Page 67 - Vibrational Spectroscopic Imaging for Biomedical Applications

Algal Cells, Cartilage, and IRENI 43

flow chamber with sub-micrometer diamond windows (Fig. 2.11f to
2.11j). ZnS windows were chosen instead of ZnSe, since the yellow
tint of the ZnSe windows makes it difficult to see the green algal cells.
This high-resolution data using an effective geometric aperture of
2
10 × 10 μm was acquired at the mid-IR beamline 031 of the Synchro-
tron Radiation Center in Wisconsin that is equipped with a commer-
cial Continuμm IR microscope and a Magna 560 FTIR spectrometer,
both from Nicolet/Thermo Fisher Scientific. This microscope is used
in a confocal arrangement, setting apertures before the condenser
2
and after the objective to image the same 10 × 10 μm area at the
sample plane. Panels (a) and (f) show visible light images taken
through a 32× refractive Schwarzschild objective, which had been
optimized with the correction collar in both cases. The corresponding
mid-IR images (Fig. 2.11b to 2.11e and 2.11g to 2.11j) depict the inte-
grated peak areas of the CH , amide II, phospholipid, and carbohy-
n
drate functional groups. As expected, the visible image of the algal
cell in the new flow chamber reveals more detail than the cell in the
conventional chamber, due to much smaller optical chromatic and
spherical aberrations. The cell wall outline, for example, that is clearly
visible in image (f) is not distinguishable in image (a). The same is
true for the mid-IR: images (Fig. 2.11g to 2.11j) of the new flow cham-
ber are much better resolved than images (Fig. 2.11b to 2.11e) of the
conventional chamber, which appear blurry and don’t reveal any
subcellular structure. The images taken through the sub-micrometer
thick diamond windows, however, show a strong correlation when
compared to the visible images. This is particularly apparent for
the CH stretch images (Fig. 2.11b, g), where minimal diffraction
n
effects allow for the best spatial resolution due to the relatively
short wavelength.
6
Compared to Fig. 2 in Heraud et al., the flow chamber presented
here yields better-resolved IR maps, which is partly due to the fact
2
2
that we use a smaller aperture of 10 × 10 μm instead of 20 × 20 μm ,
but mostly because of smaller optical aberrations due to the much
thinner windows. Furthermore, we show a representative nonaver-
aged spectrum of a single pixel and maps of the CH stretches and the
n
carbohydrates in contrast to Heraud et al.
Figure 2.12 gives an example of a typical in vivo mid-IR spec-
trum on a single Micrasteriass sp. algal cell (taken at the position of
the red marker in Fig. 2.11f to 2.11j). The noisy areas from about 3050
–1
–1
to 3700 cm and 1600 to 1700 cm are due to the absorption of the
water in the medium needed to keep the algal cells alive. This water
layer also leads to fringes visible on the spectrum due to multiple
reflections. The spectral regions marked in blue in Fig. 2.12 corre-
sponding to the functional groups of interest (CH , phospholipids,
n
amide II, and carbohydrates) do not overlap the water bands and
can successfully be extracted. The integrated peak areas of these
regions are shown as false color maps in Fig. 2.11.