
11
Fig 3.3. Light waves that pass through a narrow circular aperture, will give rise to a
diffraction pattern. This diffraction pattern contains one strong maximum (i.e. the
Airy disc) , and after that secondary maxima with less intensity.
Airy disc
12
Table 3.1.Theoretical optical resolution of different microscope objective lenses
(O = 500 nm, n = 1.0 for air, 1.34 for water and 1.52 for oil >Driscoll, 1978@ .)
Objective
N.A
0.95 air
0.75 air
0.45 air
0.25 air
1.4 oil*
1.0 oil
0.5 oil
1.2 water**
0.8 water
0.5 water
Rf (Pm)
(lateral resolution)
0.24
0.30
0.51
0.92
0.16
0.23
0.46
0.19
0.29
0.46
Rd (Pm)
(axial resolution)
0.78
1.24
3.45
11
0.54
1.1
4.25
0.65
1.47
3.75
* Oil-immersion objectives are corrected for observation of specimens close beneath
the coverslip. Thus when used for optical sectioning deep within aqueous specimens,
their performance will become severely compromised by sperical aberration.
** With a water-immersion objective, no spherical aberration is experienced when
focusing deep into an aqueous specimen >Sheppard and Shotton, 1997@. A comparison
of results obtained with dry and water immersion objectives in confocal Raman
spectroscopy is reported in Paper VII.
3.2.2 Image format
The most common format for scanning a single plane is 512 u 512 pixels. This
format results in squared pixels, ensuring that lateral resolution is equal in both x- and
y-directions. In the z-direction, the interplane spacing ideally should be chosen to
match the length of the pixel side, making the resulting voxel cubical in shape
>Béland, 1995@. However, limitations in computing power and storage capabilities
make such approaches impractical, especially for optical sectioning of thick samples.
That the microscope resolves two bright points means that there is a perceptible dark
space between them in the image. To keep them resolved after pixelation, at least two
bright pixels is needed, separated by a dark one. In practice, this means that optimum
resolution is obtained when the sampling frequency is at least 2.3 times higher than
the theoretical resolution of the objective lens. Under these conditions, the actual
resolution is not determined by the scanning but by other components in the system
>Webb, 1995@.
13
3.3 Lasers
Lasers are powerful light sources which can be used for either single- or multiplewavelength excitation. Compared with other light sources, lasers have a number of
unique properties which make them almost ideal for use in confocal microscopy.
These include high degree of mono-chromaticity, small beam divergence, high
brightness and polarized emission, all of which greatly improve the efficiency of a
confocal microscope.
The wavelengths of the light emitted from a laser are dependent upon the laser
medium within the tube. The most popular lasers are the argon ion laser, giving lines
at 488 and 514 nm, and the argon-krypton mixed gas laser, giving lines at 488, 568
and 647 nm.
Helium-neon (He/Ne) lasers have been introduced with emission wavelengths at 534,
594, 612 and 632 nm, as well as lines in the infrared, for example 1152 nm. Dye lasers
have become important light sources because they are tunable over a range of 20-50
nm, and can provide bands of radiation of a chosen wavelength. He/Ne (green) and
He/Ne (red) lasers emits light at 543 nm and 633 respectively. For a thorough review
of laser sources for confocal microscopy, see Pawely, 1995, chapter 5.
3.4 Fluorophores
Fluorophores are compounds where absorption of light causes the molecule to
fluoresce, that is to emit photons (fig 3.4). The emitted photons will have less energy
than the exciting photons and thus longer wavelengths. This effect is known as Stokes
shift (fig. 3.5) and its magnitude depends on the fluorophore excited. Some
fluorophores may change their properties in response to environmental factors such as
pH and solution compositions; in some cases the fluorescence properties might differ
between free fluorophore dye and protein-conjugated dye >Ulfhake, 1992@. Also the
concentration of fluorophore may cause slight displacements of the emission
spectrum.
Fluorescence may also be absorbed by chemicals in the specimen, including other
fluorophores. This phenomenon is often referred to as quenching. An example is
given in fig. 3.6 that shows fluorescence intensity profiles obtained from optical
sections through adsorbent particles saturated with protein containing fluorescence
labeled and unlabeled molecules in different proportions. At high fluorophore
concentration a minimum is obtained in the intensity profile, while an increased
proportion of unlabelled protein gradually results in more even profiles. Figure 3.7
shows relative solid phase concentration (Qrel ; Eq. 17 chapter 5.1.2) values obtained
with four different fluorescent dyes plotted against various dye concentrations in the
particles. At low dye concentrations there is a linear relationship between
concentration and emission intensity. However, at increased dye concentrations the
intensity curves level off. Two phenomena might be responsible for this situation
>Herman, 1998; Van Oostvelt, 1989@. One is that emitted fluorescence light is
reabsorbed by surrounding fluorophores since the absorption and emission spectra to
some extent overlap. However, as can be seen in fig 3.8, the detection range did not
have any significant effect on the intensity profiles. Another explanation, known as
the inner filter effect, is defined as a decrease in the amount of available excitation
intensity in successive layers of the fluorescing object the further the layer is from the
light source.
14
If the compound that absorbs the fluorescence is itself a fluorophore, the second
fluorophore in turn can emit undesirable fluorescence1. Another problem associated
with fluorophores is photobleaching, or fading, owing to the finite capacity of the
fluorophores to give off photons on excitation. This will show as a fluorophore signal
attenuation. The rate of fading varies with different fluorophores. Compounds that
tend to fade quickly should be avoided if possible.
When two or more fluorophores are used simultaneously, it is important to avoid
energy transfer between the fluorophores and cross-talk (bleedthrough) between
different detector channels. The choice of fluorophores should therefore be based on
their absorption/emission spectra. Spectral overlap should be avoided, or kept as low
as possible. There are different ways to avoid cross-talk. One is to record the images
in succession, another is to limit the emission spectrum that is detected. In the latter
case, the emission spectra should be separate enough to ensure detection of the
individual fluorophores using band-pass filters in front of the detectors2. New
technology for confocal fluorescence microscopy, using intensity-modulated multiple
wavelength scanning (IMS) has been described >Åslund, 1993; Carlsson, 1992 and
1995@. By using IMS, very high channel separation can be obtained, and it is possible
to improve the specificity in the detection of multiple fluorophores, i.e. to reduce the
cross-talk between the different fluorophores >Carlsson, 1998@.
Some properties of the fluorophores used in this thesis are shown in Table 3.2,
appendix A and B. For a more complete list of adsorption and emission maximum for
common fluorophores see >Herman , 1998@.
1
Transfer of excitation energy is utilized in fluorescence resonance energy transfer (FRET).
Resonance energy transfer is a process by which a fluorophore (donor) in a excited state may transfer
its excitation energy to a neighboring fluorophore (acceptor). If the donor molecule has a emission
spectrum that overlaps the absorbance spectrum of the acceptor, they can exchange energy between
each other through dipole-dipole interaction >Herman, 1998@. This energy transfer manifests itself both
by quenching of donor fluorescence in the present of acceptor and increased emission of acceptor
fluorescence. The energy transfer requires the distance between the fluorophores to be relatively close
(10-100 Å), and can be used to study interactions between cellular components as well as
conformational changes within individual molecules.
2
Leica TCS SP confocal microscope uses spectrophotometric discrimination of the fluorescence
emission.
15
Fig 3.4 Jablonski diagram demonstrating the different energy levels involved in the absorption
and emission of light >Herman, 1998; Lakowicz, 1999@.
Absorption of light occurs very quickly (a10-15 sec) and corresponds to the excitation of the
fluorophore from the ground state (S0) to an excited state (S2). Relaxation to the lowest level of
the excited state (internal conversion) occurs within approximately 10-11 sec as energy is
thermally transferred to the environment. The molecules “lives” in the lowest excited singlet
state for for approximately 10-9 sec. Relaxation from this state to the ground state with
emission of a photon is , physically, what is referred to as fluorescence. Each fluorescent
molecule (fluorophore) can repeat the excitation/emission process many times. In addition to
fluorescence, molecules which reside in the lowest excited singlet state can undergo
intersystem crossing to the triplet state from which a longed lived emission, phosphorescence,
occurs
S2
Internal conversion
Intersystem
crossing
S1
hQ
Triplett
T1
Fluorescence
hQ
S0
hQ
Phosphorescence
16
Fig 3.5 The Stokes shift is the difference between the maximal absorption and
emission wavelength of a fluorophore, here exemplified with the cyanine dye Cy5.
Stokes shift
Normalized intensity
100
Emission spectrum
Absorption
spectrum
80
60
40
20
0
500
550
600
650
700
750
800
Wavelength (nm)
Fig 3.6 Effect of fluorophore concentration on fluorescence intensity profiles.
Sample: SP Sepharose Fast Flow saturated with BSA labelled with various amounts of Cy5.
The Cy5 concentration in the particles was varied between 0.24 – 2.35 mM.
Laser power and detector voltage was kept constant. The excitation wavelength was 647 nm.
The emitted fluorescent light was detected between 660-800 nm. Cy5 concentration from top
to bottom: 0.24, 0.47, 0.94 and 2.35 mM.
Normalized intensity
100
80
60
40
20
0
0
50
xy-position (Pm)
17
100
Fig 3.7 Dependence of fluorescence intensity on fluorophore concentration and laser
power. Sample: SP Sepharose Fast Flow saturated with BSA labelled with various
amounts of fluorophores. A) Oregon Green, B) Alexa 488, C) Cy3 and D) Cy5
Laser power (Ar/Kr laser): u = 30 %, O = 50 % and ' = 100 %
B
A
200
Qrel (Units/m 3)
Qrel (Units/m3)
300
200
100
150
100
50
0
0
0
1
2
0
3
0,5
1
1,5
Conc dye (mM)
Conc dye (mM)
C
200
150
150
Qrel (Units/m 3)
Qrel (Units/m3)
D
200
100
50
100
50
0
0
0
0,25
0,5
0,75
1
1,25
0
0,5
Conc dye (mM)
1
1,5
2
2,5
Conc dye (m M )
Fig 3.8 Effect of detection range on the intensity profiles.
Excitation wavelength 649 nm. The intensity was normalized by adjusting the detector
voltage, while the dye concentration was kept constant (2.35 mM Cy5).
Detection range: 650 – 680 nm ( _ _ _ ) , 680 – 710 nm (.….) and 730 – 800 nm ( ____ ).
Normalized intencity
100
80
60
40
20
0
0
20
40
60
80
100
Xy-position (Pm)
18
120
Table 3.2. Fluorescent dyes used in this thesis
Dye
Wavelength (nm)
Excitation
Emission
max
max
Remark
.
Dyes for visualization of nucleic acids:
YOYO-1
491
509
Probe for double stranded DNA
Pico Green
502
523
Probe for double stranded DNA
TOTO-3
642
660
Probe for double stranded DNA
RiboGreen
500
525
Probe for RNA
Dyes for protein labeling:
FITC
490
518
Tends to fade; long emission tail;
pH sensitive between pH 5-8.
Oregon Green
496
524
Greater resistance to photobleaching than
FITC; pH insensitive at pH > 6.
Alexa 488
488
519
Substitute to FITC. Photostable and pHinsensitive from pH 4-10.
Cy3
552
565
Suitable in combination1 with Oregon Green or
Alexa and Cy5; pH insensitive
Cy5
650
667
Very low cross talk with Oregon Green and
Alexa 488; pH insensitive
.
