Page 63 - Vibrational Spectroscopic Imaging for Biomedical Applications

Algal Cells, Cartilage, and IRENI 39

(provided by a synchrotron light source) of aqueous samples such as
living specimen. Furthermore, the recent advent of multielement
mid-IR detectors such as line detectors or FPAs reduces the acqui-
sition times by several orders of magnitude. In combination with the
22
high brightness of a synchrotron light source, it opens the possibil-
ity for the in vivo acquisition of kinetic microspectroscopic maps of
biological samples.
For this publication we demonstrate the performance of the flow
chamber using an algal specimen (Micrasterias sp.), but the chamber
can in principle be used with any aqueous/biological, nonaqueous or
even gaseous samples.

2.3.1 Flow Chamber Design
One challenge in the design of a flow chamber is the choice of an
appropriate window material: many commonly used crystals are
water soluble (e.g., KBr, NaCl) or toxic (e.g., CdTe, KRS-5), absorb
portions of the bandwidth of interest (e.g., Si, Al O ), or exhibit dis-
2 3
persion (e.g., ZnS, CaF , BaF ) leading to optical aberrations. The lat-
2 2
ter effect is made worse by the fact that the halide materials cannot be
made into windows with a thickness of less than several millimeters
due to their brittleness. This prevents the use of high numerical aper-
ture microscope objectives with their short working distance.
Furthermore, some of these substances are tinted (e.g., ZnSe) or
opaque (e.g., Si, AMTIR) in the visible, which renders comparisons
and co-localization of features in the visible (e.g., stained sections or parts
labeled with fluorescent markers) and the IR difficult or impossible.
To circumvent the problems mentioned above, we chose diamond
as a material for our windows. This material has several considerable
advantages: it is water insoluble, nontoxic, transparent, and colorless
in the visible as well as in the entire mid-IR spectrum of interest.
Additionally, it exhibits a very low dispersion in the mid-IR region.
This is illustrated by its relatively constant refractive index over the
full mid-IR spectral range compared to other commonly used win-
dow materials (ZnS, BaF , and CaF ) as shown in Fig. 2.8.
2 2
Multiple internal reflections inside the window materials of the
order of micrometers thick can pose a serious problem because they
lead to fringes on the IR spectra. This is worst when the fringe fre-
quency is comparable to typical spectral peak widths. We overcome
this by using sub-micrometer (less than one micron) thin diamond
films such that the fringe frequency is comparable to the entire spec-
tral range. The fringe(s) can then essentially be treated as an addi-
tional baseline and subtracted from the interesting spectral features.
Diamond can be grown very thin by chemical vapor deposition
(CVD) techniques. Thin films also help to keep the flow chamber
design slim so that it can accommodate high numerical aperture
objectives with short working distances.