Living cells are highly dynamic systems responding to a large variety of biochemical and mechanical stimuli over minutes, which are well controlled by e.g. optical tweezers. However, live cell investigation through fluorescence microscopy is usually limited not only by the spatial and temporal imaging resolution but also by fluorophore bleaching. Therefore, we designed a miniature light-sheet illumination system that is implemented in a conventional inverted microscope equipped with optical tweezers and interferometric tracking to capture 3D images of living macrophages at reduced bleaching. The horizontal light-sheet is generated with a 0.12 mm small cantilevered mirror placed at 45° to the detection axis. The objective launched illumination beam is reflected by the micro-mirror and illuminates the sample perpendicular to the detection axis. Lateral and axial scanning of both Gaussian and Bessel beams, together with an electrically tunable lens for fast focusing, enables rapid 3D image capture without moving the sample or the objective lens. Using scanned Bessel beams and line-confocal detection, an average axial resolution of 0.8 µm together with a 10-15 fold improved image contrast is achieved.
In light-sheet microscopy, a confined layer in the focal plane of the detection objective is illuminated from the side. The illumination light-sheet usually has a constant beam length independent of the shape of the biological object. Since the thickness and the length of the illumination light-sheet are coupled, a tradeoff between resolution, contrast and field of view has to be accepted. Here we show that scanned Bessel beams enable object adapted tailoring of the light-sheet defined by its beam length and position. The individual beam parameters are obtained from automatic object shape estimation by low-power laser light scattered at the object. Using Arabidopsis root tips, cell clusters and zebrafish tails, we demonstrate that Bessel beam light-sheet tailoring leads to a 50% increase in image contrast and a 50% reduction in photobleaching. Light-sheet tailoring requires only binary amplitude modulation, therefore allowing a real time illumination adaptation with little technical effort in the future.
Based on an advanced silicon optical bench technology with integrated MOEMS (Micro-Opto-Electro-Mechanical-System) components, a piezo-driven fiber scanner for confocal microscopy has been developed. This highly-miniaturized technology allows integration into an endoscope with a total outer probe diameter of 2.5 mm. The system features a hydraulically-driven varifocal lens providing axial confocal scanning without any translational movement of components. The demonstrated resolutions are 1.7 μm laterally and 19 μm axially.
Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection.
Light-sheet microscopy enables fast 3D, high-contrast imaging in biology and colloidal sciences. Recently, the controlled transport of living embryos or small colloids through stable glass capillaries is manifold interesting. Although they hardly impair the sample, glass capillaries spoil the image by generating significant aberrations of the illumination and detection light. Here, we analyze the deflection of illuminating Bessel beams at the capillary by k-spectral shifting, and correct for it by a beam deflector. Using cylindrical lenses for astigmatism compensation on the detection side, we demonstrate 3D line-confocal imaging inside a glass capillary over an axial range of ±400 μm.
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