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Multifocal Multiphoton Microscopy with Adaptive Optical Correction

Student thesis: Doctoral ThesisDoctor of Philosophy

Multiphoton microscopy (MPM) is a remarkably versatile technique in biological
imaging. MPM provides increased depth over confocal imaging and can be
combined with other imaging techniques such as fluorescence lifetime
imaging microscopy (FLIM), adding functional information. FLIM read-­‐out
is relatively straightforward using time-­‐correlated single photon counting
(TCSPC). Fluorescence lifetime detection enhances the power of multiphoton
imaging to allow three dimensional, concentration independent, measurements
of environmental parameters such as pH, Oxygen tension and Ca2+ in addtion
to the interaction or conformational modification of proteins by Förster
resonant energy transfer (FRET); the latter is a particular focus of the Dimbleby
research groups at King’s College London. However, there are significant
limitations in both FLIM and MM. Limitations of TCSPC-­‐FLIM include prolonged
acquisition times along with signal and resolution degradation as a function
of depth. This thesis demonstrates advancements multiphoton fluorescence
lifetime imaging through improvements in two principal areas: speed
and resolution at depth.
In order to improve acquistion rates a multifocal multiphoton microscope
(MMM) capable of rapid, parallelized TCSPC-­‐FLIM was developed-­‐MegaFLI.
Acquisitions demonstrate rapid 3-­‐dimensional, high temporal resolution
FLIM in vivo Zebrafish. Performed by massively parallel excitation/detection
the speed is signficantly improved by a factor of 64. In parallel to the
MegaFLI project, a second microscope employing adaptive optical
correction has been developed.
The introduction of Adaptive Optics (AO) serves to improve imaging quality
by counteracting the refractive index heterogeneities introduced by the
sample, limiting the imaging depth. Incorporated with a single beam scanning
FLIM system, a pupil-­‐segmentation AO-­‐TCSPC-­‐FLIM demonstrates improved
signal-­‐to-­‐noise ratio (SNR) and resolution, permiting a more
accurate determination of fluorescent lifetime in turbid media.
Original languageEnglish
Awarding Institution
Award date2015


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