Non-Linear laser microscopy
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文摘
Non-linear laser microscopy based on multi-photon molecular excitation has reached a user-friendly maturity that offers a powerful tool for cellular and molecular biophysics. Intrinsically 3-d resoled microscopy for fluorescence imaging, photochemical micropharmacology and measurements of molecular dynamics are provided with minimal photodamage in living cells by simultaneous absorption of two or more red-infrared photons from the 100 MHz train of 100fs pulses of mode locked lasers. Two photon excitation spectra often show their largest cross sections at wavelengths far shorter than twice the one photon absorption peaks thus providing convenient red light excitation of many of the familiar visible absorbing as well as UV absorbing fluorophores. Three photon excitation provides convenient imaging of tryptophan, and polypeptide hormones and neurotransmitters that absorb below 300 nm. Our three-photon and two photon excitation cross section data and physical theory of multi-photon excitation guides experimental design. Recent developments to be illustrated include sensitivity to detect single fluorophores in solution submicron 3-d resolved Ca activity ratio imaging at speeds up to 45 microseconds per pixel, neurotransmitter release by cage activation, and applications of multicolor fluorescent markers for simultaneous imaging of 4 colours with a single excitation wave length, quantitative imaging with biochemical indicators, 3-d resolved imaging of tissue autofluorescence and green fluorescent protein genetic markers, and deep tissue fluorescence imaging.