Biomedical Engineering Reference
In-Depth Information
Figure 18.6
(A) A scanningless SD-FPM measurement of a tomogram of a dual-layer fluorescent sample.
(B) Two views of the dual-layer fluorescent sample as captured by the TD-FPM setup. Both lateral
and depth distributions of the fluorophores can be imaged effectively. Source: (A) This figure is
reproduced from figure 6(c) of Ref. [41] with permission of the Optical Society of America.
Next, SD-FPM and TD-FPM were used to image fixed biological samples consisting of the
Drosophila embryonic nervous system labeled with immunofluorescence [39] . As shown in
Figure 18.7A , the SD-FPM modality outlined the y z profile of the nervous system (top
panel) and assessed the fluorescence gradients (bottom panel) inside the Drosophila embryo
in a single shot of a few seconds without scanning. This capability might be useful in large-
scale analyses, such as those involved in the formation of morphogenetic protein gradients,
where imaging with mesoscopic resolution is sufficient and the superabundance of image
data resulting from imaging at submicroscopic resolution is to be avoided [46] .
Furthermore, 3D images of the nervous system of the Drosophila embryo were
reconstructed by the wide-field TD-FPM system ( Figure 18.2 ) as shown in Figure 18.7B .
Finally, to further investigate the capabilities of TD-FPM for wide-field fluorescence
imaging, we imaged fluorescently labeled cells (FluoCells, Invitrogen) with a conventional
epifluorescence microscope and with the wide-field TD-FPM system as shown in
Figure 18.7C . The two modalities employed identical low-aperture objectives. It is clearly
observed that the coherence gate offered by the TD-FPM system assisted in the rejection of
out-of-focus fluorescence and yielded sharper image details even with low-aperture
objectives.
Metrology with Nanometer-Level Axial Localization Precision
To demonstrate the ability of FPM to perform measurements at the nanometer scale, we
characterized the polymer swelling process in nanometer-thick polymer bilayers prior to
and following exposure to water [42] . This characterization was carried out by the
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