1
To reduce bleedthrough between Oregon Green (or Alexa), Cy3 and Cy5, a combination of limitation
of the emission spectra and recording of the fluorophores in succession is recommended
19
4. Confocal Raman spectroscopy
Confocal microscopy can also be combined with Raman Spectroscopy. Confocal
Raman Spectroscopy combines the chemical information from vibrational
spectroscopy with the spatial resolution of confocal microscopy. Depth profiles or
lateral Raman mappings can be recorded by moving the sample through the focus of
the microscope objective. In this way it is possible to monitor the distribution of
different components of a specimen >Schuster 2000@. Such Raman mappings can give
proof of homogeneous or inhomogeneous mixtures without additional staining or
other preparations >Schrof, 1998@.
The Raman effect was discovered by Sir C. V. Raman in 1928 (awarded Nobel
Prize 1930). Since then, Raman spectroscopy has become a powerful tool for
characterizing the structures of molecules. Raman spectroscopy is a technique that in
many ways complements IR-spectroscopy. In both methods vibrational energy
changes in the molecule are involved. For a molecule to absorb IR radiation, it must
have a changeable electric dipole moment. In order to obtain a Raman spectrum this is
not necessary, but there must be a change in polarizability1. The method thus makes it
possible to examine vibrational spectra of compounds that can not be examined by IR
spectroscopy. In both methods, group frequency regions are used to identify functional
groups. Raman scattering is particularly useful for such groups as: C-S , S-S , C-C ,
N=N, C=C and C{C , and IR spectroscopy for: -O-H , -C=O 2 , -P=O , -NO2 2, -N-H
and -S=O 2 >Ingle, 1988@.
Raman spectra are obtained by irradiating a sample with very intense monochromatic radiation (normally a laser beam). When the beam of radiation is passed
through the sample, a small amount of the energy is scattered. The scattered energy
will consist almost entirely of radiation of the incident frequency (the so-called
Rayleigh scattering). However, in addition certain discrete frequencies above and
below that of the incident beam will be scattered. These frequencies are referred to as
Raman scattering.
In classical terms these phenomena can be described in the following way.
The electric field (E) associated with a beam of radiation can be written
E = Em sin (2SQt)
(9)
Where Em is the amplitude of the wave, Q is the exciting frequency and t is time.
1
When a molecule is put into a static electric field, the electrons will be attracted towards the positive
pole of the field and the positively charged nuclei towards the negative pole. This separation of charge
causes an induced dipole moment in the molecule. The molecule is said to be polarized >Banwell,
1994@.
2
Groups that are approximately equivalent with Raman and IR spectroscopy
20
When this oscillating field interacts with the polarizable electron clouds of the sample
molecules, it induces a dipole moment (P) given by
P = DE = D Em sin (2SQt)
(10)
where D is the polarizability of the sample. Such an oscillary dipole emits radiation of
its own frequency, that represents the Rayleigh scattering >Ingle, 1988@. If, in addition,
the sample molecules undergo some internal motion, such as vibration which changes
the polarizability periodically, then the oscillating dipole will have superimposed upon
it the vibrational oscillation. The oscillating dipole can now be written:
P= D0Emsin2SQt + 1/2EEm (cos2S(Q-Qvib)t - cos2S(Q+Qvib)t)
(11)
where D0 is the equilibrium polarizability and E represents the rate of change of
polarizability with the vibration. The oscillating dipole will now emit radiation of
frequencies Q+Qvib and Q-Qvib (Raman scattering) as well as the exciting frequency.
The occurrence of Raman scattering may also be described in therms of the quantum
theory of radiation. In Rayleigh scattering (elastic scattering), the sample molecule is
excited by a photon to a virtual state, and then relaxes to its original vibrational state
by re-emitting a photon at the same frequency as the incident light. In this case, the
molecule does not absorb any energy from the incident radiation. The energy of the
Rayleigh frequency is given by
E = hQ = h (
c
O
)
(12)
where h is Planck´s constant, c is the speed of light and O is the wavelength.
In contrast to Rayleigh scattering, the origin of the Raman scattering is an inelastic
effect. When inelastic scattering occurs, the excited molecule relaxes to a different
vibrational level, rather than to the original state. Photons scattered with a frequency
lower than that of the incident beam is referred to as Stokes radiation, while that of
higher frequency is called anti-Stokes radiation (fig 4.1). Stokes radiation is generally
more intense than anti-Stokes, and is therefore of most interest in analytical chemistry.
The energies of the Stokes-Raman lines are given by
E - 'E = h(Q - Q´)
(13)
where Q´ is the frequency shift due to an energy change 'E. As many different
vibrational levels from molecules in the sample are involved simultaneously, this
results in several Raman lines h(Q - Q´1), h(Q - Q´2)…..h(Q - Q´n), that are recorded in
the spectrum >Lin-Vien, 1991@. In Raman (and IR) spectroscopy it is common to use
the term wave number in describing radiation.
21
Fig 4.1. Energy level diagram illustrating a) Raman lines and b) resulting Raman spectra.
In a) molecules in the ground state (Q = 0) can absorb a photon of energy hQ and reemit a photon of
energy h(Q - Q´). Molecules in a vibrational excited state (Q = 1) can return to the ground state and
emit a photon with energy h(Q + Q´). Because the ground state population is greater than that of the
excited state, the Stokes lines are more intense then the anti-Stokes lines.
a) Raman lines
Stokes scattering
hQ
Anti-Stokes scattering
h(Q - Q´)
hQ
Q=1
Q=1
Q=0
Q=0
b) Resulting Raman spectra (simplification)
Energy
Rayleigh line
Stokes line
Anti-Stokes line
Q - Q´
Q
Q + Q´
22
h(Q + Q´)
The wave number is defined as the number of waves of the radiation per centimeter,
that is wave number >cm-1@ = 1/O >cm@
In the Raman spectrum the Stokes lines are expressed as Raman shifts
'v =
v´
>cm-1@
c
(14)
Raman spectroscopy (non-confocal) is suitable for characterization and quality
control of chromatography media. A typical Raman spectrum obtained from SP
Sepharose is shown in fig 4.2. Thus, the technique has been used for characterization
of chromatography media >Doyle, 1997, 2000, Pemberton, 2001@ and for development
of cleaning in place protocol in media for expanded bed adsorption >Asplund, 2000@.
Confocal Raman spectroscopy has been used for the spatial mapping of coatings and
thin films which have been subjected to surface modifications [Schrof, 1998,
Sacristan, 2000], to obtain information about structural changes and degradation of
fuel cell membranes [Mattsson, 2000], to investigate the distribution of small
molecules in latex or PVC-films [Belaroui, 2000, Mura, 2000], to evaluate orientation
profiles of amorphous and crystalline phases across a polyethylene fiber section
[Fagano, 2001], for measurement of the chemical composition of single bacteria cells
>Reese, 2000@. Very recently the technique was utilized for measurement of the
distribution of NH2 groups in aminomethylated polystyrene divinylbenzene particles
>Kress, 2001@. In Paper VI and VII the method was used for measurement of ligand
distribution in agarose particles.
Fig 4.2 Raman spectrum of SP Sepharose™ Fast Flow.
The following bands are identified: 2928 cm-1 CH2 stretching; 1644 cm-1 C=C
stretching; 1465 cm-1 CH2 bending; 1430-1200 cm-1 C-O-H bendings + CH2 twisting;
1083 cm-1 C-O-C-/C-O-H stretching; 1049 cm-1 stretching in propyl-SO3- (Na+) .
(Courtesy of Karl-Gustav Knuuttila, Amersham Biosciences).
1.600
2928
1.50
1.40
1.30
1.20
1.10
1.00
0.90
A
0.80
0.70
0.60
1049
0.50
1083
1465
1418
0.40
851
896
932
966
795
1287
1356
0.30
530
742
1644
0.20
0.10
-0.010
3500.8
3200
2800
2400
2000
1800
1600
1400
1/cm
23
1200
1000
800
600
400 299.4
5. Chromatographic adsorbents and techniques.
5.1 The stationary phase in chromatography
Separation by chromatography depends on the differential partitioning of solutes
between a stationary phase (the chromatographic medium or the adsorbent) and a
mobile phase (the buffer solution). In liquid chromatography the stationary phase
usually consists of a porous matrix containing a stagnant volume of solvent. Typically
the solvent constitutes most of the stationary phase (often more than 90 %). Such
materials are generally referred to as gels. In protein chromatography the solvents are
normally aqueous buffers and the gel-forming materials are usually composed of
hydrophilic polymers. Gels are normally bead shaped, with average particle diameters
ranging from a few Pm to approximately 300 Pm.
Many different materials have been used for the design of chromatographic
particles. These can be classified as being inorganic (for example hydroxyapatite,
porous silica and glass), synthetic organic polymers (methacrylate, polystyrene,
polyacrylamide) or polysaccarides (cellulose, dextran, agarose) >Janson, 1998@. None
of these chromatographic materials are ideal for protein chromatography, and
compromises considering the most important qualities are necessary. On one hand,
materials consisting of polysaccarides or organic poly-acrylamide are usually more or
less compressible and will be deformed if submitted to high flow rates. On the other
hand more rigid materials like porous glass, methacrylate and polystyrene usually
suffer from high non-specific adsorption. The major disadvantage with silica is its
instability at alkaline pH. The rigidity of soft gels can be improved by chemical crosslinking, and high non-specific adsorption can be reduced by surface modification.
Composites of two matrix materials can be used to combine the best qualities of both.
5.2 Chromatographic techniques
In liquid chromatography the adsorbent is packed into a column, and the buffer and
sample are both pumped through this column. Non-interacting molecules pass rapidly
through the column and are thus separated from mulecules that are retained. The
retention is achived by exploitation of various properties of the protein. The more
important of these are listed in table 5.1, together with the corresponding
chromatographic method. The studies presented in this thesis have mainly been
developed for, and applied on, media for ion exchange and affinity chromatography.
Thus, a short introduction to these separation methods is given below.
24
Table 5.1 Protein properties and separation methods
Property
Separation method
Size and shape
Charge
Isoelectric point
Hydrophobicity
Gel filtration
Ion exchange chromatography
Chromatofocusing
Hydrophobic interaction and
reversed phase chromatography
Affinity chromatography
Covalent chromatography
Immobilized metal ion affinity
Biospecific affinities
Content of exposed thiol groups
Metal binding
5.2.1 Ion exchange chromatography (IEC).
The stationary phase of an ion exchanger consists of a matrix with either acidic or
basic groups. The acidic ion exchangers containing negative groups are called cation
exchangers and the basic ones containing positive groups anion exchagers (table 5.2).
IEC makes use of the ability of these particle-bound charges to reversibly adsorb
sample molecules of opposite charge. Thus, charged patches on the protein surface
will be attracted to a chromatography matrix provided that the ionic strength of the
surrounding buffer is low. The pH/net charge curve is a highly individual property of
a protein, and constitutes the basis for selectivity in IEC. At a pH value below the
isoelectric point of a protein it will adsorb to a cation exchanger, and above the
isoelectric point to a anion exchanger (fig 5.1). Desorption is then achieved by
increasing the ionic strengh by use of a salt gradient or by altering the pH of the
mobile phase. When the ionic strength is increased, the salt ions will compete with the
protein for the charged ligand. The higher net charge of the protein, the higher the
ionic strength is needed for desorption. The most powerful way to alter the selectivity
is to change the charge of the protein by varying the pH. To optimize selectivity a pH
value should be chosen that creates sufficiently large net charge differences among the
sample components. The second parameter to optimize is the salt gradient, which
should be within the range of 5-20 column volumes to get maximum resolution. For
further reading see Karlsson et.al. 1998 >Karlsson, 1998@.
25
Fig 5.1 Selectivity in ion exchange chromatography.
Influence of pH on sample net charge.
