Towards Chemical Imaging of Living Cells: Design and Application of a Confocal Raman Microscope


Sijtsema, Nanna Maria (1997) Towards Chemical Imaging of Living Cells: Design and Application of a Confocal Raman Microscope. thesis.

Abstract:Raman microspectroscopy is a technique that can be used to obtain
information about the chemical composition of a very small measurement
volume (0.5 fl) in a (biological) sample. Molecules present in the sample
can be identified based on their scattering characteristics and no special
treatment or preparation of the samples is necessary. Therefore, biological
samples can be measured under physiological conditions and reactions in
living cells can be monitored.
We have developed a Confocal Direct Imaging Raman Microscope
(CDIRM) which enables the measurement of both Raman microspectra of
a small measurement volume and of Raman images which show the
distribution of a specific compound over the sample. The CDIRM is the
first example in literature of a confocal microscope which is based upon
direct imaging. All currently used confocal Raman microscopes work with
image reconstruction. Direct imaging has several advantages among which
the shorter measurement times that can be used in most applications.
In chapter 2 the design of the system has been discussed and its mains
characteristics, like resolution and image quality have been described. The
resolution of the set up as determined with a 0.282 μm sphere appeared to
be 0.37 μm in the lateral direction and 1.2 μm in the axial direction (Full
Width at Half Maximum (FWHM)). The resolution for a 275 nm layer was
determined to be 1.4 μm in the axial direction. We have demonstrated that
high resolution Raman images of biological samples can be made with the
CDIRM. Raman images have been measured of the DNA and protein
distribution in a polytene chromosome. These images illustrate the
capability of our system to make Raman images of a sample with a
relatively weak Raman signal: only 0.1 photons/(second×pixel) were
detected. Further, we have shown that our system can be used to make 3-
dimensional Raman images of biological samples. 3-dimensional images of
the distribution of a drug in a living cell and of cholesterol in an eye lens
slice have been presented.
Raman microspectroscopy is one of the few techniques that enables the
monitoring of processes in single living cells, without chemical treatment
of the sample which might disturb the cellular system. In chapter 3 Raman
measurements on single activated human neutrophilic and eosinophilic
granulocytes have been shown. The granulocytes were activated by
addition of the soluble activator Phorbol Myristate Acetate or by
opsonized particles. Raman spectra were measured in the cytoplasm and
the phagosome of activated granulocytes. The resulting spectra were
compared with spectra of the native cells and clear differences could be
recognized. The results indicated an intracellular reduction of both
Myeloperoxidase and cytochrome b558, two heme-proteins which are
known to play a role in the human immune system.
An important advantage of Raman imaging compared to fluorescence
imaging is that no extrinsic labels have to be introduced to distinguish
specific molecules. However, in samples with a low concentration of weak
Raman scattering molecules it can be advantageous to introduce extrinsic
labels. These Raman labels should bind specifically to the molecules of
interest and have a relatively large Raman scattering cross section. In
certain applications it can be preferable to use such Raman labels instead
of fluorescent labels, because of their much narrower bandwidth, which
allows the detection of many more different labels in a limited wavelength
range and because they do not bleach. In chapter 4 two examples of
extrinsic Raman labeling have been demonstrated: the use of the
cholesterol specific label filipin for visualizing the cholesterol distribution
in an eye lens and the application of antibody coated polystyrene spheres
to distinguish different phenotypes of human leukocytes. Further, a
discussion is given about which molecules and structures can be used in
the development of other suitable Raman labels.
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