Virtual H&E Histology by Fiber-Based Picosecond Two-Photon Microscopy

TL;DR

This paper introduces a fiber-based, tunable two-photon microscopy system capable of creating virtual H&E histology images, expanding wavelength range, and enabling in vivo 3D imaging with fluorescence lifetime measurements.

Contribution

It presents a compact, versatile TPM system with extended wavelength capabilities and virtual histology imaging, improving in vivo histological analysis.

Findings

Achieved virtual H&E histology imaging using fiber-based TPM.

Expanded wavelength range down to 940 nm for better tissue staining compatibility.

Demonstrated in vivo 3D imaging with fluorescence lifetime measurements.

Abstract

Two-Photon Microscopy (TPM) can provide three-dimensional morphological and functional contrast in vivo. Through proper staining, TPM can be utilized to create virtual, H&E equivalent images and thus can improve throughput in histology-based applications. We previously reported on a new light source for TPM that employs a compact and robust fiber-amplified, directly modulated laser. This laser is pulse-to-pulse wavelength switchable between 1064 nm, 1122 nm, and 1186 nm with an adjustable pulse duration from 50ps to 5ns and arbitrary repetition rates up to 1MHz at kW-peak powers. Despite the longer pulse duration, it can achieve similar average signal levels compared to fs-setups by lowering the repetition rate to achieve similar cw and peak power levels. The longer pulses lead to a larger number of photons per pulse, which yields single shot fluorescence lifetime measurements (FLIM) by applying a fast 4 GSamples/s digitizer. In the previous setup, the wavelengths were limited to 1064 nm and longer. Here, we use four wave mixing in a non-linear photonic crystal fiber to expand the wavelength range down to 940 nm. This wavelength is highly suitable for imaging green fluorescent proteins in neurosciences and stains such as acridine orange (AO), eosin yellow (EY) and sulforhodamine 101 (SR101) used for histology applications. In a more compact setup, we also show virtual H&E histological imaging using a direct 1030 nm fiber MOPA.