Positive surface
net charge
Isoelectric point
Negative surface
net charge
Table 5.2 Functional groups used on ion exchangers
Name
Anion exchangers
Diethylaminoethyl (DEAE)*
Quaternary aminoethyl (QAE)
Quaternary ammonium (Q)
Trimethylaminoethyl (TMAE)
Triethyl amine (TEAE)
Functional group
-O-CH2- CH2-N+H(CH2CH3)2
-O-CH2- CH2-N+(C2H5)2- CH2- CHOH- CH3
-O-CH2-CHOH-CH2-O- CH2-CHOH- CH2-N+(CH5)3
-O-CH2- CH2- N+(CH5)3
-O-CH2-N+(CH5)3
Cation exchangers
Carboxy methyl (CM)*
Sulphopropyl (SP)
Sulphonate (S)
-O-CH2COO-O-CH2-CHOH-CH2-O-CH2- CH2- CH2SO3-O-CH2-CHOH-CH2-O-CH2-CHOH- CH2SO3-
* DEAE and CM are examples of weak ion exchangers, while quaternary amines and sulphonic groups
are strong. The names refers to the pKa values of the charged groups and does not say anything about
the strength with which they bind proteins.
26
5.2.2 Affinity chromatography (AC).
Affinity chromatography separates proteins on the basis of a reversible interaction
between a protein and a specific ligand coupled to a chromatographic matrix. The
technique offers high selectivity. Purification can be in the order of several thousandfold with high recoveries. A good affinity ligand must be able to form a reversible
complex with the protein to be isolated. The binding constant should be high enough
for the formation of stable complexes, but it should be easy to dissociate these
complexes again by simple changes in the mobile phase. The ligand should also have
chemical properties that allow easy immobilization to the chromatography matrix.
Ligands may be divided into two groups according to the specificity of the
interaction, i.e. group-specific and mono-specific. The group-specific ligands have
affinity for a group of related substances. This type of ligands includes protein A and
Protein G for purification of immunoglobulins, lectins for purification of
glycoproteins, dyes for for purification of NAD+- and NADP+-dependent enzyme,
polynucleotides for purification of oligonucleotides containing complementary
sequences etc. Other types of ligands that may be incorporated into this class are
chelating groups (e.g. iminodiacidic acid) used for immobilized metal ion affinity
chromatography (IMAC). Mono specific ligands bind to a very small number of
molecules. Examples are biotin that binds avidin, lysine that binds plasminogen and
monoclonal antibodies raised against a specific protein. The latter affinity medium is
called an immunosorbent. [Sofer and Hagel, 1997].
An ideal gel material for AC should meet the following characteristics:
1) Hydrophilic and neutral to prevent non-specific interactions. 2) The pore size
should be large enough to provide room for the, often bulky, ligand and to provide
free access to the ligand for the interacting target molecule. 3) Contain functional
groups to allow derivatization of the matrix to make it reactive towards the ligand. 4)
Chemically and physically stable. Agarose possesses most of these characteristics and
is a popular matrix for AC. For further reading see J. Carlsson et. al [J. Carlsson,
1998@.
27
6. Present investigation
6.1 Measurement of adsorption processes
6.1.1 Visualization of intraparticle protein adsorption.
Paper I describes for the first time ever the use of confocal microscopy as a tool for
studying protein adsorption to individual chromatography adsorbent particles. The
adsorption of Protein A to IgG Sepharose Fast Flow was studied by batch incubation
with varying amounts of FITC labeled Protein A. Batch uptake was allowed to
continue for 1 or 10 minutes. The reaction was then stopped by dilution and
centrifugation. The adsorbent was subsequently washed three times by repeated
dilution, centrifugation and decantation. Individual particles were analyzed by
horizontal scanning followed by translation of the confocal images into fluorescence
intensity profiles (i.e. pixel values in a digitized section along the particle diameter;
Paper II, fig 1). The result showed that, at sample amounts corresponding to the
Protein A binding capacity of the adsorbent, Protein A was adsorbed to a thin outer
layer of the particles while the interior was unused for adsorption. The depth of
adsorption was found to increase with incubation time. Furthermore, by increasing
the sample amount the adsorption depth increased.
The article also describes one important limitation of the confocal technique, namely
the attenuation of light originating from deeper layers in a sample (Paper I, fig 7). A
similar example is given in fig. 6.1 that shows a stack of confocal images through an
agarose based adsorbent particle that was partially saturated with fluorescent labeled
protein. As can be seen, confocal images at the bottom of the stack have a much
lower intensity than the corresponding images at the top. This effect is due to light
attenuation caused by absorption and scattering of both the excitation and the
emission light. Different optical pathlengths in the sample result in different degrees
of attenuation. This attenuation effect is even more pronounced in less transparent
samples, such as PS-DVB >Rademann, 2001@ or methacrylate particles (Paper V).
However, in agarose beads this is no major problem as long as one is aware of the
effect. Thus, for comparison of confocal images in a protein uptake experiment it is
important to make measurements at comparable positions, e.g. in the center of
particles of comparable size. An alternative way is to scan in xz-direction through the
particles (i.e. from top to bottom) and then compensate mathematically for light
attenuation >Abraham Lenhoff, personal communication@.
28
Fig 6.1 Effect of light attenuation when scanning deep into an agarose particle.
Optical sectioning through a SP Sepharose Fast Flow particle, partially saturated with
labeled protein. Step size between optical sections 1 Pm.
(Top of particle at top of the figure at left, bottom of particle down at the right)
6.1.2 Fractional approach to equilibrium
In Paper II, the methodology from Paper I was further developed. Confocal
microscopy was used to study protein uptake to cation exchange adsorbents during
batch experiments in a finite bath. The protein was labeled with a fluorescent dye, and
the protein solution was filled in a reaction vessel equipped with a hanging stirrer. The
adsorption experiment was started by adding gel slurry to the stirred protein solution.
Samples were then taken from the reaction vessel at fixed times, and after washing1
they were immediately analyzed by confocal scanning. The resulting confocal images
were translated into intensity profiles (Paper II, fig 1 and 3). For comparison, protein
concentration (C) was measured in the supernatant from each sample. The solid phase
concentration could then be calculated indirectly from the decrease in protein
concentration (C0 - C) according to (Eq. 15).
Q
V M C0 C VS
(15)
where VM = volume of mobile phase, VS = volume of solid phase, C0 = initial protein
concentration.
__________________________________________________________________________________
1
The washing step was excluded from the method in later studies of protein adsorption presented in the
thesis.
29
The overall fluorescence within the particles was calculated by dividing the particle
radius into defined segments - each segment being equal to one pixel unit - and
calculating the corresponding volume of a shell. By multiplying with the intensity
( I seg ) , the total fluorescence within the shell was obtained (Eq. 16). The average
fluorescence within each shell represents the concentration of adsorbed protein, and
the sum of all shells divided by the particle volume (VP) gives the relative solid phase
concentration (Qrel) expressed as arbitrary units per volume adsorbent (Eq. 17). By
subsequently relating the relative fluorescence intensity obtained at different times to
f
the value at equilibrium ( Qrel
), the degree of saturation versus time (F) could be
calculated (Eq. 18). An example is given in figure 6.2.
4 º
ª
I seg ˜ «ra3 ri 3 ˜ S »
3 ¼
¬
shell
I integr.
(16)
where ra = outer radius and ri = inner radius of a shell.
Qrel
F
¦ I
shells
shell
integr.
(17)
Vp
Qrel
f
Qrel
(18)
The results obtained by direct measurement using confocal microscopy were also
compared with the indirect measurement obtained via the fluid phase concentration
(Paper II, fig 8). A good agreement was obtained between the two uptake profiles.
However, more scatter could be observed in the data from the confocal microscope
than from the fluid phase data. This is not surprising since the confocal measurements
were made on individual particles selected visually for their similar size. The fluid
phase measurements, on the other hand, give the net result of protein uptake in all
particles and is an average of all particle diameters.
Furthermore, the correlation between the direct measurements by confocal scanning,
and the capacity calculated from the fluid phase was demonstrated for four different
protein / adsorbent combinations (fig 6.3). For each system there was a good linear
correlation. This result supports the conclusion that measuring fluorescence intensity
profiles gives a realistic picture of the kinetics of protein uptake. The development of
integrated fluorescence over time is a good representation of the fractional approach
to equilibrium, and from these data it should be possible to obtain a quantitative
description of protein uptake by mathematical analysis >Horstman, 1989; Arve, 1987;
Chang, 1998@.
30
Fig 6.2. Batch uptake of IgG to an agarose based Protein A matrix.
Sample: hIgG 2.6 mg/ml in 25 mM TRIS-HCl, 0.15 M NaCl pH 7.4
a) Confocal images
b) Fractional approach to equilibrium
1
F (-)
0,8
0,6
0,4
0,2
0
0
10
20
30
t (min)
40
50
60
Fig 6.3 Parity plott of Qrel from integrated fluorescence intensity profiles versus
capacity calculated from the decrease in fluid phase concentration according to Eg. 15.
Q = IgG / SP Sepharose Fast Flow, = IgG / SP Sepharose XL, V = lysozyme / SP
Sepharose Fast Flow and ' = lysozyme / SP Sepharose XL.
100
3
Iintegr. (relative units/m )
120
80
60
40
20
0
0
50
100
150
200
calculated capacity (kg/m³)
32
250
300
6.1.3 Dual scanning mode for simultaneous detection of two proteins.
Most modern confocal microscopes allow simultaneous detection of multiple
fluorescent labels. One such example is given in fig. 6.4, that shows confocal images
and the corresponding intensity profiles from an agarose-based particle saturated with
a mixture of BSA (Mw a 67,000 Da, pI 5.15) and E-lactoglobulin (Mw 35,000 Da, pI
5.2). The two images were obtained from the same particle, at the same time. As can
be seen, BSA bound mainly to an outer layer, while the smaller E-lactoglobulin
molecules penetrated the whole particle. In this case, BSA was labeled with Cy5 and
E-lactoglobulin with Oregon Green. These two fluorophores have different excitation
and emission wavelengths (table 3.2, appendix B). In this way energy transfer between
the fluorophores could be avoided. By using two independent detectors and
appropriate band-pass filters, almost no detectable light from either of the
fluorophores could be detected in the other’s detector channel. Thus, it was possible
to follow the two proteins independent of each other.
Fig 6.4 Simultaneous detection of two fluorescence labeled proteins.
Adsorbent: agarose based cation exchanger (prototype)
Sample: BSA/E-lactoglobulin mixture 4 mg/ml in 50 mM sodium acetate pH 4.5
BSA-Cy5 (excitation at 649 nm, emission detected > 660 nm)
E-lactoglobulin-Oregon Green (excitation at 488 nm, emission detected > 510 nm).
a) Confocal images obtained after batch incubation for 24 hours
b) Fluorecence intensity profiles
a)
b)
33
In Paper III, the technique described above allowed, for the first time, the direct
observation of two-component adsorption within individual adsorbent particles. The
batch uptake of polyclonal human IgG and BSA (in 50 mM acetate buffer pH 5.0) to
two different ion exchange adsorbents was measured using Cy5 and Oregon Green as
labels. The result revealed totally different mass transfer characteristics of IgG and
BSA, and with time IgG was displaced towards the center of the particle while BSA
dominated at the edges. This result does not correspond to the conventional picture of
protein adsorption to porous particles. Usually an equal equilibrium distribution of all
proteins adsorbed is assumed, which would be expressed by “flat” fluorescence
intensity profiles for both proteins. Control experiments performed with different
combinations of labeled and unlabelled proteins confirmed that the observed effect
was consistent irrespective of the labeling method. Experiments were also performed
with different proportions between BSA and IgG (i.e. 10:1, 1:1, 1:10 w/w). In all
cases, similar confocal images was found as above, i.e. BSA was bound to the outer
regions of the cation excanger and IgG was found in the internal regions.
