Multiphoton Confocal Microscopy
Multiphoton Confocal Microscopy - Related Videos
17:08
In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy
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Cited by 11 •
2014
Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.
10:03
Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP
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Cited by 11 •
2012
Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.
10:48
Imaging Biological Samples with Optical and Confocal Microscopy
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2023
Source: Peiman Shahbeigi-Roodposhti and Sina Shahbazmohamadi, Biomedical Engineering Department, University of Connecticut, Storrs, Connecticut Optical microscopes have been around for centuries, and while they reached their theoretical limitation of resolution decades ago, new equipment and techniques, such as confocal and digital image processing, have created new niches within the field of optical imaging. The best optical microscopes will typically have a resolution down to 200 nm in ideal...
11:55
Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy
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Cited by 6 •
2017
Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.
01:16
Confocal Fluorescence Microscopy
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2023
Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
FAQs
Localization results from the requirement that fluorescent molecules absorb photons simultaneously at the tightly focused point of the pulsed laser. Excitation therefore occurs mainly within that focal region rather than throughout the illuminated path. This spatial restriction helps produce sharper optical sections, improves image contrast, and supports reconstruction of biological structures at different depths.
A pulsed, usually near-infrared laser supplies the concentrated light needed for nonlinear excitation of fluorescent molecules. Because excitation is confined to the focus, regions outside that point experience less direct fluorescence excitation. This contributes to the method’s ability to image deeper biological samples while limiting photodamage, an important consideration for living specimens.
Confocal detection adds spatial filtering after fluorescence is generated. It rejects signals originating outside the focal region, so light from unfocused areas contributes less to the recorded image. Combined with localized nonlinear excitation, this filtering strengthens optical sectioning and contrast, allowing researchers to distinguish structures within complex cells, tissues, and other biological specimens.
The microscope records high-resolution optical sections from different positions within the specimen. These sections preserve information about structures at distinct depths, which can then be assembled into a three-dimensional reconstruction. The resulting volume helps investigators examine cell organization, tissue architecture, and biological events that are difficult to interpret from a single two-dimensional image.
A typical workflow focuses the pulsed laser within the specimen, excites fluorescent molecules at the focal region, and collects the emitted signal through the confocal detection system. Out-of-focus fluorescence is filtered, and sequential optical sections are acquired across the sample. Combining those sections produces high-resolution images or a three-dimensional representation.
The method is useful when researchers need to observe organization or activity within complex biological samples while minimizing disruption. Its applications include examining cell structure, neural activity, developmental processes, and tissue dynamics. Imaging living samples is particularly valuable because improved depth and reduced photodamage can support observation of biological events over the relevant specimen volume.