Calibrating evanescent-wave penetration depths for biological TIRF microscopy

Roughly half of a cells proteins are located at or near the plasma membrane. In this restricted space the cell senses its environment, signals to its neighbors and ex-changes cargo through exo- and endocytotic mechanisms. Ligands bind to receptors, ions flow across channel pores, and transmitters and metabolites are transported against con-centration gradients. Receptors, ion channels, pumps and transporters are the molecular substrates of these biological processes and they constitute important targets for drug discovery. Total internal reflection fluorescence microscopy suppresses background from cell deeper layers and provides contrast for selectively imaging dynamic processes near the basal membrane of live-cells. The optical sectioning of total internal reflection fluorescence is based on the excitation confinement of the evanescent wave generated at the glass-cell interface. How deep the excitation light actually penetrates the sample is difficult to know, making the quantitative interpretation of total internal reflection fluorescence data problematic. Nevertheless, many applications like super-resolution microscopy, colocalization, fluorescence recovery after photobleaching, near-membrane fluorescence recovery after photobleaching, uncaging or photo-activation-switching, as well as single-particle tracking require the quantitative interpretation of evanescent-wave excited images. Here, we review existing techniques for characterizing evanescent fields and we provide a roadmap for comparing total internal reflection fluorescence data across images, experiments, and laboratories.Comment: 18 text pages, 7 figures and one supplemental figur

High-speed in vitro intensity diffraction tomography

We demonstrate a label-free, scan-free intensity diffraction tomography technique utilizing annular illumination (aIDT) to rapidly characterize large-volume three-dimensional (3-D) refractive index distributions in vitro. By optimally matching the illumination geometry to the microscope pupil, our technique reduces the data requirement by 60 times to achieve high-speed 10-Hz volume rates. Using eight intensity images, we recover volumes of ∼350 μm  ×  100 μm  ×  20  μm, with near diffraction-limited lateral resolution of   ∼  487  nm and axial resolution of   ∼  3.4  μm. The attained large volume rate and high-resolution enable 3-D quantitative phase imaging of complex living biological samples across multiple length scales. We demonstrate aIDT’s capabilities on unicellular diatom microalgae, epithelial buccal cell clusters with native bacteria, and live Caenorhabditis elegans specimens. Within these samples, we recover macroscale cellular structures, subcellular organelles, and dynamic micro-organism tissues with minimal motion artifacts. Quantifying such features has significant utility in oncology, immunology, and cellular pathophysiology, where these morphological features are evaluated for changes in the presence of disease, parasites, and new drug treatments. Finally, we simulate the aIDT system to highlight the accuracy and sensitivity of the proposed technique. aIDT shows promise as a powerful high-speed, label-free computational microscopy approach for applications where natural imaging is required to evaluate environmental effects on a sample in real time.https://arxiv.org/abs/1904.06004Accepted manuscrip

Design and performance of a compact and stationary microSPECT system

Purpose: Over the last ten years, there has been an extensive growth in the development of microSPECT imagers. Most of the systems are based on the combination of conventional, relatively large gamma cameras with poor intrinsic spatial resolution and multipinhole collimators working in large magnification mode. Spatial resolutions range from 0.58 to 0.76 mm while peak sensitivities vary from 0.06% to 0.4%. While pushing the limits of performance is of major importance, the authors believe that there is a need for smaller and less complex systems that bring along a reduced cost. While low footprint and low-cost systems can make microSPECT available to more researchers, the ease of operation and calibration and low maintenance cost are additional factors that can facilitate the use of microSPECT in molecular imaging. In this paper, the authors simulate the performance of a microSPECT imager that combines high space-bandwidth detectors and pinholes with truncated projection, resulting in a small and stationary system. Methods: A system optimization algorithm is used to determine the optimal SPECT systems, given our high resolutions detectors and a fixed field-of-view. These optimal system geometries are then used to simulate a Defrise disk phantom and a hot rod phantom. Finally, a MOBY mouse phantom, with realistic concentrations of Tc99m-tetrofosmin is simulated. Results: Results show that the authors can successfully reconstruct a Defrise disk phantom of 24 mm in diameter without any rotating system components or translation of the object. Reconstructed spatial resolution is approximately 800 mu m while the peak sensitivity is 0.23%. Finally, the simulation of the MOBY mouse phantom shows that the authors can accurately reconstruct mouse images. Conclusions: These results show that pinholes with truncated projections can be used in small magnification or minification mode to obtain a compact and stationary microSPECT system. The authors showed that they can reach state-of-the-art system performance and can successfully reconstruct images with realistic noise levels in a preclinical context. Such a system can be useful for dynamic SPECT imaging. 2013 American Association of Physicists in Medicine