A similar experiment was performed with BSA and monoclonal IgG (in 50 mM
acetate buffer pH 4.5). The confocal images (fig 6.5a) and the intensity profiles (fig
6.5b) show how IgG is continuously displaced by BSA. The intensity profile obtained
at equilibrium shows that IgG is concentrated at the inside, while BSA dominates at
the edges. Prolonged incubation showed that the pattern was conserved for several
weeks.
The result was confirmed by a number of different control experiments:
1) Optical sectioning starting from the top and down through the middle of the
particles (fig 6.6). The stack of confocal images shows that BSA dominates at the
outer layers of the particles and IgG at the inside.
2) Adsorption experiments starting with particles pre-saturated with monoclonal IgG.
After addition of a surplus of BSA, most of the IgG molecules were displaced, and
again BSA dominated at the edges of the particles (not shown).
3) Comparison of data obtained with fluid phase measurements. A good agreement
was seen between the uptake profiles obtained by direct measurement with confocal
microscopy and by indirect measurements via the fluid phase, both for BSA and IgG.
The capacity for IgG increased to a maximum and then decreased again, while the
BSA capacity gradually increased with time (fig 6.7).
4) Equilibrium isotherms for both proteins were generated using a static micro batch
method developed by Karol Lacki (Amersham Biosciences, Uppsala), and the
association constant (Ka) was determined by fitting the experimental data to the
Langmuir model* (table 6.1).
Batch uptake experiments were also performed with a diluted hybridoma supernatant
(pH 4.5 – 5.5) supplemented with fluorescent labeled BSA and monoclonal IgG. The
ionic strength of these supernatant batches (diluted 1:2 in acetate buffer) was higher
than in the corresponding buffer experiments described above. Figure 6.8
demonstrates that the heterogeneous distribution of BSA and IgG was conserved. .
Thus, at pH 4.5, the adsorbed BSA was again predominantly bound as a sharp ring at
the outer region of the particle, while the IgG was more located at the inside. At pH
5.0 the thickness of this ring was significantly reduced, and at pH 5.5 no BSA could
be detected. In addition to the increasing volume occupied by the IgG molecules at
increasing pH, the intraparticle mass transport was much faster.
34
The confocal analysis of BSA/IgG adsorption clearly demonstrates that confocal
microscopy allows a detailed insight into kinetics and equilibrium of multi-component
protein adsorption to porous chromatography media. The results described above can
be explained as follows: At pH 4.5, BSA has much higher affinity for the adsorbent
than IgG, and will be strongly attracted to the ion exchanger. The antibodies will be
displaced by BSA both to the fluid phase and towards free ligands in the interior of
the particles. At 5.0 , i.e. closer to the isoelectric points of BSA and the monoclonal
antibodies (pI = 5.2 and 6.0 respectively), the difference in affinity is much lower and
the IgG molecules are less affected by the presence of BSA. Finally at pH 5.5 (where
the net charge of BSA is negative) IgG has much higer affinity and prevents the BSA
adsorption.
Table 6.1 Association constants for BSA and monoclonal IgG obtained from
adsorption isotherms in 50 mM acetate buffer.
pH
4.5
5.0
5.5
Ka, BSA / Ka, IgG
Ka
BSA
25.6
12.8
0.5
IgG
0.8
5.4
4.6
32
2.4
0.1
* The simpest model for adsorption chromatography assumes that the solute, S, confined in the
aqueous phase (aq) is adsorbed to the ligand, L, of a solid chromatography surface. The process is
expressed by:
k
1
o
S(aq) + L(s) 
m
k
S-L(s)
(19)
2
The adsorption process is characterized by an association constant Ka (Ka = k1/k2 where k1 is the
forward rate constant and k2 is the backward rate constant) and a dissociation constant Kd (Ka = 1/ Kd).
The association constant is given by
Ka =
>S - L@ =
>S@>L@
Q
(20)
C eq Qmax Q Were Q is the total amount of protein adsorbed per ml gel and Ceq is the protein concentration in the
supernatant. Rearranging eq. 20 yields the following relationship for the amount of adsorbed solute:
Q=
Qmax K a C eq
1 K a C eq
=
Qmax C eq
(21)
K d C eq
Even though the Langmuir isotherm is only valid for specific conditions (e.g. monolayer noncompetitive adsorption) it has been successfully applied as a first approximation to describe adsorption
in preparative chromatography of biomolecules >adopted from Sofer and Hagel, 1997@.
35
Fig 6.5 Adsorption of BSA and monoclonal IgG (equal molar ratio) to
SP Sepharose Fast Flow. Buffer: 50 mM acetate pH 4.5
a) Confocal images
b) Fluorescence intensity profiles
30 min
1h
2 h 23 min
200
200
200
150
150
150
100
100
100
50
50
50
0
0
0
20
40
60
80
100
4 h 50 min
0
0
120
20
40
60
80
100
120
6 h 18 min
0
200
200
150
150
150
100
100
100
50
50
50
0
0
20
40
60
80
100
120
100
33 h
200
0
50
0
0
20
40
60
80
100
120
36
0
25
50
75
100
Fig 6.6 Optical sectioning of an adsorbent particle.
Experimental conditions: SP Sepharose Fast Flow saturated with
BSA-OregonGreen and monoclonal IgG-Cy5 in 50 mM acetate pH 4.5.
Incubation time 6 h. Step size between optical sections: 2 Pm.
Fig 6.7 Comparison of fluid phase and confocal data (BSA/IgG adsorption)
Adsorbent: SP Sepharose Fast Flow. Buffer: 50 mM acetate pH 4.5
Squares = BSA and circles = IgG
Open symbols: capacity calculated from the fluid phase.
Closed symbols: capacity calculated from the solid phase (i.e. confocal measurements)
Qrel [Units/m3] Solid phase
100
80
80
60
60
40
40
20
20
0
0
0
10
20
30
Time (h)
37
40
Protein capacity Q [mg/ml]
Fluid phase
100
120
Fig 6.8 Influence of pH on mass transport and adsorption of BSA and monoclonal
IgG. Adsorbent: SP Sepharose Fast Flow. Sample: BSA-Alexa 150 Pg/ml +
monoclonal IgG-Cy5 150 Pg/ml in hybridoma supernatant diluted 1:2 with 50 mM
acetate buffer (conductivity a 8.5 mS/cm). a) pH 4.5, b) pH 5.0 and c) pH 5.5
38
6.1.4 Visualization of nucleic acids
Chromatography is an important technique for purification of plasmid DNA, both as a
large scale process step and as an analytical tool. For large scale chromatographic
purification of plasmid DNA, one of the major problems is the lower capacity of the
adsorbent particles in comparision to what is obtained in protein purification. One
important issue for developing improved adsorbents for plasmid purification is
knowledge about the distribution of DNA adsorbed to the particles. The aim of the
study presented in Paper IV was to evaluate the use of confocal microscopy for direct
visualization of plasmid DNA adsorbed to individual adsorbent particles.
Plasmid DNA (6.3 kilo base pairs in size) was incubated with (CTT)7 coupled NHSSepharose HP (media for triple helix affinity chromatography) and Q Sepharose XL.
Plasmids were visualized by labeling with the fluorescent dye YOYO-1, which forms
a highly fluorescent complex with double stranded DNA. The particles were then
analyzed by confocal scanning and the resulting confocal images were translated into
fluorescence intensity profiles. The thickness of the adsorption layer was measured
directly from the intensity profiles. The results show that adsorption of plasmid DNA
mainly takes place in an outer layer, while the interior of the particles remains empty.
Thus, a smaller particle diameter will result in an increased proportion of outer surface
area per unit volume, and therefore result in higher binding capacity. A comparison of
ion exchange media with different particle size distributions confirmed these
conclusions.
Similar results as above are shown in fig 6.9. Here plasmid DNA was visualized in
Q Sepharose XL (agarose based media) and Fractogel EMD DEAE (methacrylate
based) using the fluorescent probe PicoGreen and/or TOTO-3. Both these fluorescent
probes bound non-specifically to Fractogel (but not to the agarose media) and gave
strong fluorescent signals throughout the whole particle volume. To avoid this
problem, the plasmids were labeled before incubation with the adsorbent. Another
problem associated with confocal scanning in methacrylate particles is light
attenuation due to optical pathlength differences between fringe and core sections. To
avoid this problem, the confocal measurements were performed by index matching in
55 % glucose solution (See chapter 6.2.1 and Paper V).
In a similar way, RNA molecules were visualized in Q Sepharose XL by use of the
fluorescent probe RiboGreen (fig 6.10). Unfortunately RiboGreen reacts both with
DNA and RNA. Thus, to avoid crossreaction with adsorbed DNA, RNA visualization
has to be performed separately (without presence of DNA). An alternative way is to
label the different nucleic acids in advance, before saturation of the adsorbent
particles.
39
Fig 6.9 DNA visualization with PicoGreen and TOTO-3
a) Q Sepharose XL; DNA visualized with PicoGreen.
b) TOTO-3 labeled plasmid DNA adsorbed to Fractogel EMD DEAE*
Measurement performed after > 24 hours of batch incubation.
(* Confocal measurements were performed in 55 % glucose).
a) Q Sepharose XL
b) Fractogel EMD DEAE
Fig 6.10 Visualization of RNA with RiboGreen
Measurement performed after > 24 hours of batch incubation.
Adsorbent: Q Sepharose XL
a) Confocal image,
b) Fluorescence intensity profile
40
6.1.5 Observation of inner radial concentration rings.
Confocal measurements following single component adsorption to individual ion
exchange adsorbent particles have revealed a very interesting phenomenon. Thus,
under certain conditions of ionic strength and pH, adsorbed phase concentrations
have been observed that are higher at certain radial positions within the particle
compared with positions closer to the particle surface or to the center. One example is
described in figure 6.11 that shows confocal images following the adsorption of
lysozyme to two different cation exchangers (i.e. SP Sepharose Fast Flow and XL 1)
over time. On SP Sepharose Fast Flow, a concentration overshoot (below named a
“concentration ring”) is seen to form very early during the adsorption process. With
time it moves towards the center of the particle, and the protein concentration in the
ring gradually increases while the total amount of protein in the ring decreases (figs.
6.12 – 6.13). Finally at equilibrium the concentration ring has disappeared. However
under the same experimental conditions, no such concentration rings can be observed
on SP Sepharose XL 2. Another example is given in figure 6.14, that demonstrates the
effect of ionic strength on the adsorption of monoclonal IgG. By increasing the buffer
concentration from 50 to 150 mM acetate, the ring phenomenon gradually disappears.
Similar results have earlier been reported [Dziennik/Lenhoff, 1999; 2000]. The
authors also observed that no concentration ring appears at low ionic strength (i.e. 2-5
mM acetate), probably due to protein – protein repulsion. A third example is given in
figure 6.15 that shows a confocal image obtained with lactoferrin. With this protein,
multiple concentration rings can be observed on SP Sepharose XL (based on 6 %
agarose). However no such rings could be observed on a similar prototype matrix
based on 4 % agarose (with the same dextran content and ionic strength) or on SP
Sepharose Fast Flow.
To exclude the possibility that the observed concentration rings are related to optical
artifacts due to the fluorescent probes, investigations have been performed with
unlabeled proteins using a UV-laser source and by multiphoton excitation
[Dziennik/Lenhoff, 1999; 2000]. The results confirmed that concentration rings are
formed also without fluorescence labeling. However, the phenomenon can not be
explained with the current theory for ion exchange chromatography. Until now
(november 2001) no generally accepted explanation has been presented and the
suggested models are currently under debate.
