Biomedical Engineering Reference
In-Depth Information
SNR compared to that obtained using the TD scheme due to noise decorrelation in the
Fourier domain. This improved detection sensitivity may be beneficial for the localization
of dim fluorophores such as fluorescent proteins. An additional important aspect of
TD-FPM and SD-FPM is related to artifacts due to the sample motion during data
acquisition. While in TD-FPM the sample motion at a given time will affect only the
particular sampling volume that is being acquired, the effect of motion in SD-FPM is likely
to be more severe and complex because the signal is integrated over time and is obtained
by the Fourier transform integral. In general, the magnitude of motion artifacts in SD-FPM
will be governed by the total axial or transverse displacement during a single axial line
signal acquisition time. Finally, for a given sample motion, as the temporal resolution
improves, the axial and transverse displacements are decreased and so are the motion
artifacts in both TD-FPM and SD-FPM.
The detection sensitivity of FPM is determined by the distribution of the excited
fluorophores. In general, the detection sensitivity is degraded for a continuous distribution
of fluorophores that extends along the axial dimension over a large range ( B λ /2) because
fringes produced by the fluorophores are linearly combined. Therefore, the use of FPM for
imaging and ranging applications is mostly appropriate when using discrete distributions of
fluorophores. Interestingly, this characteristic of FPM is similar to that of LCI; the latter is
sensitive to well-defined specularly reflecting interfaces (produced by discontinuities in the
scattering potential). Importantly, when TD-FPM and SD-FPM systems operate in the
shot-noise- or intensity-noise- detection limited regime, a bright fluorophore at any point
inside the sample will increase the noise floor, thereby making it difficult to detect weaker
fluorophores located along the same axial line. To circumvent these limitations, FPM could
be potentially combined with fluorescence lifetime imaging techniques [44] . Similarly to
spectrally resolved interferometry, the phase ambiguity that occurs for fluorophores located
at positive and negative distances from the zero differential optical path-length point of the
interferometer (z 0 ) can be readily resolved by recording the complex spectral density,
thereby doubling the maximal imaging depth [45] .
18.4 Applications of FPM
As mentioned in the previous sections, the application of FPM is particularly attractive for
(i) optical sectioning imaging with mesoscopic resolution and (ii) metrology with
nanometer-level axial localization precision. To investigate the optical sectioning ability of
FPM, we implemented FPM in both SD and TDs. The SD-FPM setup used either a wide-
field excitation and a folded dielectric-mirror-based interferometer or a scanning line-focus
illumination and a two-opposing-lenses interferometer, depending on the application. The
TD-FPM system employed a wide-field illumination and a two-opposing-lenses
interferometer.
Search WWH ::




Custom Search