Fluorescence Phase Microscopy (FPM) and Nanoscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 380

line-focus illumination allows one to excite a single plane within thick sample and as a
result parallelizes the image-collection process, a point focus excites only a single axial line
in the specimen. On the other hand, for moderate excitation power levels, a point-scanning
illumination enables confocal detection that further improves the rejection of scattered
fluorescent light from undesired points within the sample. We note that scanning of the
illumination can be avoided by using 3D imaging spectrometers such as a computed-
tomography-based imaging spectrometer. The self-referencing interferometer may consist of
a two-opposing-lenses interferometer or a common-path-folded interferometer employing a
reflecting surface depending on the application. Low-aperture lenses/mirrors are preferred
when a large depth of field is required and moderate transversal resolutions are sufficient,
whereas higher lens/mirror apertures can be used for obtaining a higher transversal
resolution with a smaller confocal length. Importantly, phase-sensitive fluorescence
measurements by planar reflectors [33] and two-opposing-lenses interferometers [2,4,32]
have been performed with nanometer-level sensitivity. Advantages of the planar reflectors
include simple alignment and high sensitivity due to the common-path characteristic of the
interference process, though with limited transversal resolution. To circumvent the
transversal resolution problem, the two-opposing-lenses interferometers have been
employed. However, experiments with these interferometers require the implementation of
careful and complex alignment procedures.
18.3.3 Time Domain/Spectral Domain-FPM—Capabilities and Limitations
TD-FPM and SD-FPM can be considered as a hypothetical optical low-coherence
interferometric system [18 23,26,28] in which the sample under observation also acts as a
spatially incoherent source with low-temporal coherence. Since the light source in FPM is
spatially incoherent, coherent crosstalk that degrades measurement quality in low-coherence
interferometry (LCI) is suppressed in FPM. However, unlike LCI, FPM suffers from the
relatively limited light-collection efficiency (dictated by the aperture of the objective
lenses) because it employs self-referencing interferometry where the reference signal results
from fluorophores in the sample itself and not from a separate strong reference signal as in
LCI. Therefore, the heterodyne gain commonly employed by LCI to place the detection
system in the shot-noise limited regime cannot be utilized by FPM, which consequently
requires the use of low-noise, high-sensitivity CCD cameras and relatively bright
fluorescent tags. Moreover, the absence of heterodyne gain results in an SNR curve that
monotonically increases with fluorescence power, in fundamental contrast to SNR curves of
LCI that achieves a global maximum at a particular reference power level.
Unlike TD-FPM, SD-FPM can acquire the entire fluorescence profile along a specific depth
without any mirror or sample scanning. Moreover, when operating in the shot-noise- or
intensity-noise-limited detection regime, SD-FPM should theoretically provide an increased