One explanation of the phenomenon might be due to variation of the electrostatic
potential along the radial direction of the particles [Dziennik/Lenhoff, 1999; 2000;
Liapis 2001]. When positively charged proteins are adsorbed to the negatively charged
surface on the ion exchanger, the magnitude of the negative surface potential will be
reduced. This will cause the avarage pore potential with protein present to become less
negative compared to pores without protein. This pore potential will be a function of
the concentration of adsorbed protein. Since there is a radial gradient in protein
concentration during the adsorption process, there will also be a radial gradient in the
average pore potential, i.e. an electrical field in the direction of protein adsorption.
Therefore, an additional contribution to the protein flux caused by electrophoretic
migration has been suggested [Dziennik/Lenhoff, 1999; 2000@. Such contribution to
the protein flux could result in a local concentation overshoot in the protein front.
__________________________________________________________________________________
1
Sepharose XL media has the same base construction as Sepharose Fast Flow, that is 6 % highly crosslinked agarose beads. The XL beads have been derivatized by binding dextran spacers to the agarose
backbone, before coupling of the ion exchange groups. 2 The same result was obtained both with and
without the washing step before confocal measurement (6.1.2)
41
Recently, a theoretical model describing the formation of an intraparticel
concentration hump in a case of adsorption of a single charge analyte was presented
>Liapis, 2001@. By accounting for the presence of an electrical double layer1 in the
pores of adsorbent particle, the mechanism of the model considers the induced
interaction of electrostatic potential distribution inside the pore and the mechanism of
mass transfer by diffusion, electrophoretic migration and adsorption. According to the
model, a single pore is divided into two regions, namely the electrical double layer,
and the electroneutral core region around the pore centre line. The interplay between
mass transport steps in these regions leads to a formation of a hump in the radial
profile of adsorbate concentration, which subsequently results in a hump in the radial
concentration profile of the adsorbate in the adsorbed phase. It was suggested that this
hump could be considered to be the concentration ring observed in confocal scanning
laser microscopy experiments.
The models presented above might at least partially explain the development of
inner radial concentration rings. However for a more accurate explanation of the
phenomenon also explaining the multiple ring effect, more experimental work and an
extended theoretical model are needed. Nevertheless, observation of concentration
rings is a good example of the strength of confocal microscopy compared with a finite
bath or column experiment. The latter methods can not provide this kind of
information.
1
When a surface with ionisable groups is immersed in an electrolyte solution, a charged surface is
created. Due to thermal motion (i.e. entropy) the counter ions corresponding to the oppositely charged
surface group are not bound as stoichiometric 1:1 complexes to the surface. Instead, they are distributed
in a diffuse layer close to the surface, the so-called diffuse double layer. The final distribution of the
counter ions in the double layer is a result of the balance between the electrostatic attraction to the
charged surface, the way the counter ions shield each other, and the effect of thermal motion >Ståhlberg,
1999@.
42
Fig 6.11 Development of concentration rings during protein uptake (confocal images).
Sample: Lysozyme (Cy5) 2 mg/ml in 50 mM glycin buffer pH 9.0
A) SP Sepharose Fast Flow, B) SP Sepharose XL
Fig 6.12 Development of concentration rings during protein uptake (intensity
profiles). Adsorbent: SP Sepharose Fast Flow. Sample: Lysozyme (experimental
conditions as in fig 6.11)
15 m in
200
Intensity (arb. units)
Intensity (arb. units)
5 m in
150
100
50
200
150
100
50
0
0
0
50
0
100
30 m in
100
45 m in
200
Intensity (arb. units)
Intensity (arb. units)
50
xy-position ( P m)
xy-position ( P m)
150
100
50
0
0
50
100
200
150
100
50
0
0
xy-position ( P m)
50
xy-position ( P m)
43
100
Fig 6.13 Protein concentration in “concentration rings” at different times during batch
uptake. Upper diagram: Fluorescence intensity / volume of the ring.
Lower diagram: Total intensity in the concentration rings.
(All points are related to the value obtained after 5 minutes)
Adsorbent: SP Sepharose Fast Flow.
Sample: Lysozyme 2 mg/ml in 50 mM 50 mM glycin buffer pH 9.0.
2
Irel / V ring
1,5
1
0,5
0
0
15
30
45
60
Time (min)
1
I tot, ring
0,75
0,5
0,25
0
0
15
30
45
Time (min)
44
60
Fig 6.14 Development of concentration rings during protein uptake
Adsorbent: SP Sepharose Fast Flow. Sample: Monoclonal IgG (Oregon Green)
Buffer: 50 mM, 100 mM and 150 mM acetate pH 5.0
Fig 6.15 Concentration rings observed with lactoferrin.
Adsorbent: SP Sepharose XL. Sample: lactoferrin (Cy5) 4 mg/ml in 100 mM acetate
pH 4.5. Confocal image obtained after 24 hours of batch incubation.
45
6.2 Measurement of the spatial distribution of immobilized ligands
Developing methods for covalent immobilization of ligands to surfaces is a key
activity when designing media for bioprocessing and chromatography. To be
successful, knowledge about the immobilization process is necessary. In general, the
immobilization of a ligand consists of three different steps: activation of the surface to
make it reactive towards the functional group of the ligand, coupling of the ligand,
and deactivation and blocking of residual active groups. An overview of various
immobilization methods is given by Carlsson et. al. >J. Carlsson, 1998@.
One issue of great interest is knowledge about the resulting ligand distribution in the
chromatographic matrices. This information is important not only for evaluation of the
immobilization process itself, but also to provide data for modeling and simulation of
chromatography processes. Too high concentration at the outer layer of the particles
might lead to crowding, i.e., the functional groups are situated so close to each other
that simultaneous binding to all groups is impossible for sterical reasons. If crowding
occurs the capacity of the gel will be reduced. A high density of ligands may also have
an influence on the kinetics, promote avidity effects in the binding of large molecules,
or lead to association of smaller ones [Björklund, 1996; Woodbury, 1999]. Examples
with an uneven distribution has been reported [Subramanian, 1994, McAlpine, 1999].
Subramanian et al. studied the role of antibody density effects on immunosorbent
efficiency. Fluorescent-labeled beads were sectioned using a microtome, and labeled
antibodies were then visualized by immunofluorescence. McAlpine et al. used optical
analysis for indirect measurement of the distribution of a fluorescent dye covalently
attached to the particles. Detection at the wavelength-specific emission of the
fluorophore allowed visualization of the dye distribution. However, faster methods for
direct measurement of ligand distribution in individual particles are desirable.
6.2.1 Studies of trypsin immobilization on porous glycidyl methacrylate beads
A major limitation to the use of confocal microscopes to image thick samples lies in
the reduction of signal intensity when focusing deep into refractive-index-mismatched
specimens. In spherical particles, attenuation due to optical pathlength differences
between fringe and core sections can be observed as a false minimum in the intensity
profiles >Visser, 1991; Roerdink, 1993; Liljeborg, 1996@. An example is given in
Paper V, where the immobilization of FITC-labeled trypsin on porous glycidyl
methacrylate (GMA-GDMA) beads was investigated.
Preliminary results indicated that the immobilized trypsin was most concentrated at
the bead surfaces. Thus, fluorescence intensity profiles were characterized by a
marked minimum at the center of the particles. It was also observed that the minimum
depended on the observation depth, with more pronounced minima displayed at larger
observation depth. The same effect could also be observed for immobilized FITC and
FITC-dextran in the absence of protein. With the aim to reduce the refractive index
difference between the bead (approximately 1.44 – 1.48) and the surrounding solution,
solutions containing different concentrations of D-glucose were added to the aqueous
samples. In this way the refractive index in the solution could be increased from about
1.34 to 1.42 (the latter value given by the solubility limit of D-glucose). The
magnitude of the intensity minimum was found to decrease with an increasing glucose
concentration (fig 6.16a), and at 55 wt% sugar in the aqueous solution, the intensity
minimum was essentially eliminated (fig 6.16b).
46
Fig 6.16 Effects of glucose concentration on intensity profiles. A) Relative intensity
lost in the center of the beads compared with the fringe intensity. B) Intensity profiles
obtained from porous glycidyl methacrylate beads with immobilized FITC-trypsin at a
glucose concentration of 0% (filled symbols) and 55% (open symbols) in the aqueous
solution. The bead size was a 120 Pm, whereas the observation depth was 20 Pm.
Thus, in the presence of a high concentration of glucose in the measuring solution the
intensity profiles for both FITC, FITC-trypsin and FITC-dextran were quite
homogenous (Paper VI, fig 3-5)
For comparison, also poly(styrene-co-divinylbenzene) (PS-DVB) beads (110-130
Pm) with immobilized trypsin were investigated. In this case the intensity distribution
was nonuniform also at high glucose concentration (Paper V, fig 6). However, the
refractive index of PS (| 1.6) is significally higher then that of acrylate polymers, and
therefore the uneven distribution is probably artifactual and due to an insufficient
contrast matching.
6.2.2. Measurement of ligand distribution in affinity- and ion exchange media.
In Paper VI, two independent methods, i.e. confocal scanning laser microscopy and
confocal micro-Raman spectroscopy, were used to analyze the spatial distribution of
IgG antibodies immobilized on BrCN-activated agarose beads. Both these
measurement methods indicate an even distribution of immobilized antibodies within
Sepharose 4 Fast Flow and Sepharose 6 Fast Flow.
In the first method the internal distribution profile of fluorescence labeled Protein A
was used as an indirect measure of the distribution of IgG. The influence of protein
concentration was investigated by determining the adsorption isotherms for Protein A
(fig 6.17). Furthermore, adsorbent particles incubated with varying Protein A
concentration were analyzed with confocal microscopy. The results reveal that a
Protein A concentration below the saturated part of the isotherm leads to
inhomogeneous adsorption to the particles (fig 6.18) Thus, for indirect measurement
of ligand distribution through adsorption of fluorescence labeled molecules, it is very
important to make sure that the fluid phase concentration is sufficient for saturation of
the whole particles.
In the second method, confocal micro-Raman spectroscopy was used to detect
vibrations originating directly from the immobilized antibodies. The advantage of this
method is that no sample preparation is required. However, the measurement times are
longer and the sensitivity is lower. Furthermore, for direct measurement with Raman
spectroscopy it is necessary to identify a significant band originating from the ligand,
that gives a sufficiently strong signal in relation to the base matrix.
47
Fig 6.17 Adsorption isotherms for Protein A on IgG Sepharose 4 Fast Flow
(diamonds) and IgG Sepharose 6 Fast Flow (sqares).
10
Q [ mg/mL bed ]
8
6
4
2
0
0
0,5
1
1,5
C [ mg/mL ]
2
2,5
Fig 6.18 Fluorescence intensity profiles obtained from IgG Sepharose Fast Flow
after 72 hours of batch incubation with varying fluid phase concentrations.
A) 0.8, B) 0,4, c) 0.2 and D) 0.1 mg Protein A / ml.
A)
B)
150
150
100
100
50
50
0
0
0
50
100
0
C)
50
100
D)
150
150
100
100
50
50
0
0
50
0
100
0
48
50
100
In paper VII, confocal Raman and fluorescence spectroscopy were applied for
measurement of the spatial distribution of cation exchange- and chelating- groups,
using Nd3+ ions as fluorescent probes. A comparison was also made between
measurements performed with different microscope objectives (i.e. a dry metallurgical
and a water immersion objective).
Agarose particles with a surface layer of sulphopropyl groups were used to
investigate the applicability of the confocal spectroscopic method. Nd3+ was attached
to the negatively charged sulphopropyl groups, which made it possible to follow the
distribution of sulphopropyl groups by the distribution of Nd3+. Measurements were
performed both from side to side and from bottom to top (fig 6.19). As can been seen
the distribution shows the expected appearance with a decrease of sulphopropyl
groups in the middle of the particle.
