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Related Concept Videos

Total Internal Reflection Fluorescence Microscopy01:05

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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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Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy
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Data Analysis for Total Internal Reflection Fluorescence Microscopy.

Charles L Asbury1

  • 1Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195.

Cold Spring Harbor Protocols
|May 4, 2016
PubMed
Summary
This summary is machine-generated.

We developed semiautomated image analysis programs for total internal reflection fluorescence microscopy. These tools quantify particle behavior, such as brightness and mobility, on single microtubules.

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Area of Science:

  • Biophysics
  • Microscopy
  • Cell Biology

Background:

  • Total internal reflection fluorescence (TIRF) microscopy utilizes dichroic filters to split emitted fluorescence into multiple color channels for simultaneous imaging.
  • Existing TIRF instruments can be configured for two or three color channels, accommodating various fluorescent labels like green fluorescent protein (GFP) and Alexa dyes.
  • Simultaneous multicolor imaging in TIRF microscopy enables the study of dynamic molecular interactions within cells.

Purpose of the Study:

  • To describe the development of semiautomated image analysis programs for TIRF microscopy.
  • To enable the quantification of individual particle properties, including brightness, residence time, and mobility, when bound to single microtubules.
  • To provide a protocol for analyzing single microtubule data, emphasizing the need for comprehensive data collection for robust conclusions.

Main Methods:

  • Utilized two-color and three-color TIRF microscopy setups with specific wavelength ranges for different fluorescent probes (e.g., GFP, mCherry, Alexa dyes).
  • Developed semiautomated image analysis programs using LabView for particle tracking and property extraction.
  • Collected images at 10 frames/sec for approximately 200 seconds, with options for alternative analysis software like ImageJ or Matlab.

Main Results:

  • Successfully implemented semiautomated analysis to extract quantitative data on particle brightness, residence time, and mobility.
  • Demonstrated the capability of simultaneous multicolor imaging in TIRF microscopy for analyzing particle dynamics.
  • Established a protocol for analyzing data from single microtubules, highlighting the importance of extensive data aggregation.

Conclusions:

  • The developed semiautomated image analysis programs provide an efficient method for quantifying particle behavior in TIRF microscopy.
  • Simultaneous multicolor imaging and robust data analysis are crucial for understanding complex molecular interactions on microtubules.
  • The protocol facilitates the study of single particle dynamics, contributing to a deeper understanding of cellular processes.