Exploiting multimode waveguides for pure fibre-based imaging

We acknowledge support from the UK Engineering and Physical Science Research CouncilThere has been an immense drive in modern microscopy towards miniaturisation and fibre based technology. This has been necessitated by the need to access hostile or diffcult environments in-situ and in-vivo. Strategies to date have included the use of specialist fibres and miniaturised scanning systems accompanied by ingenious microfabricated lenses. We present a novel approach for this field by utilising disordered light within a standard multimode optical fibre for lensless microscopy and optical mode conversion. We demonstrate the modalities of bright-field and dark-field imaging and scanning fluorescence microscopy at acquisition rates allowing observation of dynamic processes such as Brownian motion of mesoscopic particles. Furthermore, we show how such control can realise a new form of mode converter and generate various types of advanced light fields such as propagation-invariant beams and optical vortices. These may be useful for future fibre based implementations of super-resolution or light sheet microscopy.Publisher PDFPeer reviewe

Three-dimensional measurements with a novel technique combination of confocal and focus variation with a simultaneous scan

The most common optical measurement technologies used today for the three dimensional measurement of technical surfaces are Coherence Scanning Interferometry (CSI), Imaging Confocal Microscopy (IC), and Focus Variation (FV). Each one has its benefits and its drawbacks. FV will be the ideal technology for the measurement of those regions where the slopes are high and where the surface is very rough, while CSI and IC will provide better results for smoother and flatter surface regions. In this work we investigated the benefits and drawbacks of combining Interferometry, Confocal and focus variation to get better measurement of technical surfaces. We investigated a way of using Microdisplay Scanning type of Confocal Microscope to acquire on a simultaneous scan confocal and focus Variation information to reconstruct a three dimensional measurement. Several methods are presented to fuse the optical sectioning properties of both techniques as well as the topographical information. This work shows the benefit of this combination technique on several industrial samples where neither confocal nor focus variation is able to provide optimal results.Postprint (author's final draft

Master slave en-face OCT/SLO

Master Slave optical coherence tomography (MS-OCT) is an OCT method that does not require resampling of data and can be used to deliver en-face images from several depths simultaneously. As the MS-OCT method requires important computational resources, the number of multiple depth en-face images that can be produced in real-time is limited. Here, we demonstrate progress in taking advantage of the parallel processing feature of the MS-OCT technology. Harnessing the capabilities of graphics processing units (GPU)s, information from 384 depth positions is acquired in one raster with real time display of up to 40 en-face OCT images. These exhibit comparable resolution and sensitivity to the images produced using the conventional Fourier domain based method. The GPU facilitates versatile real time selection of parameters, such as the depth positions of the 40 images out of the set of 384 depth locations, as well as their axial resolution. In each updated displayed frame, in parallel with the 40 en-face OCT images, a scanning laser ophthalmoscopy (SLO) lookalike image is presented together with two B-scan OCT images oriented along rectangular directions. The thickness of the SLO lookalike image is dynamically determined by the choice of number of en-face OCT images displayed in the frame and the choice of differential axial distance between them