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Virtual HE histology by fiber-based picosecond two-photon microscopy
Author(s): 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
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Paper 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.

Paper Details

Date Published: 22 February 2019
PDF: 8 pages
Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 108822F (22 February 2019); doi: 10.1117/12.2507866
Show Author Affiliations
Jan Philip Kolb, Medizinisches Laserzentrum Lübeck GmbH (Germany)
Daniel Weng, Univ. zu Lübeck (Germany)
Hubertus Hakert, Medizinisches Laserzentrum Lübeck GmbH (Germany)
Matthias Eibl, Univ. zu Lübeck (Germany)
Wolfgang Draxinger, Univ. zu Lübeck (Germany)
Tobias Meyer, Friedrich-Schiller-Univ. Jena (Germany)
Thomas Gottschall, Friedrich-Schiller-Univ. Jena (Germany)
Ralf Brinkmann, Medizinisches Laserzentrum Lübeck GmbH (Germany)
Reginald Birngruber, Univ. zu Lübeck (Germany)
Jürgen Popp, Friedrich-Schiller-Univ. Jena (Germany)
Jens Limpert, Friedrich-Schiller-Univ. Jena (Germany)
Sebastian Nino Karpf, Univ. zu Lübeck (Germany)
Robert Huber, Univ. zu Lübeck (Germany)

Published in SPIE Proceedings Vol. 10882:
Multiphoton Microscopy in the Biomedical Sciences XIX
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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