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Fluorescence and confocal microscopes operating principle. Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. [1]
Confocal and two-photon microscopes provide optical sectioning ability so that calcium signals can be resolved in microdomains such as dendritic spines or synaptic boutons, even in thick samples such as mammalian brains. Images are analyzed by measuring fluorescence intensity changes for a single wavelength or two wavelengths expressed as a ...
The three-dimensional point spread functions (a,c) and corresponding modulation transfer functions (b,d) of a wide-field microscope (a,b) and confocal microscope (c,d). In both cases the numerical aperture of the objective is 1.49 and the refractive index of the medium 1.52.
A subclass of confocal microscopes are spinning disc microscopes which are able to scan multiple points simultaneously across the sample. A corresponding disc with pinholes rejects out-of-focus light. The light detector in a spinning disc microscope is a digital camera, typically EM-CCD or sCMOS.
Endomicroscopy is a technique for obtaining histology-like images from inside the human body in real-time, [1] [2] [3] a process known as ‘optical biopsy’. [4] [5] It generally refers to fluorescence confocal microscopy, although multi-photon microscopy and optical coherence tomography have also been adapted for endoscopic use.
Similar to confocal microscopy, the laser in CLE filtered by the pinhole excites the fluorescent dye through a beam splitter and objective lens. The fluorescent emission then follows similar paths into the detector. A pinhole is used to select emissions from the desired focal plane. Two categories of CLE exist, namely probe-based (pCLE) and the ...
By virtue of the linearity property of optical non-coherent imaging systems, i.e., . Image(Object 1 + Object 2) = Image(Object 1) + Image(Object 2). the image of an object in a microscope or telescope as a non-coherent imaging system can be computed by expressing the object-plane field as a weighted sum of 2D impulse functions, and then expressing the image plane field as a weighted sum of the ...
STED microscopy is one of several types of super resolution microscopy techniques that have recently been developed to bypass the diffraction limit of light microscopy to increase resolution. STED is a deterministic functional technique that exploits the non-linear response of fluorophores commonly used to label biological samples in order to ...