Raman Imaging and Raman Mapping - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 96

5.2.2 Line Scanning
Line scanning offers a more rapid image generation method. In this method
the laser is focused on a line, usually with a cylinder lens. Because the lens
focuses a Gaussian beam to a high aspect ratio, only the central portion of
the beam is usable. This problem can be overcome with a Powell lens [3],
which does focus the beam to a uniform intensity high aspect rectangle. In
the most popular scanning configuration, the line-focused beam is stationary
and the object is translated beneath it. Less commonly, a line-focused beam
is scanned across a stationary object [4].
The reduction in total measurement time from point-by-point scanning is
significant, because 100 or more spectra are obtained simultaneously. The mea-
surement time to acquire these spectra may be a short as or only a little longer
than the measurement time for a single spectrum with a point-focused laser.
The reason is that total laser power can be much larger in the line-focused
case. However, because each charge-coupled device (CCD) row contains the
spectrum at 1 pixel, the detector must be read out pixel by pixel. To minimize
detector noise CCDs are read out at low rates (16-50 kHz), so that 1-3 s is
required to read the detector, depending on the number of pixels in the detec-
tor. This time can be reduced by synchronizing stage movement with readout
of the bottom row of the detector, followed by shifting the remaining spectral
image one row toward the readout register [5]. An alternative, at least for use
with green lasers, is to use a fast-readout detector. The electron-multiplying
CCD (EMCCD) has been used for this purpose [4, 6]. It can be read out at
1 MHz or faster. Because the avalanche gain mechanism of the EMCCD intro-
duces very little excess noise, the amplification also allows reduced acquisition
times for objects containing weak scatterers.
In line-scanning Raman imagers, the spectrograph entrance slit is often
used as the spatial filter that enables confocal operation. However, the sec-
tioning provided by a slit is less strong than the sectioning provided by the
more common pinhole. Stronger sectioning can be obtained if the point spread
function of the objective is deconvoluted pixel by pixel along the slit direction.
5.2.3 Global Illumination
A narrowband filter can be used for Raman imaging. The first successful
modern instrumentation employed an interference filter, which could be tilted
to vary the passband [7]. Subsequently, acousto-optic tunable filters (AOTF)
[8] and liquid crystal tunable filters (LCTF) [9] were introduced into Raman
imaging and provided electronic tunabililty. The tunable filter approach has
proven most useful for measurement of isolated bands. If only a few frames
are needed to define the band, global Raman imaging can be quite rapid.
Where there are many overlapping bands or a non-linear background, many
images must be taken at different Raman shifts, and the time advantage
disappears.