Wavefront shaping of a Bessel light field enhances light sheet microscopy with scattered light

The project was supported by the UK Engineering and Physical Sciences Research Council, RS MacDonald Charitable Trust, SULSA, and the St. Andrews 600th anniversary BRAINS appeal. K. D. is a Royal Society Wolfson Merit Award holder.Light sheet microscopy has seen a resurgence as it facilitates rapid, high contrast, volumetric imaging with minimal sample exposure. Initially developed for imaging scattered light, this application of light sheet microscopy has largely been overlooked but provides an endogenous contrast mechanism which can complement fluorescence imaging and requires very little or no modification to an existing light sheet fluorescence microscope. Fluorescence imaging and scattered light imaging differ in terms of image formation. In the former the detected light is incoherent and weak whereas in the latter the coherence properties of the illumination source, typically a laser, dictate the coherence of detected light, but both are dependent on the quality of the illuminating light sheet. Image formation in both schemes can be understood as the convolution of the light sheet with the specimen distribution. In this paper we explore wavefront shaping for the enhancement of light sheet microscopy with scattered light. We show experimental verification of this result, demonstrating the use of the propagation invariant Bessel beam to extend the field of view of a high resolution scattered light, light sheet microscope and its application to imaging of biological super-cellular structures with sub-cellular resolution. Additionally, complementary scattering and fluorescence imaging is used to characterize the enhancement, and to develop a deeper understanding of the differences of image formation between contrast mechanisms in light sheet microscopy.Publisher PD

Light sheet microscopy with acoustic sample confinement

Contactless sample confinement would enable a whole host of new studies in developmental biology and neuroscience, in particular, when combined with long-term, wide-field optical imaging. To achieve this goal, we demonstrate a contactless acoustic gradient force trap for sample confinement in light sheet microscopy. Our approach allows the integration of real-time environmentally controlled experiments with wide-field low photo-toxic imaging, which we demonstrate on a variety of marine animal embryos and larvae. To illustrate the key advantages of our approach, we provide quantitative data for the dynamic response of the heartbeat of zebrafish larvae to verapamil and norepinephrine, which are known to affect cardiovascular function. Optical flow analysis allows us to explore the cardiac cycle of the zebrafish and determine the changes in contractile volume within the heart. Overcoming the restrictions of sample immobilisation and mounting can open up a broad range of studies, with real-time drug-based assays and biomechanical analyses.</p

Silver stain for electron microscopy

Ammoniacal silver stain used for light microscopy was adapted advantageously for use with very thin biological sections required for electron microscopy. Silver stain can be performed in short time, has more contrast, and is especially useful for low power electron microscopy

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume

Planar Airy beam light-sheet for two-photon microscopy

We demonstrate the first planar Airy light-sheet microscope. Fluorescence light-sheet microscopy has become the method of choice to study large biological samples with cellular or sub-cellular resolution. The propagation-invariant Airy beam enables a ten-fold increase in field-of-view with single-photon excitation; however, the characteristic asymmetry of the light-sheet limits its potential for multi-photon excitation. Here we show how a planar light-sheet can be formed from the curved propagation-invariant Airy beam. The resulting symmetric light sheet excites two-photon fluorescence uniformly across an extended field-of-view without the need for deconvolution. We demonstrate the method for rapid two-photon imaging of large volumes of neuronal tissue.Comment: 7 pages, 4 figure

Light-sheet microscopy: a tutorial

This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.Peer ReviewedPostprint (published version

The role of light microscopy in aerospace analytical laboratories

Light microscopy has greatly reduced analytical flow time and added new dimensions to laboratory capability. Aerospace analytical laboratories are often confronted with problems involving contamination, wear, or material inhomogeneity. The detection of potential problems and the solution of those that develop necessitate the most sensitive and selective applications of sophisticated analytical techniques and instrumentation. This inevitably involves light microscopy. The microscope can characterize and often identify the cause of a problem in 5-15 minutes with confirmatory tests generally less than one hour. Light microscopy has and will make a very significant contribution to the analytical capabilities of aerospace laboratories