Related Experiment Video
Updated: May 2, 2026
12:28
Imaging Plasma Membrane Deformations With pTIRFM
Published on: April 2, 2014
15.0K
Polarization-controlled TIRFM with focal drift and spatial field intensity correction
Daniel S Johnson1, Ricardo Toledo-Crow2, Alexa L Mattheyses1
1Laboratory of Cellular Biophysics, The Rockefeller University, New York, New York.
Biophysical Journal
|March 11, 2014
Summary
A new total internal reflection fluorescence microscopy (TIRFM) technique overcomes laser interference fringes for faster, clearer imaging of cellular processes like exocytosis and membrane dynamics.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- Total internal reflection fluorescence microscopy (TIRFM) is crucial for studying cellular dynamics like exocytosis and membrane processes.
- TIRFM improves signal-to-noise ratios in various advanced imaging techniques.
- Laser interference fringes in TIRFM hinder accurate quantification and imaging of large areas.
Purpose of the Study:
- To present a novel TIRFM technique that compensates for spatial fringes.
- To enable simultaneous rapid image acquisition with specific polarization.
- To develop a back reflection detection scheme for improved alignment and focus drift compensation.
Main Methods:
- Developed a new TIRFM method to compensate for spatial fringes.
- Enabled rapid (~25 ms) image acquisition for both parallel and perpendicular excitation.
- Implemented a back reflection detection scheme for laser alignment and focus drift compensation.
Main Results:
- Successfully compensated for spatial fringes in TIRFM imaging.
- Achieved rapid acquisition of parallel and perpendicular excitation data simultaneously.
- Demonstrated the technique's utility in imaging membrane orientation and exocytic vesicle dynamics.
Conclusions:
- The new TIRFM technique offers improved imaging quality and speed.
- It overcomes limitations of previous fringe-reduction methods by allowing simultaneous polarization excitation.
- The instrument is effective for studying cellular membrane dynamics and vesicle transport.
More Related Videos
Related Concept Videos
01:15Insensitive Nuclei Enhanced by Polarization Transfer (INEPT)
1.2K
Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) is an advanced Nuclear Magnetic Resonance (NMR) technique specifically designed to detect and enhance the signals of low-abundance nuclei, such as carbon-13 and nitrogen-15, in small molecules. The fundamental principle behind INEPT is the transfer of polarization from a more abundant and highly polarizable nucleus, typically hydrogen-1, to the low-abundance nucleus of interest. This process effectively boosts the NMR signal of the...
1.2K
01:29Potential Due to a Polarized Object
946
A neutral atom consists of a positively charged nucleus surrounded by a negatively charged electron cloud. When placed in an external electric field, the external electric force pulls the electrons and nucleus apart, opposite to the intrinsic attraction between the nucleus and the electrons. The opposing forces balance each other with a slight shift between the center of masses of the nucleus and the electron cloud, resulting in a polarized atom. On the other hand, a few molecules, like water,...
946
01:05Total Internal Reflection Fluorescence Microscopy
11.0K
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.
11.0K
01:20¹³C NMR: Distortionless Enhancement by Polarization Transfer (DEPT)
1.3K
When proton-coupled carbon-13 spectra are simplified by a broadband proton decoupling technique, structural information about the coupled protons is lost. Distortionless enhancement by polarization transfer (DEPT) is a technique that provides information on the number of hydrogens attached to each carbon in a molecule. While the DEPT experiment utilizes complex pulse sequences, the pulse delay and flip angle are specifically manipulated. The resulting signals have different phases depending on...
1.3K