3D adaptive optics in a light sheet microscope

Bourgenot, Cyril; Saunter, Christopher D.; Taylor, Jonathan; Girkin, John M.; Love, Gordon D.

doi:10.1364/oe.20.013252

Cited by 105 publications

(76 citation statements)

References 25 publications

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“…In agreement with previously reported zebrafish optical characterization, spherical aberration is a main contributor to resolution and SNR degradation (Bourgenot et al, 2012;Gould et al, 2012;Rueckel et al, 2006). As Zebrafish generate less aberration than the oncological sample (Figure 71), the system corrections (Z5--Z14) contributed Zebrafish produce substantial second--harmonic (SHG) signal (Huang et al, 2011).…”

Section: Adaptive Optics In Vivosupporting

confidence: 90%

Multifocal multiphoton microscopy with adaptive optical correction

et al. 2013

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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...

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Section: Adaptive Optics In Vivosupporting

confidence: 90%

Multifocal multiphoton microscopy with adaptive optical correction

et al. 2013

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“…Tilted illumination makes total aberration more severe, as new aberrations will be introduced. Using some optical optimization and wavefront modulation methods, these complicated aberrations could be corrected to some extent [27][28][29][30]. However, those methods were mostly based on simplified models, it was difficult to make an ideal illumination beam in a heterogeneous sample.…”

Section: Aberrations Caused By Mismatch Of Refractive Indexmentioning

confidence: 99%

High axial resolution imaging system for large volume tissues using combination of inclined selective plane illumination and mechanical sectioning

Zhang

Yang

et al. 2017

Biomed. Opt. Express

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Abstract:To resolve fine structures of biological systems like neurons, it is required to realize microscopic imaging with sufficient spatial resolution in three dimensional systems. With regular optical imaging systems, high lateral resolution is accessible while high axial resolution is hard to achieve in a large volume. We introduce an imaging system for high 3D resolution fluorescence imaging of large volume tissues. Selective plane illumination was adopted to provide high axial resolution. A scientific CMOS working in sub-array mode kept the imaging area in the sample surface, which restrained the adverse effect of aberrations caused by inclined illumination. Plastic embedding and precise mechanical sectioning extended the axial range and eliminated distortion during the whole imaging process. The combination of these techniques enabled 3D high resolution imaging of large tissues. Fluorescent bead imaging showed resolutions of 0.59 μm, 0.47μm, and 0.59 μm in the x, y, and z directions, respectively. Data acquired from the volume sample of brain tissue demonstrated the applicability of this imaging system. Imaging of different depths showed uniform performance where details could be recognized in either the near-soma area or terminal area, and fine structures of neurons could be seen in both the xy and xz sections.

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“…In [39], the aberration created by a cylindrical glass pipette in a water immersion medium has been theoretically and experimentally studied. Astigmatism is in this configuration, the main aberration.…”

Section: Refractive Index Mismatchmentioning

confidence: 99%

“…In [39], the variation of sample-induced aberrations has been characterised with depth and corrected with AO in an ex-vivo Zebrafish embryo pectoral fin. In [41], aberrations are corrected, at different depths in the terminal pharyngeal bulb of a C elegans sample.…”

Section: Sample Induced Aberrationsmentioning

confidence: 99%

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Light sheet adaptive optics microscope for 3D live imaging

Bourgenot

Taylor

Saunter

et al. 2013

Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XX

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3D adaptive optics in a light sheet microscope

Cited by 105 publications

References 25 publications

Multifocal multiphoton microscopy with adaptive optical correction

Multifocal multiphoton microscopy with adaptive optical correction

High axial resolution imaging system for large volume tissues using combination of inclined selective plane illumination and mechanical sectioning

Light sheet adaptive optics microscope for 3D live imaging

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