Virtual HE histology by fiber-based picosecond two-photon microscopy

Jan Philip Kolb; Daniel Weng; Hubertus Hakert; Matthias Eibl; Wolfgang Draxinger; Tobias Meyer; Thomas Gottschall; Ralf Brinkmann; Reginald Birngruber; Jürgen Popp; Jens Limpert; Sebastian Nino Karpf; Robert Huber

doi:10.1117/12.2507866

22 February 2019 Virtual H&E histology by fiber-based picosecond two-photon microscopy

Jan Philip Kolb, Daniel Weng, Hubertus Hakert, Matthias Eibl, Wolfgang Draxinger, Tobias Meyer, Thomas Gottschall, Ralf Brinkmann, Reginald Birngruber, Jürgen Popp, Jens Limpert, Sebastian Nino Karpf, Robert Huber

Author Affiliations +

Proceedings Volume 10882, Multiphoton Microscopy in the Biomedical Sciences XIX; 108822F (2019) https://doi.org/10.1117/12.2507866
Event: SPIE BiOS, 2019, San Francisco, California, United States

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, HE 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 HE histological imaging using a direct 1030 nm fiber MOPA.

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Jan Philip Kolb, Daniel Weng, Hubertus Hakert, Matthias Eibl, Wolfgang Draxinger, Tobias Meyer, Thomas Gottschall, Ralf Brinkmann, Reginald Birngruber, Jürgen Popp, Jens Limpert, Sebastian Nino Karpf, and Robert Huber "Virtual H&E histology by fiber-based picosecond two-photon microscopy", Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 108822F (22 February 2019); https://doi.org/10.1117/12.2507866

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KEYWORDS

Luminescence

Femtosecond phenomena

Picosecond phenomena

Fiber lasers

Optical filters

Laser systems engineering

Objectives

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