Two-Photon Microscopy: Imaging Tissue with Lasers

Two-photon fluorescence microscopy allows for imaging deep within biological tissue without making an incision. Two-photon fluorescence occurs when two photons are simultaneously absorbed by a dye molecule and a single higher... [ view full abstract ]

Two-photon fluorescence microscopy allows for imaging deep within biological tissue without making an incision. Two-photon fluorescence occurs when two photons are simultaneously absorbed by a dye molecule and a single higher energy photon is released. The low probability of two-photon absorption ensures that fluorescence will only occur where many photons are in the same place at the same time, i.e., at the focus of a laser beam. By moving the focus of the laser and recording the induced fluorescence, a 3D image of the fluorescing medium is produced with micron level resolution. We have built an upright microscope to allow for water immersion samples, and by experimentally measuring its lateral point spread function (PSF) have found its resolution to be (2.2 ± 0.2) μm in the focal plane of the microscope. Current work involves expanding the measurement of the PSF to a third dimension, and consolidating control of the various instruments of the microscope into a single computer program.