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Related Concept Videos

Two-Dimensional Microscopy in Microbiology01:29

Two-Dimensional Microscopy in Microbiology

Two-dimensional (2D) microscopy encompasses a range of optical techniques that capture images within a single focal plane, offering detailed representations of microscopic structures. These techniques are essential in biological and medical research, enabling the visualization of cellular and subcellular structures with different levels of contrast and specificity.There are several major types of 2D microscopy, each with strengths and applications.Bright-Field MicroscopyBright-field microscopy...
Phase Contrast and Differential Interference Contrast Microscopy01:26

Phase Contrast and Differential Interference Contrast Microscopy

Phase-Contrast Microscopes
In-phase-contrast microscopes, interference between light directly passing through a cell and light refracted by cellular components is used to create high-contrast, high-resolution images without staining. It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen. Altered wavelength paths are created using an annular stop in the condenser. The annular stop produces a hollow cone of...
Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.
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Fluorometers and spectrofluorometers are two types of instruments used for measuring molecular fluorescence. These instruments differ in how they select excitation and emission wavelengths and the type of light sources they utilize. Fluorometers use absorption interference filters to choose excitation and emission wavelengths. The excitation source in a fluorometer is typically a low-pressure mercury vapor lamp that emits intense lines distributed throughout the ultraviolet and visible regions.
Confocal Fluorescence Microscopy01:16

Confocal Fluorescence Microscopy

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,...
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Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope
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Published on: October 9, 2014

Diffraction phase and fluorescence microscopy.

Yongkeun Park, Gabriel Popescu, Kamran Badizadegan

    Optics Express
    |June 17, 2009
    PubMed
    Summary
    This summary is machine-generated.

    We developed a new live-cell imaging technique called diffraction phase and fluorescence (DPF) microscopy. This method combines quantitative phase imaging with fluorescence microscopy for detailed cell analysis.

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    Area of Science:

    • Biophysics
    • Cell Biology
    • Microscopy

    Background:

    • Live-cell imaging requires techniques that can provide high-resolution, quantitative data without perturbing cellular processes.
    • Simultaneous acquisition of different types of information, such as morphology and molecular localization, is crucial for understanding complex cellular dynamics.

    Purpose of the Study:

    • To introduce and validate a novel microscopy technique, diffraction phase and fluorescence (DPF) microscopy.
    • To demonstrate the capability of DPF microscopy for simultaneous quantitative phase imaging and epi-fluorescence investigation of live cells.

    Main Methods:

    • Development of a DPF instrument integrating an interference microscope with a conventional inverted fluorescence microscope.
    • Quantitative phase imaging with sub-nanometer optical path-length stability.
    • Application of DPF microscopy to red blood cells for nanoscale motion analysis and mitotic kidney cells for composite imaging.

    Main Results:

    • Achieved sub-nanometer optical path-length stability in quantitative phase images over extended periods.
    • Successfully quantified rapid nanoscale motions in red blood cells using DPF microscopy.
    • Demonstrated the utility of the composite phase-fluorescence imaging mode with live mitotic kidney cells.

    Conclusions:

    • DPF microscopy offers a powerful new tool for simultaneous quantitative phase and fluorescence imaging of live cells.
    • The technique enables high-resolution, stable imaging for studying cellular dynamics and nanoscale movements.
    • DPF microscopy has broad potential applications in cell biology and biophysics research.