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


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.

Original languageEnglish
Title of host publicationMultiphoton Microscopy in the Biomedical Sciences XIX
EditorsAmmasi Periasamy, Peter T. C. So , Karsten König
Number of pages8
Publication date22.02.2019
Article number108822F
ISBN (Print)978-151062406-1
Publication statusPublished - 22.02.2019
EventMultiphoton Microscopy in the Biomedical Sciences XIX 2019 - San Francisco, United States
Duration: 03.02.201906.02.2019
Conference number: 148723

Research Areas and Centers

  • Academic Focus: Biomedical Engineering


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