Fig 6.19 Intensity profile of Nd3+ fluorescence in a Sepharose particle with a surface
layer of sulphopropyl groups .
1400
side to side
bottom to top
Intensity
1200
1000
800
600
400
200
0
0
20
40
µm
60
80
100
In Chelating Sepharose Fast Flow Nd3+ was attached to the chelating iminodiacetic
groups. Measurements were performed both from side to side through the center and
from bottom to top of the particles. The depth profiles obtained with the dry
metallurgical objective showed a maximum of the Nd3+ signal in the middle of the
particles whereas flat intensity profiles were obtained with the immersion objective
(fig 6.20). The increase of the Nd3+-signal in the middle of the particle when using the
dry metallurgical objective could be explained by the increase of the focal volume
when focusing into a material of a different refractive index [Everall, 2000]. The
effect can be seen in the measurement from side to side. In the edge of the particle a
small amount of the particle and a larger amount of the surrounding water is included
in the focal volume and the measurement gives a low Nd3+-signal. In the middle of the
particle the focal volume is exclusively from the particle and hence a large Nd3+-signal
can be obtained. When the immersion objective is used the focal volume is smaller
and no differences of the middle and the edge can be found. These results show the
importance of using an immersion objective when wet adsorbent particles are
examined.
49
Fig 6.20 Intensity profile of Nd3+ fluorescence in Chelating SepharoseTM Fast Flow
a. particle measured with a dry metallurgical 50x-objective.
b. particle measured with a water immersion 63x-objective.
8000 a)
7000
Intensity
6000
5000
4000
side to side
bottom to top
3000
2000
0
10
20
30
40
50
60
µm
7000
6500
b)
6000
Intensity
5500
5000
4500
4000
bottom to top
side to side
3500
3000
0
20
40
60
µm
50
80
100
7. Future work
The results presented in this thesis demonstrate the great potential offered by
confocal microscopy and confocal Raman spectroscopy for direct observation of
adsorption processes, and for measuring the spatial distribution of immobilized
functional groups. The methods are at this point by no means fully explored in all
their modifications. The work is being continued both in Uppsala and elsewhere. The
following issues ought to be further investigated:
x The methodology presented in the thesis has mainly been developed and applied on
media for ion exchange and affinity chromatography. However, similar measurements
should be performed with adsorbent particles for hydrophobic interaction and reversed
phase chromatography. Furthermore, development of the technique is needed for
confocal measurements in less transparent samples, like methacrylate and PS-DVB
based particles, and for scanning deep into thick samples. In these cases, numerical
methods are needed to compensate for optical artifacts due to adsorption and
scattering of both the excitation and fluorescent light. Another possibility might be to
use multiphoton excitation, which makes it possible to image twice as deep into a
sample compared with conventional confocal microscopy.
x The studies of batch uptake in a finite bath can be supplemented by direct
measurement of adsorption- and desorption processes under dynamic conditions. By
continuous scanning of individual particles in a flow chamber over time, it should be
possible to study the effect of flow rate on protein uptake. It might also be possible to
measure the occurrence and effect of convective mass transport through single
adsorbent particles. Furthermore, this approach allows studies of desorption processes
using pH, salt gradients or displacers, where the batch method described in this thesis
is too slow.
x Modeling and simulation are important tools which can be used to optimize
chromatography processes. In order to perform accurate simulations, experimental
methods are required which allow a reliable determination of the model parameters.
Confocal microscopy allows the intraparticle protein concentration profile and the
diffusion distance to be determined in real time. Utilization of these data will certainly
result in more accurate modeling and simulations.
x The use of different fluorescent dyes for protein labeling and independent detectors,
allows direct observation of a multi-component adsorption processes. The twocomponent study with BSA and IgG should be extended by varying experimental
conditions like pH and ionic strength. These studies should also be performed with
other protein systems. The technique offers the possibility for simultaneous
visualization of up to four different components.
x Observation of the development of inner radial concentration rings is a good
example of the strength of confocal microscopy. Although experimental observations
have been shown and theoretical models have been suggested, more experimental
work (including other proteins and a variety of experimental conditions) and an
extended theoretical model are needed in order to fully explain the phenomenon.
51
8. Abbreviations and nomenclature
Airy disc
Three-dimensional diffraction pattern of object formed by each
imaging point in the specimen. The overlap of neighboring Airy
discs determines the resolution of the microscope.
Aperture
The detector aperture, or pinhole, positioned in front of
the detector, allowing detection of in-focus light only.
Band-pass filter
Filter that passes light of a certain restricted range
of wavelengths.
Coherent light
Light beam where the photons are in phase. A laser is an
example of such a light source.
Detector
The detection device can be a photomultiplier tube
(PTM), CCD camera (charge coupled devise) or photo-diode.
The voltage between the anode and cathode in a PTM controls
the brightness of the image.
Dichroic mirror
Mirror that reflects light below a certain wavelength (usually
excitation light) but transmits light above a certain wavelength
(usually emission light).
Fluorescence
Property of certain molecules to absorb energy in the form of light and
then release this energy at longer wavelength than the wavelength of
absorption (i.e. at lower energy level).
Fluorescence
intensity profile
Pixel values in a digitized section along a user-defined
area (in this work along the particle diameter), displayed
in a diagram.
FWHM
Full-Width-Half-Maximum. The full width of a light intensity
curve measured at 50% of the intensity maximum. FWHM is
often referred to as the optical section thickness.
Horizontal section
A single, two-dimensional confocal image perpendicular
to the optical axis.
Isoelectric point
The isoelectric point (pI) is the pH at which there is no net electric
charge on a protein
Intensity
How bright an object is. Intensity is proportional to amplitude
squared.
Laser
A laser is a device that produces an intense, concentrated, and
parallel beam of coherent light. The name laser is an acronym of
Light Amplification by Stimulated Emission of Radiation.
52
Magnification
Relationship between the size of an image and the size of the
original object.
Mass transfer
A general term covering the mechanisms and speed of transport
for “moving” a solute into a column, into the bead and towards
the ligand – and vice versa. The mass transfer mechanisms can
be divided in four different steps: mass transfer in the mobile
phase, in the stagnant film layer around the particles,
intraparticle mass transfer and finally adsorption to (or
desorption from) the ligand.
Multi-photon
microscopy
In multi-photon fluorescence microscopy, two or more photons, which
individually have insufficient energy to excite the fluorescence
molecule, interact co-operatively to achieve excitation. The
experimental benefits are reduced photobleaching, reduced cytotoxic
effects and improved sensitivity and optical sectioning. Furthermore it
is possible to image twice as deep into a sample compared with
conventional confocal imaging.
Numerical aperture
A measure of the light collecting ability of the objective, defined as
N.A. = n sin(D), where n is the refractive index of the medium
between objective and object, and Dis the half angle of the light cone
probing the object. A large aperture allows in more light and gives
higher resolution, however at the expense of a shorter working
distance.
Optical section
A confocal image represents a focus plane of a certain
thickness. This plane is called an optical section.
Photobleaching
Photochemical reaction of fluorophore, light and oxygen that causes
the intensity of the fluorescence emission to decrease with time.
Photomultiplier tube Converts incoming photons of light into electrons and then amplifies
the number of electrons
Pixel
A two-dimensional picture element in a confocal image.
The pixel size is related to the distance between scanned points.
Quenching
Any process that decreases the quantum efficiency of a fluorophore.
Refractive index
Relationship between velocity of light in a material of interest
and the velocity of light in vacuum.
Resolution
Smallest distance by which two objects can be separated and still
be resolved as separate objects.
53
Section series
A stack of confocal images separated in space to describe a
three-dimensional volume.
Spherical aberration Inaccurate focusing of light due to the curved surface of a lens.
THAC
Triple helix Affinity chromatography. Triple helix affinity interaction
involves the formation of a three stranded structure that comprises at
least one strand of the target DNA and one or two strands of an
affinity recognition probe. This probe can either be a polynucleotide
or a nucleic acid analogue.
Vertical section
A single, two-dimensional confocal image parallel to the optical
axis (perpendicular to a horizontal section).
Voxel
A three-dimensional pixel in a confocal image. The voxel size is
related to the pixel size, times the step size between sections in a
section series.
54
9. Acknowledgments
First, I would like to thank my supervisors:
Docent Lars Hagel, Amersham Biosciences, for critical reading of all my manuscripts
and for many useful discussions. Professor Karin Caldwell, Center for Surface
Biotechnology, for accepting me as a graduate student, and for critical reading of the
thesis. Professor Jan-Christer Janson, Center for Surface Biotechnology, for his
support and for taking care of the theoretical part of my graduate studies, especially
during the time before the Center for Surface Biotechnology was established. My
colleague and co-author Rolf Hjorth. It was Rolf that originally gave me the idea to
evaluate the use of confocal microscopy for characterization of chromatography
media.
I also want to thank:
Ove Öhman (at the time at Pharmacia Biotech) who gave me my first practical
experience in how to handle the confocal microscope. Jörg Thömmes and Thomas
Linden, Heinrich Heine University Jülich, Germany, Martin Malmsten and Kezhao
Xing, Institute for Surface Chemistry, Stockholm, Kjell Carlsson and Anders
Liljeborg, the Royal Institute of Technology, Stockholm, Mina Larsson and Jan
Lindgren at the Ångström laboratory, Uppsala University, Stefan Gunnarsson and
Gary Wife, Center for Evolution Biology, Uppsala University. All my colleagues and
friends at Amersham Biosciences.
Finally, I want to thank my family: Annika, Åsa and Tomas for all their understanding
and patience for the time I have spent at home reading books and scientific papers and
in front of the computer.
55
10. References
Amir, H. N., Moronne, M. and Ferrari, M. 1998 ”Detection of functional groups and
antibodies on microfabricated surfaces by confocal microscopy.” Biotechnol.
Bioeng. 60, 137-146
Arve, B. H. and Liapis, A. I. 1987 ”Modeling and analysis of biospecific adsorption in
a finite bath.” AIChE J. 33, 179-193
Asplund, M., Ramberg, M. And Johansson, B-L. 2000 ”Development of a cleaning in
place protocol and repetitive application of Escherichia coli homogenate on
STREAMLINE™ Q XL.” Process Biochemistry 35, 1111-1118
Bancel, S. and Hu, W-S 1996 ”Topographical imaging of macroporous microcarriers
using laser scanning confocal microscopy.” J. Ferment. Bioeng. 81, 437-444.
Banwell, C. N. and McCash, E. M. 1994 ”Fundamentals of molecular spectroscopy”
McGraw-Hill publishing company, Maidenhead, England
Béland, M-C. and Mangin, P.J. 1995 ”Three-dimensional evaluation of paper
Surfaces using confocal microscopy.” Surface analysis on paper, chapter 1.
Editors: T. E. Conners and S. Banerjee, CRC Press, Inc
Belaroui, F., Grohens, Y., Boyer, H. and Holl, Y. 2000 “ Depth profiling of small
molecules in dry latex films by confocal Raman spectroscopy” Polymer 41, 7641
Björklund, M. and Hearn, M.T.W. 1996 “Synthesis of silica-based heparin-affinity
adsorbents” J. Chromatogr. A, 728, 149-169
Booth, M. J., Neil, M.A.A. and Wilson, T. 1998 ”Aberration correction for confocal
imaging in refractive-index-mismatched media.” J. Microscopy 192, vol 90, 90-98
Brewer, L. R., Davidson, J. C., Balch, J. B. and Carrano, A. V. 1995 ”Threedimensional imaging of DNA fragments during elecrophoresis using a confocal
detector.” Electrophoresis 16, 1846-1850
Brocks, C. a: and Cramer, S. 1992 “Steric mass-action ion exchange: Displacement
profiles and induced salt gradients”. AIChE Journal 38(12), xx-xx
Carlsson, J., Janson, J-C. and Sparrman, M. 1998 “Affinity chromatography”
In: Janson, J-C and Ryden, L.(Editors) “Protein purification – principles, high
resolution methods and applications” Wiley-VCH, New York 1998, Chapter 5.
