Two-Photon Imaging in Turbid Media to a Few MM Depths

Abstract

We present a new detection method for two-photon fluorescence imaging of turbid media that extends imaging depth to few mm. Compared to conventional two-photon microscopy, the imaging depth enhancement is achieved by use of a more efficient detection of fluorescence, propagating in multiple-scattering media. The imaging depth in turbid media depends on two major factors: the ability of the imaging system to deliver necessary excitation light power to a certain depth to induce two-photon fluorescence and the ability of the detection system to collect and detect fluorescence photons scattered by the media. In two-photon imaging, usually, fluorescence is collected by the same microscope objective that is used for excitation. This optical scheme has its principal limitation in efficiency of fluorescence detection because of the narrow angle and area from which fluorescence photons can be collected. As a result, the maximum depth of tissue imaging that was reached so far by two-photon fluorescence microscopy is about 1mm. Our system utilizes for excitation the conventional two-photon microscopy scheme, however, for fluorescence detection we used 1” cathode area PMT directly placed on the turbid sample from the opposite side of excitation. The PMT was equipped with a mechanical shutter and optical filters to reject excitation light and transmit only fluorescence. All optical components of the detector were coupled with index matching compounds to assure refractive index continuity from the sample to the PMT photocathode to minimize light losses due to reflection at boundaries. This detection scheme, while simple in construction and practically not requiring any optics, was proved to be very efficient in the collection of weak fluorescence from a wide area of a turbid sample and allowed to obtain high resolution images at depths up to 3mm.