Page 266 - Vibrational Spectroscopic Imaging for Biomedical Applications
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as high as 250 μg/μl, and DNA and RNA concentrations in the range
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of 100 μg/μl have been reported. These values depend on the cell
type, the phase of the cell cycle and the location within the cell.
Data acquisition of small objects with high spatial resolution such
as single cells and their subcellular organelles require coupling of
Raman spectrometers with microscopes. The coupling of a Raman
spectrometer with a microscope offers two main advantages. First,
lateral resolutions can be achieved up to the Abbe’s limit of diffrac-
tion below 1 μm according to the formula δ = 061 ⋅ λ NA/ . High
.
lat
axial resolutions are obtained by confocal microscopes. The diffrac-
tion limit in the axial dimension can be calculated as δ =⋅ λn/ NA 2
2
lat
(wavelength λ, refractive index n, numerical aperture NA). Second,
maximum sensitivity can be achieved because the photon flux (pho-
ton density per area) at the focused laser beam onto the sample is at
maximum and the collection efficiency of scattered photons from the
sample is at maximum. The lateral resolution, the diameter of the focused
laser and the collection efficiency depend on the NA of the microscope
objective which is the product of the sine of the aperture angle and the
diffraction index. High NA objectives have small working distances
below 1 mm in air. The numerical aperture of water immersion objec-
tives can be enlarged due to the larger diffraction index of water. Oil
immersion objectives in combination with oil should be avoided
because spectral contributions of oil overlap with the Raman spectrum
of the sample.
8.3.1 Lung Fibroblast Cells
Lung fibroblast cells were studied because cell cultivation can easily
be controlled, cell geometry and morphology seemed comparatively
suitable for depicting and discrimination of visible compartments,
they grow adherent to quartz slides and they keep their shape over
time after fixation by formalin. Although living cells can be studied
by Raman spectroscopy, fixation prevents the cell changes morphol-
ogy or composition during time-consuming acquisition of images by
linear Raman spectroscopy. Furthermore, the cell environment
should be carefully controlled in long-term experiments, e.g., cell
culture media, temperature, or carbon dioxide. Using the Raman
microspectrometer HoloSpec with 785 nm excitation (Kaiser Optical
System, United States), Raman images were collected with a step size
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of 1 μm. Acquisition of a 60 × 60 raster with 1 second exposure time
per spectrum takes more than 1 hour. Beside exposure time, addi-
tional time is needed to move the sample stage. Using the confocal
Raman microscope CRM300 (Witec, Germany), a Raman image of a
single cell was recorded with 0.2 second exposure time per spectrum
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and 300 nm step size. Various cell compartments could be identified
in this highly resolved Raman image. In spite of the low-exposure
time, Raman spectra of reasonable signal to noise ratio were obtained
after k-means cluster analysis. Calculating cluster averaged spectra