Carlsson, K., Danielsson, P. E., Lenz, R., Liljeborg, A., Majlöf, L. and Åslund, N.
1985 ”Three-dimensional microscopy using a confocal laser scanning microscope.
Opt. Lett. 10, 53-55
Carlsson, K, and Åslund, N. 1987 ”Confocal imaging for 3-D digital microscopy.”
Appl. Opt. 26, 3232-3238
Carlsson, K. 1994 ”An introduction to conventional and confocal microscopy.”
The Royal Institute of Technology, Stockholm, Sweden
Carlsson, K. and Mossberg, K. 1992 ”Simultaneous confocal recording of multiple
fluorescent labels with improved channel separation.” J. Microscopy 176, 287-299
Carlsson, K. and Ulfhake, B. 1995 ”Improved fluorophore separation with IMS
confocal microscopy.” J NeuroReport 6, 1169-1173.
Carlsson, K. and Liljeborg, A. 1998 ”Confocal Fluorescence microscopy using IMS:
Evaluation of results from spectral and lifetime imaging.” SPIE vol 3261A, 30-36
56
Chang, C. And Lenhof, A. M. 1998 ”Comparison of protein adsorption isotherms and
uptake rates in preparative cation-exchange materials.”
J. Chromatogr. A 827, 281-293
Cutts, L. S., Roberts, P. A., Adler, J., Davies, M. C. and Melia, C. D. 1995
”Determination of localized diffusion coefficients in gels using confocal scanning
laser microscopy. J. Microscopy 180, 131-139
de Beer, D., Stoodley, P. and Lewandowski, Z. 1997 ”Measurment of local diffusion
coefficients in biofilms by microinjection and confocal microscopy.” Biotechnol.
Bioeng. 53, 151-158
DeLeo, P. C., Baveye, P. and Ghiorse, W. C. 1997 ”Use of confocal microscopy on
soil thin-sections for improved characterization of microbial growth in
unconsolidated soils and aquifer materials.” J. Microbiological Meth. 30, 193-203
De Smedt, S. C., Meyvis, K. L., Demeester, J., Van Oostvelt, P., Blonk, J.C.G. and
Hennink, W. E. 1997 ”Diffusion of macromolecules in dextra methacrylate
solutions and gels as studied by confocal scanning laser microscopy.”
Macromolecules 30, no 17, 4863-4870
Doyle, C. A., Vickers, T. J., Mann, C. K. And Dorsey, J. G. 1997 ”Characterization of
liquid chromatography stationary phases by Raman spectroscopy. Effect of ligand
type.” J. Chromatogr. A 779, 91-112
Doyle, C. A., Vickers, T. J., Mann, C. K. And Dorsey, J. G. 2000 ”Characterization of
C18-bonded liquid chromatographic stationary phases by Raman spectroscopy.”
J. Chromatogr. A 877, 25-39 and 41-59
Driscoll, W. G. and Vaughan, W. (Editors) ”Handbook of optics.” Optical society of
America. McGraw-Hill, Inc 1978
Dziennnik, S. R. and Lenhoff, A. M. 1999 “Use of confocal microscopy to resolve
Protein transport mechanisms in chromatographic particles” Oral presentation at
AIChE meeting, Dallas, 1999
Dziennnik, S. R. and Lenhoff, A. M. 2000 “The use of confocal microscopy to resolve
protein transport maechanisms in chromatographic particles” Oral presentation at
PREP 2000, Washington D.C.
Egner, B. J., Rana, S., Smith, H., Bouloc, N., Frey, J. G., Brocklesby, SW. S. and
Bradley, M. 1997 ”Tagging in combinatorial chemistry: the use of coloured and
fluorescent beads.” Chem. Commun. 735-736
Everal, N.J. 2000 “Modeling and measuring the effect of refraction on the depth
resolution of confocal Raman microscopy” Applied Spectroscopy 54 (6), 773 –782
Everal, N.J. 2000 “Confocal Raman Microscopy: Why the depth resolution and spatial
accuracy can be much worse than you think” Applied Spectroscopy 54 (10), 1515-20
Fagnano, C., Rossi, M., Porter, R. S. and Ottani, S. 2001 “A study of solid-state
Drawn fibers of polyethylene by confocal Raman microspectrometry: evaluation of
the orientation profiles of amorphous and crystalline phases across the fiber
section” Polymer 42, 5871
Go, W.G., Roettger, B. F., Holicky, E. L., E. M. Hadac and Miller, L. J. 1997
”Quantitative dynamic multicompartment analysis of cholecystokinin
receptor movement in a living cell using dual fluorophores and reconstruction of
confocal images.” Anal. Biochem 247, 210-215
Herman, B. 1998 Fluorescence Microscopy. BIOS Scientific Publishers, Oxford, UK.
57
Horstman, B. J. and Chase, H. A. 1989 ”Modeling the affinity adsorption of
immunoglobulin G to Protein A immobilized to agarose matrixes.”
Chem. Eng. Res. Des. 67, 243-254
Ingle, J. D. And Crouch, S. 1988 Spectrochemical analysis. Prentice Hall Inc.
Janson, J-C. and Jönsson, J-Å. 1998 “Introduction to chromatography” In:Janson, J-C
and Ryden, L.(Editors) “Protein purification – principles, high resolution methods
and applications” Wiley-VCH, New York 1998, Chapter 2.
Karlsson, E., Ryden, L. and Brewer J 1998 “Ion exchange chromatography” In:
Janson, J-C and Ryden, L.(Editors) “Protein purification – principles, high
resolution methods and applications” Wiley-VCH, New York 1998, Chapter 4.
Kim, H. B., Hayashi, M., Nakatani, K. and Kitamura, N. 1996 ”In situ measurments of
ion-exchange processes in single polymer particles: Laser trapping micro
spectroscopy and confocal fluorescence microspectroscopy.” Anal. Chem.
68, 409-414
Kress, J., Rose, A., Feey, J. G., Brocklesby, S. W., Ladlow, M., Mellor, W. G. and
Bradley, M. 2001 “Site distribution in resin beads as determined by confocal
Raman spectroscopy” Chem. Eur. J. 7, 3880-3883
Kågedal, L. 1998 “Immobilized metal ion affinity chromatography” In: Janson, J-C
and Ryden, L.(Editors) “Protein purification – principles, high resolution methods
and applications” Wiley-VCH, New York 1998, Chapter 8.
Laca, A., Garcia, L. A., Argueso, F. And Dias, M. 1999 ”Protein diffusion in alginate
beads monitored by confocal microscopy. The application of wavelets for data
reconstruction and analysis.” J. Industrial Microbiol. & Biotechnol. 23, 155-165
Lakowicz, J. R. 1999. “Principles of fluorescence spectroscopy” Kluver Academic
/Plenum Publichers, New York
Liapis, A. I., Grimes, B. A., Lacki, K. and Neretnieks, I. 2001 “Modeling and
analysis of the dynamic behavior of mechanisms that result in the
development of inner radial humps in the concentration of a single adsorbate in the
adsorbed phase of porous adsorbent particles observed in confocal scanning laser
microscopy experiments: diffusional mass transfer and adsorption in the presence
of an electrical double layer.” J Chromatogr. A 921,no 2, 135-145
Liu, J. 1997 Dissertion: ”The roles of surface heterogenity in protein-biomaterial
interactions and platelet adhesion.” Dept. of Material Science and Engineering,
The University of Utah.
Liljeborg, A. 1996 ”Simulation of light attenuation within fluorescent microsperes
Used for liquid fraction separation recorded by a CSLM.” SPIE vol 2655, 11-17
Lin-Vien, D., Colthup, N. B., Fately, W. G. And Grassel, J. G. 1991 ”The handbook
of Infrared and Raman characteristic frequencies of organic molecules” Academic
press, Inc, San Diego.
Lundqvist, A., Ocklind, G., Haneskog, L. and Lundahl , P. 1998 ”Freeze-thaw
immobilization of liposomes in chromatographic gel beads: Evaluation by confocal
microscopy and effects of freezing rate.” J. Mol. Recogn. 11, 1-6
Majlof, L. and Forsgren, P-O. 1993 ”Confocal microscopy: Important considerations
for accurate imaging.” Methods in cell biology, vol 38, 79-95.
Editor B. Matsumoto, Academic Press 1993.
Masters, B. R. (Editor) ”Selected Papers on Confocal Microscopy” The International
Society for Optical Engineering, Washington, 1996
58
Mattisson, C. 1999 Dissertion: ”Diffusion studies in gels using holographic laser
interferometry.” Dept. of Chemical Engineering I. Lund University.
Mattsson, H., Torell, L. M. and Sundholm, F. 2000 “Degradation of a fuel cell
membrane, as revealed by micro-Raman spectroscopy” Electrochimica Acta
45, 1405
McAlpine, S.R. and Screiber, S.L. 1999 “Visualizing functional group distribution in
solid-support beads using optical analysis” Chem. Eur. J. 12, 5-x
Minsky, M. "Microscopy apparatus" US Patent 3,013,467 (December 19, 1961)
Minsky, M. 1988. ”Memoir on inventing the confocal scanning microscope”
Scanning vol 10, no 4, 128-38
Mura, C., Yarwood, J, Swart, R. and Hodge, D.. 2000 “ Raman microscopic studies of
the distribution of the fungicide fluorfolpet in plasticised PVC films”
Polymer 41, 8659
Pawley, J. B. (Editor) ”Handbook of Biological Confocal Microscopy”, Plenum
Press,New York, 1995
Pemberton, J. E., Ho, M., Orendorff, C. J. and Ducey, M. W. 2001 “Raman
spectroscopy of octadecylsilane stationary phase conformational order. Effect of
solvent” J. Chromatogr. A 913, 243-252
Poliz, J. C., Browne, E. S., Wolf, D. E. and Pederson, T. 1998 ”Intranuclear
diffusion and hybridization state of oligonucleotides measured by fluorescence
correlation spectroscopy in living cells.” Proc. Natl. Acad. Sci. 95, 6043-6048
Rademann, J., Barth, M., Brock, R., Egelhaaf, M. and Jung, G. 2001 “Spatially
resolved single bead analysis: homogeneity, diffusion and adsorption in crosslinked polystyrene” Chem. Eur. J. 7, 3884-3889
Reese, I., Urlaub, E., Grapes, J. R and Lendl, B. 2000 “Multidimensional information
on the chemical composition of single bacteria cells by confocal Raman microspectroscopy.” Anal. Chem. 72, no 22, 5529-5534
Ribbe, A. E. 1997 ”Laser scanning confocal microscopy in polymer science.
Trends Polymer Sci. 5, 333-337
Roerdink, J. B. T. M. and Bakker, M. 1993 ”An FFT-based method for attenuation
correction in fluorescence confocal microscopy.” J. Microscopy 169, 3-14
Sacristan, C,, Reinecke, S., Spells, S. and Yarwood, J. 2000 “Selective surface
modification of PVC films revealed by confocal Raman microspectroscopy”
Macromolecules 33, 6134
Schaeberle, M. D., Morris, H. R., Turner, J. F. and Treado, P. J. 1999 ”Raman
Chemical Imaging Spectroscopy.” Anal. Chem. 71, no 5, 175-181
Schrof, W., Klingler, J., Heckmann, W. and Horn, D. 1998 ”Confocal fluorescence
And Raman microscopy in industrial research.” Colloid Polym Sci. 276, 577-588.
