Biomedical Engineering Reference
In-Depth Information
Figure 14.3
(A) SD-DIC system; (B and C) possible sample configurations for surface profiling and live cell
imaging, respectively. SLD, superluminescence diode; SPM, spectrometer; L 1 and L 2 , lenses.
Source: From Ref. [23] .
reflected from the sample surface are collected. When imaging thin, transparent samples,
such as cells, they are plated on a reflective surface to enable double-pass transmission
measurement, as shown in Figure 14.3C . A small glass chamber is typically used to keep
cells alive in culture media. In either mode, reflected o- and e-waves return through the
same optical path to be mixed in the fiber coupler to interfere, before entering a
spectrometer (OceanOptics: HR4000) for detection. A polarization controller may be used
in the detection arm to maximize the fringe visibility.
Although the system shown in Figure 14.3A resembles a conventional reflected-light DIC
microscope, the fundamental differences are the PM fiber and the spectral-domain
detection, which enable the high-resolution operation. The PM fiber functions as a phase
retarder, which adds a large bias, approximately 215
m (single pass) in this system, to the
OPL gradient between the o- and e-waves. Hence, the sample signal intensity received by
the spectrometer can be written as:
μ
I t ðk; x; yÞ ~ r o ðx; yÞ 1 r e ðx; yÞ 1
2
ηr o ðx; yÞr e ðx; yÞ cos ½ 2 kðΔL PR 1δL NP 1δL DIC ðx; yÞÞ
(14.3)
where r o and r e represent the reflectivities of a surface or the transmissivities of a cell at
position ( x , y ) for the o- and e-waves, respectively; k is the wavenumber; η is the
interference visibility; ΔL PR , δL NP , and δL DIC are single-pass OPL differences generated by
the phase retarder, the Nomarski prism, and the sample gradient, respectively.
Equation (14.3) shows that the phase retarder functions similarly to the frequency shifter
used to generate a carrier frequency in a heterodyne system. Without the phase bias, the
submicron/nanometer magnitudes of
L DIC will generate only low-frequency
interferometric fringes in the spectrum. Such modulation makes it difficult to accurately
δ
L NP and
δ
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