@Conference{Lamminger2021,
  author    = {P. Lamminger, M. Loop, J. Klee, D. Weng, J.P. Kolb, M. Strauch, S. Karpf and R. Huber},
  booktitle = {Multimodal Biomedical Imaging XVI},
  title     = {Combination of two-photon microscopy and optical coherence tomography with fully fiber-based lasers for future endoscopic setups},
  year      = {2021},
  publisher = {SPIE},
  doi       = {10.1117/12.2578679},
  keywords  = {AG-Huber_NL, AG-Huber_OCT},
}

@InProceedings{Strauch2020,
  author    = {M. Strauch, J.P. Kolb, D. Weng, M. Wacker, W. Draxinger, N. Merg, J. Hundt, S. Karpf and R. Huber},
  booktitle = {104. Jahrestagung der Deutschen Gesellschaft fuer Pathologie},
  title     = {Two-photon microscopy for sectioning-free virtual {H&E} imaging},
URL = {https://www.pathologie-dgp.de/media/Dgp/user_upload/Verhandlungsband_2020_final__kompr._.pdf},
  year      = {2020},
  keywords  = {AG-Huber_NL},
}

@INPROCEEDINGS{8872571,
  author={Weng, Daniel and Hakert, Hubertus and Blömker, Torben and Kolb, Jan Philip and Strauch, Matthias and Eibl, Matthias and Lamminger, Philipp and Karpf, Sebastian and Huber, Robert},
  booktitle={2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC)}, 
  title={Sub-Nanosecond Pulsed Fiber Laser for 532nm Two-Photon Excitation Fluorescence (TPEF) Microscopy of UV Transitions}, 
  year={2019},
  volume={},
  number={},
  pages={1-1},
  abstract={Summary form only given. Two-photon microscopy is a powerful technique for in vivo imaging, due to its high penetration depth and axial sectioning. Usually excitation wavelengths in the near infrared are used. However, most fluorescence techniques for live cell imaging require labeling with exogenous fluorophores. It has been shown that shorter wavelengths can be used to excite the autofluorescence of endogenous proteins, e.g. tryptophan. Recently we demonstrated a fully fiber-based laser source built around a directly modulated, ytterbium amplified 1064 nm laser diode with sub-nanosecond pulses for two-photon imaging [2]. The overall system enables to capture high-speed fluorescence lifetime imaging (FLIM) with single pulse excitation. Here, we extend the spectral range of this laser source by frequency doubling it to 532nm to achieve two-photon excited fluorescence microscopy (TPM) in the ultraviolett (UV) range to harness endogenous autofluorescence. In this presentation we explore first TPM results of tryptophan to investigate signal levels and fi delity before transitioning to biological tissues. It has been shown that TPM of endogenous tryptophan can be used to visualize immune system activity in vivo. Our laser source could be a cheap, flexible and fiber-based alternative to the OPO-based Ti:Sa Lasers currently employed. The basic concept of our design is to shift the wavelength of the pulsed fiber-based master oscillator power amplifier (MOPA) by second-harmonic generation (SHG) using phase-matching in a KTP crystal. This generates a coherent output at 532nm at a maximal peak power of 500W. We achieved a maximum conversion efficiency of about 17%. After the SHG module, the 532nm light is coupled into a single-mode fiber and delivered to a home built microscope. A 40x microscope objective is used to excite the sample and epi-collect the fluorescence. The fluorescence is recorded on a UV-enhanced photomultiplier tube (PMT). For a proof of concept measurement, crystalized tryptophan was imaged. Here we show signals of pure tryptophan, with laser parameters of 1MHz repetition rate and 100ps pulse duration. We used spectral bandpass fi lters in order to detect only fluorescence signal, however, from crystalized tryptophan we observed an unexpected short lifetime. We have recently shown that we can shift our laser output from 1064nm to longer wavelengths. By shifting to 1180nm and frequency doubling to 590nm a more efficient fluorescence excitation of tryptophan can be achieved. In the future we aim at in vivo imaging with our setup.},
  keywords={},
  doi={10.1109/CLEOE-EQEC.2019.8872571},
  ISSN={},
  month={June}}

@inproceedings{10.1117/12.2507866,
author = {Jan Philip Kolb and Daniel Weng and Hubertus Hakert and Matthias Eibl and Wolfgang Draxinger and Tobias Meyer and Thomas Gottschall and Ralf  Brinkmann and Reginald Birngruber and J{\"u}rgen Popp and Jens Limpert and Sebastian Nino Karpf and Robert Huber},
title = {{Virtual HE histology by fiber-based picosecond two-photon microscopy}},
volume = {10882},
booktitle = {Multiphoton Microscopy in the Biomedical Sciences XIX},
editor = {Ammasi Periasamy and Peter T. C. So and Karsten K{\"o}nig},
organization = {International Society for Optics and Photonics},
publisher = {SPIE},
pages = {108822F},
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.},
keywords = {Multiphoton Microscopy, Four Wave Mixing, FWM, Histology, Laser, Non Linear Microscopy, Two Photon Microscopy, JenLab Young Investigator Award},
year = {2019},
doi = {10.1117/12.2507866},
URL = {https://doi.org/10.1117/12.2507866}
}

