Time-resolved fluorescence microscopy using two-photon excitation
2003
We have developed a high sensitivity time-resolved two-photon scanning microscope. At an excitation wavelength of 960 nm, a spatial point spread function of 0.3 μm (FWHM) radially and 0.9 μm (FWHM) axially is measured for an 1.25 N.A. objective. The light source is a mode-locked titanium-sapphire laser. The time resolution is 400 ps with common chromophores used in microscopy. Time resolution is obtained using the frequency-domain heterodyning technique in which the laser is synchronized at a very high cross-correlation frequency to the rest of the electronics. We demonstrate spatial and time resolution using well-characterized fluorescent microspheres. We show two applications of two-photon time-resolved fluorescence microscopy: time-resolved imaging of multiple dye labeled cells and quantitative cellular calcium concentration using a lifetime indicator.
Keywords:
- Resonance fluorescence
- Two-photon excitation microscopy
- Fluorescence microscope
- Photoactivated localization microscopy
- Microscopy
- Fluorescence cross-correlation spectroscopy
- Fluorescence-lifetime imaging microscopy
- Analytical chemistry
- Super-resolution microscopy
- Chemistry
- Materials science
- Nuclear magnetic resonance
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