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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

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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...
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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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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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Conducting Multiple Imaging Modes with One Fluorescence Microscope
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Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging.

Jaime Ortega Arroyo1, Daniel Cole1, Philipp Kukura1

  • 1Physical and Theoretical Chemistry Laboratory, Oxford, UK.

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Interferometric scattering microscopy (iSCAT) provides fast, precise imaging for single molecules without fluorescent labels. This protocol details building an iSCAT microscope using available parts, compatible with fluorescence microscopy.

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Area of Science:

  • Optical microscopy
  • Nanoscale imaging
  • Single-molecule biophysics

Background:

  • Interferometric scattering microscopy (iSCAT) offers high speed and precision for tracking nanoscale objects.
  • Unlike fluorescence microscopy, iSCAT avoids limitations of dye photochemistry and labeling requirements.
  • iSCAT enables label-free optical sensing at the single-molecule level.

Purpose of the Study:

  • To present a protocol for constructing an iSCAT microscope.
  • To demonstrate the compatibility of iSCAT with simultaneous single-molecule fluorescence microscopy.
  • To provide a resource for researchers with intermediate optics experience.

Main Methods:

  • Construction of an iSCAT microscope using commercially available optical components.
  • Integration and simultaneous operation with objective-type total internal reflection fluorescence microscopy.
  • Characterization of iSCAT performance for nanoscale tracking and label-free sensing.

Main Results:

  • A functional iSCAT microscope was successfully constructed from standard optical parts.
  • The iSCAT microscope demonstrated compatibility with simultaneous total internal reflection fluorescence microscopy.
  • The protocol is feasible for researchers with graduate-level optics and microscopy experience within two weeks.

Conclusions:

  • This protocol enables the construction of a versatile iSCAT microscope.
  • The iSCAT system facilitates advanced single-molecule studies, combining scattering and fluorescence detection.
  • The developed iSCAT microscope serves as a valuable tool for nanoscale imaging and sensing.