@InProceedings{Strauch2019,
  author    = {Strauch, Matthias and Kolb, Jan Philip and Weng, Daniel and Wacker, Melanie and Draxinger, Wolfgang and Karpf, Sebastian and Huber, Robert},
  booktitle = {31st Congress of the ESP},
  title     = {Sectioning-Free Virtual H&E Imaging of Tissue Samples with Two-Photon Microscopy},
  year      = {2019},
  keywords  = {AG-Huber_NL},
}

@article{Eibl2018,
  doi = {10.1364/boe.9.006273},
  url = {https://doi.org/10.1364/boe.9.006273},
  year = {2018},
  month = nov,
  publisher = {The Optical Society},
  volume = {9},
  number = {12},
  pages = {6273},
  author = {Matthias Eibl and Daniel Weng and Hubertus Hakert and Jan Philip Kolb and Tom Pfeiffer and Jennifer E. Hundt and Robert Huber and Sebastian Karpf},
  title = {Wavelength agile multi-photon microscopy with a fiber amplified diode laser},
  journal = {Biomedical Optics Express}
}

@inproceedings{10.1117/12.2286035,
author = {Matthias Eibl and Sebastian Karpf and Hubertus Hakert and Daniel Weng and Tom Pfeiffer and Jan Philip Kolb and Robert Huber},
title = {{Single pulse two-photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate and an all fiber based setup }},
volume = {10414},
booktitle = {Advances in Microscopic Imaging},
editor = {Emmanuel Beaurepaire and Francesco Saverio Pavone and Peter T. C. So},
organization = {International Society for Optics and Photonics},
publisher = {SPIE},
pages = {1041403},
abstract = {Newly developed microscopy methods have the goal to give researches in bio-molecular science a better understanding of processes ongoing on a cellular level. Especially two-photon excited fluorescence (TPEF) microscopy is a readily applied and widespread modality. Compared to one photon fluorescence imaging, it is possible to image not only the surface but also deeper lying structures. Together with fluorescence lifetime imaging (FLIM), which provides information on the chemical composition of a specimen, deeper insights on a molecular level can be gained. However, the need for elaborate light sources for TPEF and speed limitations for FLIM hinder an even wider application. In this contribution, we present a way to overcome this limitations by combining a robust and inexpensive fiber laser for nonlinear excitation with a fast analog digitization method for rapid FLIM imaging. The applied sub nanosecond pulsed laser source is perfectly suited for fiber delivery as typically limiting non-linear effects like self-phase or cross-phase modulation (SPM, XPM) are negligible. Furthermore, compared to the typically applied femtosecond pulses, our longer pulses produce much more fluorescence photons per single shot. In this paper, we show that this higher number of fluorescence photons per pulse combined with a high analog bandwidth detection makes it possible to not only use a single pulse per pixel for TPEF imaging but also to resolve the exponential time decay for FLIM. To evaluate our system, we acquired FLIM images of a dye solution with single exponential behavior to assess the accuracy of our lifetime determination and also FLIM images of a plant stem at a pixel rate of 1 MHz to show the speed performance of our single pulse two-photon FLIM (SP-FLIM) system.},
keywords = {Nonlinear microscopy, Fluorescence microscopy, Fiber optics imaging, Lifetime-based sensing, Lasers, fiber, Nonlinear optics, fibers},
year = {2017},
doi = {10.1117/12.2286035},
URL = {https://doi.org/10.1117/12.2286035}
}

@inproceedings{10.1117/12.2251965,
author = {Matthias Eibl and Sebastian Karpf and Hubertus Hakert and Daniel Weng and Torben Bl{\"o}mker and Robert Huber},
title = {{Pulse-to-pulse wavelength switching of diode based fiber laser for multi-color multi-photon imaging}},
volume = {10083},
booktitle = {Fiber Lasers XIV: Technology and Systems},
editor = {Craig A. Robin and Ingmar Hartl},
organization = {International Society for Optics and Photonics},
publisher = {SPIE},
pages = {100831C},
abstract = {We present an entirely fiber based laser source for non-linear imaging with a novel approach for multi-color excitation. The high power output of an actively modulated and amplified picosecond fiber laser at 1064 nm is shifted to longer wavelengths by a combination of four-wave mixing and stimulated Raman scattering. By combining different fiber types and lengths, we control the non-linear wavelength conversion in the delivery fiber itself and can switch between 1064 nm, 1122 nm, and 1186 nm on-the-fly by tuning the pump power of the fiber amplifier and modulate the seed diodes. This is a promising way to enhance the applicability of short pulsed laser diodes for bio-molecular non-linear imaging by reducing the spectral limitations of such sources. In comparison to our previous work [1, 2], we show for the first time two-photon imaging with the shifted wavelengths and we demonstrate pulse-to-pulse switching between the different wavelengths without changing the configuration.},
keywords = {stimulated raman scattering, two-photon imaging, fiber amplifier, four-wave-mixing, wavelength conversion, non-linear imaging},
year = {2017},
doi = {10.1117/12.2251965},
URL = {https://doi.org/10.1117/12.2251965}
}

