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Updated: Jun 26, 2026
14:09
High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip
Published on: November 16, 2019
Time-gated total internal reflection fluorescence microscopy with a supercontinuum excitation source.
Pierre Blandin1, Sandrine Lévêque-Fort, Sandrine Lécart
1Laboratoire de Photophysique Moléculaire, CNRS, Université Paris-Sud, Bat 210, 91405 Orsay, France.
Applied Optics
|January 20, 2009
Summary
We developed a versatile total internal reflection fluorescence lifetime imaging microscopy setup. This advanced microscopy offers subwavelength axial resolution, ideal for dynamic neurobiology research.
Area of Science:
- Biophysics
- Microscopy
- Neuroscience
Background:
- Fluorescence lifetime imaging microscopy (FLIM) is crucial for biological research.
- Achieving high axial resolution in FLIM is challenging, limiting dynamic studies.
Purpose of the Study:
- To develop and present a novel total internal reflection fluorescence lifetime imaging microscopy (TIRF-FLIM) setup.
- To achieve subwavelength axial resolution for wide-field FLIM.
- To enable dynamic neurobiological applications.
Main Methods:
- Instrumental development of a versatile TIRF-FLIM system.
- Illumination using a supercontinuum laser source.
- Characterization of imaging performance across various fluorophores.
Main Results:
- Demonstrated wide-field FLIM with subwavelength axial resolution.
- The setup accommodates a broad spectrum of fluorophores.
- Achieved short overall acquisition times suitable for dynamic processes.
Conclusions:
- The developed TIRF-FLIM system is a versatile tool for advanced biological imaging.
- Its high axial resolution and speed are advantageous for dynamic neurobiology.
- This instrumental advancement expands the capabilities of FLIM in life sciences.
Related Concept Videos
01:05Total Internal Reflection Fluorescence Microscopy
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.
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.