Multifocal multiphoton microscopy with adaptive optical correction

Abstract

Multiphoton microscopy (MPM) is a remarkably versatile technique in biologicalimaging. 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 Ca 2+ 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 CollegeLondon. 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. 3 DECLARATION OF AUTHORSHIPThe work present is my own work with the exception of TRI2, which was developed The introduction (Chapter 1) concentrates on optical methods developed in the context of multiphoton microscopy (MPM) or more specifically two--photon microscopy (TPM) (Denk et al., 1990). TPM enables long--term imaging of in vivo biological specimens, image generation at increased tissue depth, and higher signal--to--noise images compared to wide--field and confocal approaches (Helmchen and In comparison to confocal microscopy, two--photon microscopy (TPM) offers considerable benefits since excitation is confined to a femtolitre volume in the vicinity of the focal plane, reducing overall photobleaching and phototoxicity of thick samples (Centonze and White, 1998;Denk et al., 1990;Helmchen and Denk, 2005;Williams et al., 2001). Out--of--focus excitation is avoided due to the localization of the excitation making the confocal pinhole obsolete (Amos and Where is the numerical aperture of the obj...

Homogeneous multifocal excitation for high-throughput super-resolution imaging

“…We implemented the mfFIFI module into an iSIM microscope, since its fast acquisition speeds and resolution doubling give it good potential for high-throughput super-resolution imaging. However, the mfFIFI module could be extended to any multi-focal excitation microscope such as a spinning disk confocal microscope 6 using a Nipkow disk configuration, but not with other flat-fielding solutions using diffractive optical elements 20 , spatial light modulators (SLM) 21 or beam splitters 23 . Furthermore, combining mfFIFI with phase masks required for donut beam shaping could extend its application to parallelized STED and RESOLFT microscopes.…”

“…While this gives the iSIM good potential for high-throughput super-resolution imaging, spatially varying illumination produces a spatially dependent signal to noise ratio while restricting the imaging FOV to ∼50×50 µm 2 . Although solutions for producing homogeneous multi-focal excitation exist using diffractive optical elements 20 , spatial light modulators (SLM) 21 , multi-mode fibers 22 or beam splitters 23 , each suffers from some combination of a limited number of excitation spots 20,21,23 , heterogeneity 21,23 or low transmission efficiency 20 .…”

An adaptive approach for uniform scanning in multifocal multiphoton microscopy with a spatial light modulator

“…For the task of exciting multiple fluorescence spots (MFS), some devices are considered as potential candidates, e.g., a microlens array [3], a diffractive optical element (DOE) [4,5], a beam splitter [6], and a spatial light modulator (SLM) [7,8]. The SLM can adaptively excite MFS of arbitrary number, positions and intensities by applying an appropriate computergenerated hologram (CGH).…”

“…In ref. [8], Coelho et al clarified that the MFS uniformity was affected by CGH design algorithm. The uniformity in their experiment was 0.10 to 0.15 for a few tens of spots.…”

Adaptive optics for a time-resolved Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) in vivo

“…AO relies on introducing a dynamic optical correction element, such as a spatial light modulator (SLM), into the imaging path. The purpose is to introduce an equal but opposite distortion to the wavefront, therefore negating aberrations within the specimen [ 5 – 7 ]. The wavefront can be modeled as phase variations in the pupil of the imaging objective.…”