KEYWORDS: Photons, Fluorescence lifetime imaging, Luminescence, Monte Carlo methods, Fluorescence resonance energy transfer, Spatial resolution, In vivo imaging, Hassium, Microscopy, Smoothing
Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique which gives access to the local environment
of fluorophores in living cells. However, to correctly estimate all lifetime parameters, time domain FLIM
imaging requires a high number of photons and consequently a long laser exposure time which is not compatible
with the observation of dynamic molecular events and which induces cellular stress phenomena. For reducing this
exposure time, we have developed an original approach to statistically inflate the number of collected photon.
This approach called Adaptive Monte Carlo Data Inflation (AMDI) combines the well-known bootstrap technique
with an adaptive Parzen kernel. We have evaluated its potential on experimental FLIM data in vivo. We
have demonstrated that our robust method allows estimating precisely fluorescence lifetime with exposure time
reduced up to 50 times for mono-exponential (corresponding to a minimum of 20 photons/pixel) and 10 times
for bi-exponential decays (corresponding to a minimum of 5000 photons/pixel) in comparison with the standard
fitting method. Furthermore, thanks to AMDI, we demonstrate that it becomes possible to estimate accurately
all fitting parameters in FRET experiments without constraining any parameter. An additional benefit of our
technique is that it improves the spatial resolution of the FLIM images by reducing the commonly used spatial
binning factor.
We report our progress in the development of Differential Aberration Imaging (DAI), a technique that enhances twophoton
fluorescence (TPEF) microscopy by improving rejection of out-of-focus background by means of a deformable
mirror (DM). The DM is used to intentionally add aberrations to the imaging system, which causes dramatic losses to
in-focus signal while preserving the bulk of the out-of-focus background. By taking the difference between TPEF
images with and without added aberrations, the out-of-focus portion of the signal is further rejected. We now introduce
an implementation of DAI using a new type of DM that can be produced at much lower cost.
We present a simple and robust way to reject out-of-focus background when performing deep two-photon excited
fluorescence (TPEF) imaging in thick tissue. The technique is based on the use of a deformable mirror (DM)
to introduce illumination aberrations that preferentially degrade TPEF signal while leaving TPEF background
relatively unchanged. A subtraction of aberrated from unaberrated images leads to background rejection. We
present a heuristic description of our technique, which we corroborate with experiment. Images of a labeled
mouse olfactory bulb are compared with standard TPEF microscopy images, demonstrating significant out of
focus TPEF background rejection with our technique. Finally we improve our technique by developing a faster
aberration modulation mechanism that performs background subtraction line by line rather than frame by
frame. In this manner, the overall image acquisition rate of our technique is the same as that of a standard
TPEF microscope.
We present a simple and robust way to reject out-of-focus background when performing deep two-photon excited
fluorescence (TPEF) imaging in thick tissue. The technique is based on the use of a deformable mirror (DM)
to introduce illumination aberrations that preferentially degrade TPEF signal while leaving TPEF background
relatively unchanged. A subtraction of aberrated from unaberrated images leads to background rejection. We
present a heuristic description of our technique, which we corroborate with experiment. Images of a labeled
mouse olfactory bulb are compared with standard TPEF microscopy images, demonstrating significant out of
focus TPEF background rejection with our technique. Finally we improve our technique by developing a faster
aberration modulation mechanism that performs background subtraction line by line rather than frame by
frame. In this manner, the overall image acquisition rate of our technique is the same as that of a standard
TPEF microscope.
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