Schuster, K. C., Rese, I., Urlab, E., Grapes, J. R and Lendl, B. 2000
“Multidimensional information on the chemical composition of single bacterial
cells by confocal Raman microspectroscopy” Anal. Chem. vol 72, issue 22,
5529-5534
Sing, A. and Gopinathan, K. P. 1998 “Confocal microscopy: A powerful technique for
biological research.” Current Science 74, no 10, 841-851
Sofer, G and Hagel, L. 1997 “Handbook of process chromatography” Academic press,
London
59
Spiess, A. S. and Kasche, V. 2001 “Direct measurement of pH profiles in
immobilized enzyme carriers during kinetically controlled synthesis using CLSM”
Biotechnol. Prog. 17, 294-303.
Sheppard, C. J. R. and Shotton, D. M. 1997 ”Confocal laser scanning microscopy”.
BIOS Scientific Publishers, Oxsford, UK
Skidmore, G., Horstmann, B. J. And Chase, H. 1990 ”Modelling single-component
protein adsorption to the cation exchanger Sepharose FF.”
J. Chromatogr. A 498, 113-128
Ståhlberg, J. 1999 “ Retention models for ions in chromatography” J. Chromatogr. A
855, 3-55.
Subramanian, A., Van Cott, K.E., Milbrath, D.S. and Velander, W.H. 1994
”Role of local antibody density effects on immunosorbent efficiency”
J. Chromatogr. A 672, 11
Tata, B.V.R. and Baldev, R. 1998 “Confocal laser scanning microscopy: Applications
in material science and technology” Bull. Mater. Sci. 21, no 4, 263-278
Thill, A., Simon, V. B., Wiesner, M., Bottero, J. Y., and Snidaro, D. 1988
”Determination of structure of aggregates by confocal scanning laser microscopy.”
J. of Colloid and Interface Sci. 204, 357-362
Ulfhake, B., Mossberg, K., Carlsson, K., and Arvidsson, U. 1992 ”Confocal
fluorescence microscopy in three-dimensional analysis of axon terminal
distribution, neuronal connectivity, and colocalization of messenger molecules in
nervous tissue.” Computers and Computation in the Neurosciences, chapter 7.
Editor: P. Conn, Academic Press, INC, San Diego
Van Oostvelt, P. and Bauwens, S. 1989 “Quantitative fluorescence in confocal
microscopy” J. Microscopy 158, 121-132
Visser, T. D., Groen, F. C. A and Brakenhoff, G. J. 1991 ”Absorption and scattering
correction in fluorescence confocal microscopy.” J. Microscopy 163, 189-200.
Wallén, P., Carlsson, K. and Mossberg, K. 1992 ”Confocal laser scanning
Microscopy as a tool for studying the 3-morphology of nerve cells.” Visualization
in Biomedical Microscopes. 3-D Imaging and Computer applications, chapter 5.
Editor: A. Kriete, VCH Verlagsgesellschaft mbH, Weinheim, 1992
Webb, R.H. and Dorey, C.K. 1995. ”Handbook of Biological Confocal Microscopy”,
Pawley, J. B. (Editor) Plenum Press, New York,1995.
Wilson, T. 1989 ”Optical sectioning in confocal microscopes.”
J. Microscopy 154(2), 143-156
Wilson, T. (Editor) ”Confocal Microscopy” Academic Press Inc, San Diego, 1990
Woodbury, C.P. and Venton, D. L. 1999 “Methods of screening combinatorial
libraries using immobilized or restrained receptors” J. Chromatogr. B 725, 113137
Åslund, N. and Carlsson, K. 1993 ”Confocal scanning microfluoromertry of duallabelled specimens using two excitation wavelength and lock-in detection
technique.” Micron 24, 603-609
60
Appendix A
Molecular structure of fluorescent dyes used for protein labelling
FITC (fluorescein-5-isothiocyanate)
Mw: 389 g/mol
Alexa 488
SO3-
Mw: 643 g/mol
SO3-
O
H 2N
+
NH2
O
O
O
N
O
O
O
61
Oregon Green 488
O
HO
Mw: 509 g/mol
O
F
F
O
O
O
N
O
O
O
Cy3
SO3-
SO3-
Mw: 778 g/mol
N
N
+
O
N
O
O
O
Cy5
Mw: 792 g/mol
SO3-
SO3-
N
O
N
O
O
O
62
N
+
Appendix B
Excitation and emission spectra of fluorescent dyes
Fluorescence excitation and emission spectra were acquired with a SPEX Fluorolog-3
spectrofluorometer (ISA Instruments, New Jersey, USA) equipped with double grating
monochromators on both the excitation and emission side and with a Xe lamp as
excitation source. Each fluorescent dye was diluted in 50 mM acetate buffer pH 5.0 to
a concentration of a10-7 M. Excitation spectra were recorded by scanning the
excitation wavelength while keeping the emission wavelength (Oem) fixed; in analogy,
emission spectra were recorded at a fixed excitation wavelength (Oexc).
Excitation spectra for Alexa och Oregon Green were recorded from 400-540 nm (with
Oem=560 nm), Cy3 was scanned from 400-600 nm (Oem=610 nm) and Cy5 from 500700 nm (Oem=710 nm). In the same way emission spectra for Alexa and Oregon Green
were recorded from 470-700 nm (Oexc=460 nm), for Cy3 from 525-700 (Oexc=515 nm)
and for Cy5 from 610-800 nm (Oexc=600 nm).
B) Oregon Green
100
100
80
80
Normalized intensity
Normalized intensity
A) Alexa 488
60
40
20
60
40
20
0
0
400
450
500
550
600
650
700
400
450
500
Wavelength (nm)
C) Cy3
600
650
700
D) Cy5
100
Normalized intensity
100
Normalized intensity
550
Wavelength (nm)
80
60
40
80
60
40
20
20
0
0
400
450
500
550
600
650
700
500
550
600
650
Wavelength (nm)
Wavelength (nm)
63
700
750
800
Appendix C
Influence of fluorescent dyes on mass transport kinetics.
A) Retention time of labelled and unlabelled proteins in cation exchange
gradient elution
Small samples (0.02 mL, 2 mg/ml) of unlabelled protein and protein/dye conjugates
were analysed with a Shimadzu HPLC 10ADvp system (Duisburg, Germany) with a
flow rate of 0.5 mL/min. After loading the samples on a Sepharose Fast Flow column
(vol. 1 mL) equilibrated with 50 mM acetate buffer pH 5.0 the samples were eluted in
a linear salt gradient of 20 column volumes up to 50 mM acetate buffer supplemented
with 1 M NaCl. The elution was monitored by detecting the absorbance at 220 nm
with the UV/VIS SPD10Avp Detector. As a characteristic value the average of three
retention times per unlabelled protein and protein/dye conjugate was used to
investigate and quantify the deviations between retention of the different molecular
species. The maximum deviation between the retention time of labelled and
unlabelled protein was 3 %. Thus, the adsorption of neither IgG nor BSA was
significantly influenced by the coupling of fluorescent dyes.
Table 1. Retention time of labelled and unlabelled proteins in cation exchange
gradient elution on SP Sepharose Fast Flow (data from Paper VIII).
Sample
IgG Alexa
IgG Oregon
IgG Cy3
IgG Cy5
IgG unlabelled
Retention time (min)
18.59
18.02
18.37
18.5
18.63
% deviation
0.2
3
1.4
0.7
-
BSA Alexa
BSA Oregon
BSA Cy3
BSA Cy5
BSA unlabelled
13.78
14.06
13.68
13.81
13.93
1
1
1.8
0.9
-
B) Influence of protein labelling on the protein uptake pattern
The influence of different fluorescent dyes on the protein uptake pattern was
investigated by sequential incubation of different BSA protein/dye conjugates. Batch
incubation is started with protein labeled with one fluorescent dye. After a certain
time, the supernatant is removed and replaced with the same protein labeled with
another fluorophor, with the same protein concentration as the the end of the first
incubation. After a second incubation, this procedure is repeted once again with a
third fluorophore labeling (Paper VIII).
64
Figure 1 shows a control experiment performed with different combinations of
labelled proteins. All combinations gave the same result. Thus, different dyes does
not have any major impact on the adsorption pattern.
Fig. 1 Influence of different fluorescent dyes on the protein uptake pattern.
Adsorbent: SP Sepharose Fast Flow.
Lane 1: BSA-Alexa; Cy3 and Cy5. Lane 2: BSA-Cy3, Cy5 and Alexa
Lane 3: BSA-Cy5, Alexa and B Cy3. Lane 4: Control with BSA-Cy3.
(Cuortesy of Thomas Linden)
A) BSA in 50 mM acetate pH 5.0
B) BSA in 150 mM actetate pH 5.0
65
Appendix D
Experimental
Confocal microscopy analysis presented in Paper I, II, IV and V was performed with a
MultiProbe 2001 from Molecular Dynamics, and in Paper III and IV with Leica TCS
SP confocal scanning laser microscope. Both instruments where supplied with
argon/krypton lasers. Confocal micro-Raman spectroscopy was performed with a
Renishaw System 2000 micro-Raman spectrometer equipped with near-infrared diode
laser.
Sepharose™ 6 Fast Flow, IgG Sepharose, SP Sepharose Fast Flow, SP Sepharose XL,
NHS-Sepharose High Performance, CrBr-activated Sepharose, Sephadex™ G-25 M,
SOURCE™ S30, PS-DVB- methacrylate- and agarose particles, FITC-Protein A,
Cy3 and Cy5™ reactive dye were obtained from Amersham Biosciences (Uppsala,
Sweden). Fractogel EMD DEAE was purchased from Merck (Dramstadt, Germany),
and Oregon Green and Alexa 488 protein labeling kit, YOYO-1, PicoGreen,
RiboGreen and TOTO-3 from Molecular Probes Europe BV (Leiden, The
Netherlands). Lysozyme and BSA were purchased from SIGMA-Aldrich (Stockholm,
Sweden), and polyclonal human Immunoglobulin G (hIgG) from Pharmacia & Upjohn
(Stockholm, Sweden). Hybridoma cell culture supernatant and monoclonal IgG
(purified from hybridoma culture supernatant) was a kind gift from Thomas Linden,
Heinrich Heine University, Jülich, Germany. Plasmid DNA was kindly donated by
Rhone Poulenc Rorer, France. All other chemicals were of analytical grade and were
from commercial sources.
66
DIRECT OBSERVATION OF BIOMOLECULE ADSORPTION AND SPATIAL DISTRIBUTION
OF FUNCTIONAL GROUPS IN CHROMATOGRAPHIC ADSORBENT PARTICLES
ANDERS LJUNGLÖF 2002
ERRATA
Page 26 ,Table 5.2 row 5
Quaternary ammonium (Q)
Reads:
-O-CH2-CHOH-CH2-O- CH2-CHOH- CH2-N+(CH5)3
Should Read: -O-CH2-CHOH-CH2-O- CH2-CHOH- CH2-N+(CH3)3
Page 26, Table 5.2 row 6
Trimethylaminoethyl (TMAE)
Reads:
-O-CH2- CH2- N+(CH5)3
Should Read: -O-CH2- CH2- N+(CH3)3
Page 26, Table 5.2 row 7
Triethyl amine (TEAE)
Reads:
-O-CH2-N+(CH5)3
Should Read: -O-CH2-N+(C2H5)3
Page 49, row 6
Missing references: J.Bergström, R.Berglund, L.Söderberg,
international publication number, WO 98/39364 and WO 98/39094