In Vitro Raman and Fluorescence Laboratory System
Our laboratory system is versatile, capable of collecting both Raman and fluorescence
spectra from both macroscopic and microscopic samples. Raman excitation is provided
by an argon ion laser-pumped Ti:sapphire laser, tunable between 720 and 1000 nm, with
output as high as 1 W (usually 50-300 mW for tissue studies, depending on how tightly
focused the excitation spot is on the sample). The excitation laser beam traverses a
bandpass filter and can either be focused onto a bulk sample (~1 mm diameter spot) or
launched into the microscope (resolution ~2 m) via a prism that can be moved in and
out of position. If the bulk tissue path is followed, the Raman scattered light is collected
and collimated by a f/1.2 camera lens, filtered by a holographic Raman edge filter, and
launched into a 0.25 m f/4 imaging spectrograph attached to a liquid nitrogen cooled
CCD detector. Alternatively, for microscopic measurements, an epi-illuminated Zeiss
Axioscope 50 microscope is used. The objective both focuses the excitation and collects
the Raman scattered light in a backscattering geometry. A dichroic beamsplitter and
mirror combination redirect the Raman-scattered light to the spectrograph system along
the same optical path used for the bulk tissue system (minus the camera lens), after
passing through a confocal pinhole to increase axial resolution.
Argon ion
pump laser
Ti: sapphire
laser
Dichroic
beam-splitter
CCD
f/4
Spectrograph
Confocal
pinhole
UV, Visible Excitation
(352, 476, 647 nm)
filter
Band-pass filters
Notch filter
NIR
excitation
(830 nm)
CCD Camera
Motorized translation stage
Collimating lenses
We determined that at the smallest confocal aperture diameter (~300 m) the spatial
resolution of the microscope is ~2 m3. This value was obtained by focusing the laser
spot onto a single, 1 m diameter polystyrene bead. Moving the sample stage laterally by
1 m caused a signal decrease of about 90 %. Axially, we determined the signal to
decrease that much after about 2 m translation. This resolution is comparable to what
has been described for other confocal Raman systems. In practice, the lateral resolution
obtained from tissue samples is approximately 2 m. This sampling volume is smaller
than the dimensions of all morphological features of interest for developing a
spectroscopic model (typical cell diameter ~10 m).
The spectrograph itself has an adjustable slit and a turret, which holds three gratings for a
range of measurements. For the Raman studies a 600 grooves/mm grating blazed at 1 m
is used along with the 140 m spectrograph entrance slit setting, providing ~ 8 cm-1
resolution. As most biological samples do not exhibit Raman bandwidths narrower than
10 cm-1, a 140 m spectrograph entrance slit makes sense, providing increased optical
throughput.
A CCD camera atop the microscope allows for registration of the focused laser spot with
a white light trans-illuminated image. The microscope itself is equipped with a range of
objectives, both normal and phase contrast. Typically, for Raman studies we used a 63x
infinity-corrected water immersion objective (0.9 NA). As both the detector and the
microscope translation stage are computer controlled, spectral maps of the tissue can be
created by moving the translation stage in a raster-scan pattern under the microscope
objective.
Simply by changing the excitation source, the filters/beamsplitters, and spectrograph
grating, the system can be adapted for collecting fluorescence measurements. Three
additional wavelengths, 352 (UV argon), 477 (argon), and 647 (krypton) nm, are
delivered to the system via fiber optics from lasers in adjacent laboratories. A 600
grooves/mm grating blazed at 500 nm is often used (the third grating available to us is
300 grooves/mm blazed at 1 m). The switch between Raman and fluorescence can be
made in less than two minutes.
REFERENCES
Brennan JF, Wang Y, Dasari RR and Feld MS, “Near-Infrared Raman Spectrometer
Systems for Human Tissue Studies”, Appl Spectrosc, 51(2), 201-208 (1997b).