Search results for "optical sectioning"
showing 10 items of 26 documents
Two simple criteria to estimate an objective's performance when imaging in non design tissue clearing solutions
2019
Tissue clearing techniques are undergoing a renaissance motivated by the need to image fluorescent neurons, and other cells, deep in the sample without physical sectioning. Optical transparency is achieved by equilibrating tissues with high refractive index (RI) solutions. When the microscope objective is not perfectly matched to the RI of the cleared sample, aberrations are introduced. We present two simple-to-calculate numerical criteria predicting: (i) the degradation in image quality (brightness and resolution) from optimal conditions of any clearing solution/objective combination; (ii) which objective, among several available, achieves the highest resolution in a given medium. We deriv…
Flexible multi-beam light-sheet fluorescence microscope for live imaging without striping artifacts
2018
The development of light-sheet fluorescence microscopy (LSFM) has greatly expanded the experimental capabilities in many biological and biomedical research fields, enabling for example live studies of murine and zebrafish neural activity or of cell growth and division. The key feature of the method is the selective illumination of a sample single plane, providing an intrinsic optical sectioning and allowing direct 2D image recording. On the other hand, this excitation scheme is more affected by absorption or scattering artifacts in comparison to point scanning methods, leading to un-even illumination. We present here an easily implementable method, based on acousto-optical deflectors (AOD),…
Investigating the performance of reconstruction methods used in structured illumination microscopy as a function of the illumination pattern's modula…
2016
Surpassing the resolution of optical microscopy defined by the Abbe diffraction limit, while simultaneously achieving optical sectioning, is a challenging problem particularly for live cell imaging of thick samples. Among a few developing techniques, structured illumination microscopy (SIM) addresses this challenge by imposing higher frequency information into the observable frequency band confined by the optical transfer function (OTF) of a conventional microscope either doubling the spatial resolution or filling the missing cone based on the spatial frequency of the pattern when the patterned illumination is two-dimensional. Standard reconstruction methods for SIM decompose the low and hi…
Optical sectioning microscopy through single-shot Lightfield protocol
2020
Optical sectioning microscopy is usually performed by means of a scanning, multi-shot procedure in combination with non-uniform illumination. In this paper, we change the paradigm and report a method that is based in the light field concept, and that provides optical sectioning for 3D microscopy images after a single-shot capture. To do this we fi rst capture multiple orthographic perspectives of the sample by means of Fourier-domain integral microscopy (FiMic). The second stage of our protocol is the application of a novel refocusing algorithm that is able to produce optical sectioning in real time, and with no resolution worsening, in the case of sparse f luorescent samples.We provide the…
Optical sectioning by two-pinhole confocal fluorescence microscopy.
2003
A two-pinhole axially superresolving confocal fluorescence imaging system is presented. Based on the concept of subtractive incoherent imaging, the system described here is equipped with a zero-focus complex-transmittance pupil filter in one of the collector paths. The optical sectioning capacity of the system is 25% superior to that of a free-pupil one-pinhole instrument.
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.
Optical-sectioning microscopy by patterned illumination
2010
We propose a very simple method for the flexible production of 1D structured illumination for high resolution 3D microscopy. Specifically, we propose the insertion of a Fresnel biprism after a monochromatic point source for producing a pair of twin, fully coherent, virtual point sources. The resulting interference fringes are projected into the 3D sample and, by simply varying the distance between the biprism and the point source, one can tune the period of the fringes, while keeping their contrast, in a very versatile and efficient way.
Digital slicing of 3D scenes by Fourier filtering of integral images
2008
We present a novel technique to extract depth information from 3D scenes recorded using an Integral Imaging system. The technique exploits the periodic structure of the recorded integral image to implement a Fourier-domain filtering algorithm. A proper projection of the filtered integral image permits reconstruction of different planes that constitute the 3D scene. The main feature of our method is that the Fourier-domain filtering allows the reduction of out-of-focus information, providing the InI system with real optical sectioning capacity.
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.