Related Experiment Video
Updated: May 15, 2026
20:00
Single Molecule Fluorescence Microscopy on Planar Supported Bilayers
Published on: October 31, 2015
Development of new photon-counting detectors for single-molecule fluorescence microscopy
X Michalet1, R A Colyer, G Scalia
1Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095-1547, USA. michalet@chem.ucla.edu
Summary
Researchers are improving single-molecule fluorescence spectroscopy by developing parallel arrays of single-photon-counting avalanche diodes (SPADs) for higher throughput and large-area photon-counting cameras for advanced imaging.
Area of Science:
- Optics and Photonics
- Biophysical Techniques
- Spectroscopy
Background:
- Single-molecule fluorescence microscopy utilizes distinct optical configurations: point-like detection for diffusing molecules and wide-field detection for immobilized ones.
- Current detectors, single-photon-counting avalanche diodes (SPADs) and electron-multiplying charge-coupled devices (EMCCDs), have limitations in throughput and frame rate, respectively.
Purpose of the Study:
- To enhance the throughput of single-molecule fluorescence spectroscopy in solution.
- To develop advanced detectors for improved single-molecule imaging applications.
Main Methods:
- Development of parallel arrays of SPADs to increase data acquisition speed.
- Engineering of large-area photon-counting cameras with sub-nanosecond resolution.
Main Results:
- Achieved increased throughput in single-molecule fluorescence spectroscopy using parallel SPAD arrays.
- Demonstrated sub-nanosecond resolution in fluorescence lifetime imaging with new camera technology.
Conclusions:
- Parallel SPAD arrays offer a pathway to higher throughput in solution-based single-molecule spectroscopy.
- Advanced photon-counting cameras enable high-resolution fluorescence lifetime imaging at the single-molecule level.
Related Concept Videos
01:37Super-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.
01:25Fluorescence and Phosphorescence: Instrumentation
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.
01:19Protein 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...
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...