Abstract

Molecular excitation by simultaneous absorption of two or more infrared photons front strongly focused 100-fs pulses front mode-locked lasers provides intrinsic 3D resolution for fluorescence microscopy and photochemical micro pharmacology in living biological cells. Confinement of nonlinear excitation to the focal volume illuminated by ~10¹⁹ photons/nm²s eliminates out-of-focus photobleaching and photodamage. New methods for accurate measurements of multiphoton excitation cross sections and spectra have revealed useful two-photon cross sections, some with blue shifts, that facilitate application of a wide range of UV-absorbing fluorophores, including indicators such as Indo-1 for soluble ions like Ca²⁺, selective DNA stains, such as DAPI, green fluorescent protein (GFP), fluorogens, and NADH as a metabolic indicator and efficient visible fluorophores including flurocein, rhodamine, Texas Red, and rhodamine 123 a membrane potential indicator for mitochondria. Three-photon excitation of the amino acid tryptophan provides imaging of protein distributions and of secretory granules containing certain neurotransmitters. Applications to living biological cells and 200-μm deep into thick tissues are illustrated. Manipulation of excitation pulse patterns is providing new diagnostic data on dynamics of molecular intersystem crossings and mechanisms of photobleaching. Temporal resolution for 3-D resolved linear traces exceeds 1 ms with ~1 millisecond per voxel and full video rate precision fluorescence imaging with 3-D resolution is realized in living tissues.

© 1996 Optical Society of America