Point Spread Function (PSF)
RSS feed-
Methods to Calibrate and Scale Axial Distances in Confocal Microscopy as a Function of Refractive Index
Application example of HyVolution Super-Resolution - Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive-index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high-end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented.Read article -
Measuring the 3D STED-PSF with a new Type of Fluorescent Beads
A new type of fluorescent bead is presented by GATTAquant. These beads, called GATTA-Beads, are characterized by a small diameter (23 nm), high intensity and size uniformity. In combination with state-of the-art STED microscopes such as the Leica TCS SP8 STED 3X and high-end image restoration methods available in the Huygens Software, it is shown that these new beads can be used for accurate STED PSF characterization in 3D. Furthermore, it is shown that the measured 3D STED-PSF can be used to improve image restoration quality in combination with STED deconvolution methods available in the Huygens Software.Read article -
Mirror-Enhanced Super-Resolution Microscopy
Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes.Read article -
Translation Microscopy (TRAM) for Super-Resolution Imaging
Super-resolution microscopy is transforming our understanding of biology but accessibility is limited by its technical complexity, high costs and the requirement for bespoke sample preparation. We present a novel, simple and multi-color super-resolution microscopy technique, called translation microscopy (TRAM), in which a super-resolution image is restored from multiple diffraction-limited resolution observations using a conventional microscope whilst translating the sample in the image plane.Read article -
Quantifying the Resolution of a Leica SR GSD 3D Localization Microscopy System with 2D and 3D Nanorulers
DNA origami based nanorulers produced by GATTAquant are common standards to test the achievable spatial resolution of super-resolution microscopes. Recently the nanorulers were used to test the performance of the Leica SR GSD 3D microscope.Read article -
Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media
Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope.Read article -
Super-Resolution – On a Heuristic Point of View About the Resolution of a Light Microscope
Since super-resolution has become one of the most favored methods in biomedical research, the term has become increasingly popular. Still, there is much of confusion about what is super-resolution and what is resolution at all. Here, the classical view of microscopic resolution is discussed and some techniques that resolve better than classical are briefly introduced. The picture on the right shows the intensity distribution of an image of two points whose distance is just the Rayleigh criterion (false color coding).Read article -
Video Talk by Jeff Lichtman: Point Spread Function
An infinitesimally small point appears in the microscope as a spot with a certain size, blurred in the z-direction and with concentric rings around it. This "point spread function" reveals many of the optical properties of your microscope. This lecture explains why and how the microscope images a point as a point spread function.Read article -
Video Tutorial: STED Parameters and STED Deconvolution
Scientific Volume Imaging has a leading role in deconvolution with the Huygens STED deconvolution option, that takes the specific properties of the STED PSF into account. Huygens is compatible with data from pulsed, CW, and CW-gated STED systems, and reads the microscopic parameters automatically from these Leica LIF files. Most recently, Huygens is also able to handle data from the novel Leica TCS SP8 STED 3X, which can obtain super-resolution in both lateral (x,y) and axial (z) directions.Read article -
Huygens STED Deconvolution Increases Signal-to-Noise and Image Resolution towards 22 nm
STED microscopy has proven to be a valuable super-resolution technique, resolving objects that are smaller than the diffraction-limited resolution. Deconvolution of STED images with Huygens pushes the resolution even further. In a recent publication (see link below), we demonstrate that Huygens offers a two-fold improvements of STED images in X, Y, and Z resolution, and increases signal-to-noise ratios eight times. The presented data also shows that a lateral resolution increase from 50 nm towards 22 nm was obtained by applying Huygens deconvolution on (biological) gated-STED images. Furthermore, we describe that stabilization of 3D STED images is essential for optimal deconvolution, as it corrects for lateral drift which would normally distort the structure of the STED PSF.Read article -
Pinhole Geometry: Four Corners are Perfect
Square and hexagonal pinholes provide identical image signal levels, if the geometries are compared in a sensible manner. The amount of light passing the pinhole depends on the area of that aperture, consequently the area is the parameter that must be compared when discussing brightness of focus images. The use of incommensurable edge lengths is meant to confuse the reader and thus dishonest and reprehensible. In this article, the signal level as a function of geometry and size in confocal microscopes is described.Read article -
Deconvolution
Fluorescence microscopy is a modern and steadily evolving tool to bring light to current cell biological questions. With the help of fluorescent proteins or dyes it is possible to make discrete cellular components visible in a highly specific manner. A prerequisite for these kinds of investigations is a powerful fluorescence microscope. One special aim is the three-dimensional illustration of a structure to get an impression of full plasticity. This poses a certain problem for the experimenter using a classical light microscope.Read article