@inproceedings{10.1117/12.2250831,
author = {Matthias Eibl and Sebastian Karpf and Hubertus Hakert and Daniel Weng and Robert Huber},
title = {{Two-photon-excited fluorescence (TPEF) and fluorescence lifetime imaging (FLIM) with sub-nanosecond pulses and a high analog bandwidth signal detection}},
volume = {10069},
booktitle = {Multiphoton Microscopy in the Biomedical Sciences XVII},
editor = {Ammasi Periasamy and Peter T. C. So and Karsten K{\"o}nig and Xiaoliang S. Xie},
organization = {International Society for Optics and Photonics},
publisher = {SPIE},
pages = {100691F},
abstract = {Two-photon excited fluorescence (TPEF) microscopy and fluorescence lifetime imaging (FLIM) are powerful imaging techniques in bio-molecular science. The need for elaborate light sources for TPEF and speed limitations for FLIM, however, hinder an even wider application. We present a way to overcome this limitations by combining a robust and inexpensive fiber laser for nonlinear excitation with a fast analog digitization method for rapid FLIM imaging. The applied sub nanosecond pulsed laser source is synchronized to a high analog bandwidth signal detection for single shot TPEF- and single shot FLIM imaging. The actively modulated pulses at 1064nm from the fiber laser are adjustable from 50ps to 5ns with kW of peak power. At a typically applied pulse lengths and repetition rates, the duty cycle is comparable to typically used femtosecond pulses and thus the peak power is also comparable at same cw-power. Hence, both types of excitation should yield the same number of fluorescence photons per time on average when used for TPEF imaging. However, in the 100ps configuration, a thousand times more fluorescence photons are generated per pulse. In this paper, we now show that the higher number of fluorescence photons per pulse combined with a high analog bandwidth detection makes it possible to not only use a single pulse per pixel for TPEF imaging but also to resolve the exponential time decay for FLIM. To evaluate the performance of our system, we acquired FLIM images of a Convallaria sample with pixel rates of 1 MHz where the lifetime information is directly measured with a fast real time digitizer. With the presented results, we show that longer pulses in the many-10ps to nanosecond regime can be readily applied for TPEF imaging and enable new imaging modalities like single pulse FLIM.},
keywords = {FLIM, TPEF, fiber laser, endoscope, MOPA, Nonlinear microscopy, Fluorescence microscopy, Lifetime-based sensing},
year = {2017},
doi = {10.1117/12.2250831},
URL = {https://doi.org/10.1117/12.2250831}
}

@article{Eibl:17,
author = {Matthias Eibl and Sebastian Karpf and Daniel Weng and Hubertus Hakert and Tom Pfeiffer and Jan Philip Kolb and Robert Huber},
journal = {Biomed. Opt. Express},
keywords = {Fiber optics imaging; Nonlinear optics, fibers; Lasers, fiber; Lifetime-based sensing; Fluorescence microscopy; Nonlinear microscopy; Fourier domain mode locking; Image quality; Imaging techniques; Laser sources; Pulsed fiber lasers; Three dimensional sensing},
number = {7},
pages = {3132--3142},
publisher = {Optica Publishing Group},
title = {Single pulse two photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate},
volume = {8},
month = {Jul},
year = {2017},
url = {https://opg.optica.org/boe/abstract.cfm?URI=boe-8-7-3132},
doi = {10.1364/BOE.8.003132},
abstract = {Two-photon-excited fluorescence lifetime imaging microscopy (FLIM) is a chemically specific 3-D sensing modality providing valuable information about the microstructure, composition and function of a sample. However, a more widespread application of this technique is hindered by the need for a sophisticated ultra-short pulse laser source and by speed limitations of current FLIM detection systems. To overcome these limitations, we combined a robust sub-nanosecond fiber laser as the excitation source with high analog bandwidth detection. Due to the long pulse length in our configuration, more fluorescence photons are generated per pulse, which allows us to derive the lifetime with a single excitation pulse only. In this paper, we show high quality FLIM images acquired at a pixel rate of 1 MHz. This approach is a promising candidate for an easy-to-use and benchtop FLIM system to make this technique available to a wider research community.},
}