Alexander D. Corbett

Porphyrins for Probing Electrical Potential Across Lipid Bilayer Membranes by Second Harmonic Generation

Neurons communicate by using electrical signals, mediated by transient changes in the voltage across the plasma membrane. Optical techniques for visualizing these transmembrane potentials could revolutionize the field of neurobiology by allowing the spatial profile of electrical activity to be imaged in real time with high resolution, along individual neurons or groups of neurons within their native networks.1, 2 Second harmonic generation (SHG) is one of the most promising methods for imaging membrane potential, although so far this technique has only been demonstrated with a narrow range of dyes.3 Here we show that SHG from a porphyrin-based membrane probe gives a fast electro-optic response to an electric field which is about 5–10 times greater than that of conventional styryl dyes. Our results indicate that porphyrin dyes are promising probes for imaging membrane potential.

Fast Measurement of Sarcomere Length and Cell Orientation in Langendorff-Perfused Hearts Using Remote Focusing Microscopy

Rationale: Sarcomere length (SL) is a key indicator of cardiac mechanical function, but current imaging technologies are limited in their ability to unambiguously measure and characterize SL at the cell level in intact, living tissue. Objective: We developed a method for measuring SL and regional cell orientation using remote focusing microscopy, an emerging imaging modality that can capture light from arbitrary oblique planes within a sample. Methods and Results: We present a protocol that unambiguously and quickly determines cell orientation from user-selected areas in a field of view by imaging 2 oblique planes that share a common major axis with the cell. We demonstrate the effectiveness of the technique in establishing single-cell SL in Langendorff-perfused hearts loaded with the membrane dye di-4-ANEPPS. Conclusions: Remote focusing microscopy can measure cell orientation in complex 2-photon data sets without capturing full z stacks. The technique allows rapid assessment of SL in healthy and diseased heart experimental preparations.

Multi-viewer autostereoscopic display with dynamically addressable holographic backlight

— The Multi-User 3-D Television Display (MUTED), designed to provide three-dimensional television (3-D TV) by the display of autostereoscopic imagery to multiple viewers, each of whom should enjoy freedom of movement, is described. Such an autostereoscopic display system, which allows multiple viewers simultaneously by the use of head tracking, was previously demonstrated for TV applications in the ATTEST project. However, the requirement for a dynamically addressable, steerable backlight presented several problems for the illumination source. The MUTED system demonstrates significant advances in the realization of a multi-user autostereoscopic display, partly due to the provision of a dynamic backlight employing a novel holographic laser projector. Such a technology provides significant advantages in terms of brightness, efficiency, laser speckle, and the ability to correct for optical aberrations compared to both imaging and scanned-beam projection technologies.

Caveolae in Rabbit Ventricular Myocytes: Distribution and Dynamic Diminution after Cell Isolation

Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account.

Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen

Remote focussing microscopy allows sharp, in-focus images to be acquired at high speed from outside of the focal plane of an objective lens without any agitation of the specimen. However, without careful optical alignment, the advantages of remote focussing microscopy could be compromised by the introduction of depth-dependent scaling artifacts. To achieve an ideal alignment in a point-scanning remote focussing microscope, the lateral (XY) scan mirror pair must be imaged onto the back focal plane of both the reference and imaging objectives, in a telecentric arrangement. However, for many commercial objective lenses, it can be difficult to accurately locate the position of the back focal plane. This paper investigates the impact of this limitation on the fidelity of three-dimensional data sets of living cardiac tissue, specifically the introduction of distortions. These distortions limit the accuracy of sarcomere measurements taken directly from raw volumetric data. The origin of the distortion is first identified through simulation of a remote focussing microscope. Using a novel three-dimensional calibration specimen it was then possible to quantify experimentally the size of the distortion as a function of objective misalignment. Finally, by first approximating and then compensating the distortion in imaging data from whole heart rodent studies, the variance of sarcomere length (SL) measurements was reduced by almost 50%.

