Abstract
We constructed an advanced detection system for two-photon fluorescence microscopy that allows us to image in biological tissue and tissue phantoms up to the depth of a few mm with micron resolution. The innovation lies in the detection system which is much more sensitive to low level fluorescence signals than the fluorescence detection configuration used in conventional two-photon fluorescence microscopes. A wide area photocathode photomultiplier tube (PMT) was used to detect fluorescence photons directly from a wide (1 inch diameter) area of the turbid sample, as opposed to the photon collection by the microscope objective which can only collect light from a relatively small area of the sample. The optical path between the sample and the photocathode is refractive index matched to curtail losses at the boundaries due to reflections. The system has been successfully employed in the imaging of tissue phantoms simulating brain optical properties and in biological tissues, such as murine small intestine, colon, tumors, and other samples. The system has in-depth fluorescence lifetime imaging (FLIM) capabilities and is also highly suitable for SHG signal detection, such as collagen fibers and muscles, due to the intrinsically forward-directed propagation of SHG photons.
Highlights
The ability to visualize features in deep layers of biological tissue with high resolution is a very sought-after feature of an imaging system employed in medical diagnostics and in clinical and biological applications
The system is equipped with a second photomultiplier tube (PMT) (Hamamtsu H7422P-40) that works in the epi-fluorescence configuration to compare the results between the conventional 2-photon fluorescence microscope configuration and our system
We have demonstrated the capabilities of our system to acquire fluorescence lifetime imaging (FLIM) images in depths exceeding those achievable by conventional microscopes
Summary
The ability to visualize features in deep layers of biological tissue with high resolution is a very sought-after feature of an imaging system employed in medical diagnostics and in clinical and biological applications. Deep-tissue imaging has many applications, all based on the necessity of exploring cells and molecules in the intact organism, a sort of “optical pathology” that removes the need of biopsies and fixing, and grants access to structures and functions in the native physiological environment Both the geometrical and optical properties of the sample and the characteristics of the microscope system affect the achievable imaging depth. While transparent specimens can be imaged with a traditional microscope, biological tissue is an intrinsically turbid medium, which produces a strong multiple scattering, absorption and exhibits inhomogeneity of the refractive index These features make traditional light and fluorescence microscopy, even with the aid of staining and the addition of fluorescent markers to improve contrast, ineffective past the 100-200 μm surface layer of the sample [1]. The phasorapproach [15] introduced by Digman et al, performs FLIM data analysis in the frequency domain and it has greatly simplified data processing
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