Search results for " Fluorescence microscopy"
showing 10 items of 35 documents
Fluorescence and spin properties of defects in single digit nanodiamonds
2009
International audience; This article reports stable photoluminescence and high-contrast optically detected electron spin resonance (ODESR) from single nitrogen-vacancy (NV) defect centers created within ultrasmall, disperse nanodiamonds of radius less than 4 nm. Unexpectedly, the efficiency for the production of NV fluorescent defects by electron irradiation is found to be independent of the size of the nanocrystals. Fluorescence lifetime imaging shows lifetimes with a mean value of around 17 ns, only slightly longer than the bulk value of the defects. After proper surface cleaning, the dephasing times of the electron spin resonance in the nanocrystals approach values of some microseconds, …
Optical-sectioning improvement in two-color excitation scanning microscopy
2004
We present a new beam-shaping technique for two-color excitation fluorescence microscopy. We show that by simply inserting a properly designed shaded-ring filter in the illumination beam of smaller wavelength, it is possible to improve the effective optical sectioning capacity of such microscopes by 23%. Such an improvement is obtained at the expense of only a very small increasing of the overall energy in the point-spread-function sidelobes. The performance of this technique is illustrated by a numerical imaging simulation.
Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector
2019
Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line detection of a complementary metal-oxide-semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acoustooptic deflector. Such a simple solution enables us t…
Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes
2009
Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guid…
Unlocked Concanavalin A Forms Amyloid-like Fibrils from Coagulation of Long-lived "Crinkled'' Intermediates
2013
Understanding the early events during amyloid aggregation processes is crucial to single out the involved molecular mechanisms and for designing ad hoc strategies to prevent and reverse amyloidogenic disorders. Here, we show that, in conditions in which the protein is positively charged and its conformational flexibility is enhanced, Concanavalin A leads to fibril formation via a non-conventional aggregation pathway. Using a combination of light scattering, circular dichroism, small angle X-ray scattering, intrinsic (Tryptophan) and extrinsic (ANS) fluorescence and confocal and 2-photon fluorescence microscopy we characterize the aggregation process as a function of the temperature. We high…
Spatial calibration of structured illumination fluorescence microscopy using capillary tissue phantoms.
2008
Quantitative assessment of microvascular structure is relevant to the investigations of ischemic injury, reparative angiogenesis and tumor revascularization. In light microscopy applications, thick tissue specimens are necessary to characterize microvascular networks; however, thick tissue leads to image distortions due to out-of-focus light. Structured illumination confocal microscopy is an optical sectioning technique that improves contrast and resolution by using a grid pattern to identify the plane-of-focus within the specimen. Because structured illumination can be applied to wide-field (nonscanning) microscopes, the microcirculation can be studied by sequential intravital and confocal…
Three-ring filters increase the effective NA up to 1.46 in optical sectioning fluorescence microscopy
2003
Single-photon fluorescence confocal microscopy techniques can be combined with the use of specific binary filters in order to increase their optical sectioning capability. We present a novel class of axially super-resolving binary pupil filters specially designed to reach this aim. These filters let us to obtain a relevant compression of the z-response together with the reduction of the photo-bleaching effect typically inherent to apodization techniques. The fact of joining both the three-ring filters we propose in the illumination path, and the confocal detection gives rise to an important effective increase of lenses of effective numerical aperture.
Axial gain resolution in optical sectioning fluorescence microscopy by shaded-ring filters.
2009
We present a new family of pupil masks to control the axial component of the intensity distribution in the focal region of tightly focused light fields. The filters, which consist of a circular clear pupil with a single shaded ring, allow to control the width of the central lobe of the axial spot together with the residual sidelobes energy. The filters can be applied to improve the optical sectioning capacity of different scanning microscopes.
Removing striping artifacts in light-sheet fluorescence microscopy: a review
2022
Abstract In recent years, light-sheet fluorescence microscopy (LSFM) has found a broad application for imaging of diverse biological samples, ranging from sub-cellular structures to whole animals, both in-vivo and ex-vivo, owing to its many advantages relative to point-scanning methods. By providing the selective illumination of sample single planes, LSFM achieves an intrinsic optical sectioning and direct 2D image acquisition, with low out-of-focus fluorescence background, sample photo-damage and photo-bleaching. On the other hand, such an illumination scheme is prone to light absorption or scattering effects, which lead to uneven illumination and striping artifacts in the images, oriented…
Subtractive imaging in confocal scanning microscopy using a CCD camera as a detector
2012
[EN] We report a scheme for the detector system of confocal microscopes in which the pinhole and a large-area detector are substituted by a CCD camera. The numerical integration of the intensities acquired by the active pixels emulates the signal passing through the pinhole. We demonstrate the imaging capability and the optical sectioning of the system. Subtractive-imaging confocal microscopy can be implemented in a simple manner, providing superresolution and improving optical sectioning. (C) 2012 Optical Society of America