Mirror-mode sensing with a holographic modal wavefront sensor

Currently, in most adaptive optical systems, the control loop between the wavefront sensor and the deformable mirror involves intense mathematical calculations, both during calibration and operation of the system. Although thorough research has been done to optimise the control loop, some issues like error propagation and system bandwidth will always be ultimately limited by the coupling between the mirror and the wavefront sensor. Closed-loop by direct feedback from the wavefront sensor to the deformable mirror was proposed by F. Roddier in his well-quoted curvature wavefront sensing paper. However, due to the natural properties of the defocused-image, this direct feed-back method is limited to bimorph mirror applications only. Recently, M.A.A Neil et al proposed a new modal wavefront sensor (MWFS), which can detect several Zernike modes by a simple intensity subtraction operation. One drawback of this method is that it can only handle a limited number of modes. However, in this paper, we refine this method to detect the orthogonal modes of a deformable mirror instead of Zernike modes in a to-be corrected wavefront. Since the number of actuators of a deformable mirror limits the number of mirror modes, the drawback is minimised in this application. Considering the mirror modes can be directly transformed to the deformable mirror control command set by a proper gain coefficient, it is reasonable to construct a direct-feed back adaptive optical system with the modal wavefront sensing. We will report our first stage investigation on direct feedback adaptive optical system which is to understand the response of MWFS to mirror modes.

25.1: Distinguished Paper: Multi-Viewer Autostereoscopic Display with Dynamically-Addressable Holographic Backlight

An autostereoscopic display system, which allowed multiple viewers simultaneously by use of head-tracking, was previously demonstrated for TV applications in the ATTEST project. However, the requirement for a dynamically addressable, movable backlight presented several problems for the illumination source. In this paper, the authors demonstrate how the use of a novel laser-based holographic projection system can be used to address these problems.

Characterising a holographic modal phase mask for the detection of ocular aberrations

The accurate measurement of the double-pass ocular wave front has been shown to have a broad range of applications from LASIK surgery to adaptively corrected retinal imaging. The ocular wave front can be accurately described by a small number of Zernike circle polynomials. The modal wave front sensor was first proposed by Neil et al. and allows the coefficients of the individual Zernike modes to be measured directly. Typically the aberrations measured with the modal sensor are smaller than those seen in the ocular wave front. In this work, we investigated a technique for adapting a modal phase mask for the sensing of the ocular wave front. This involved extending the dynamic range of the sensor by increasing the pinhole size to 2.4mm and optimising the mask bias to 0.75λ. This was found to decrease the RMS error by up to a factor of three for eye-like aberrations with amplitudes up to 0.2μm. For aberrations taken from a sample of real-eye measurements a 20% decrease in the RMS error was observed.

Laser written fluorescence in plastic slides for microscope calibration (Conference Presentation)

Fluorescence microscopy is an essential tool in bio-imaging, yet there are no widely adopted standards for the calibration of fluorescent microscopes. Calibration provides a wide range of information relating to microscope performance. Without calibration, images taken on two separate microscopes cannot be directly compared as they may have differing magnifications, illumination intensities or detector sensitivities. As the range of microscopy techniques capturing 3D information continues to increase, the need for standardisation becomes ever greater. Widely used methods for determining microscope performance are currently limited to basic techniques such as fluorescent beads, which don’t form a regularly spaced pattern and reflective etched gratings, which are limited to being two-dimensional and require changes to the microscope filter sets. Using ultrafast laser processing inside plastic substrates, we demonstrate the generation of bright fluorescent patterns in three dimensions offering new possibilities for calibration in fluorescence microscopy. The fabricated calibration slides can be used to quantify a range of parameters that determine microscope performance. For example, spatial distortions within the field of view can be quantified by a regular array of bright fluorescent points. Other patterns can determine factors such as detector linearity, field flatness and changes in the point spread function across the field of view and over depth. The patterns can additionally be used to calibrate spatial length-scales and for colour channel registration.

Toward multi-focal spot remote focusing two-photon microscopy for high speed imaging

Optical sectioning techniques using two-photon excitation of fluorescent indicators are central to diverse imaging applications. The limitations of the technique are low speed and undesirable specimen agitation. In our design, high-speed axial scanning is carried out by moving a reference objective to axially displace the focal spot without introducing significant spherical aberration and any agitation of the specimen. Further, the system is configured to allow switching between single spot and multiple focal spot remote focusing to allow either high dynamic range or high speed imaging.