Nichtlineare Mirkoskopie

Nichtlineare optische Bildgebung und Spektroskopie an biologischen Proben

Die nichtlineare optische Mikroskopie ist eine leistungsstarke Technik für die Biomolekularwissenschaft, die es ermöglicht, biochemische Prozesse auf zellulärer Ebene besser zu verstehen. Das Hauptziel ist es, nicht nur die Morphologie, sondern auch die chemische Zusammensetzung einer Probe zu identifizieren.

Wir arbeiten derzeit an drei Ansätzen um optisch Kontrast zu erzeugen:

  • Zeitcodierte Ramanspektroskopie- und mikroskopie (TICO Raman)
  • Zweiphotonen-Fluoreszenzmikroskopie (TPEF)
  • Einzelphotonen Fluoreszenzlebenszeit Bildgebung (SP-FLIM)

zugehörige Publikationen

2023

Philipp Lamminger, Hubertus Hakert, Simon Lotz, Jan Philip Kolb, Tonio Kutscher, Sebastian Karpf, and Robert Huber,
Four-Wave Mixing Fast Wavelength Sweeping FDML Laser with kW Peak Power at 900 nm and 1300 nm, in 2023 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) , 072023. pp. 1.
DOI:10.1109/CLEO/Europe-EQEC57999.2023.10232141
Bibtex: BibTeX
@INPROCEEDINGS{10232141,
  author={Lamminger, Philipp and Hakert, Hubertus and Lotz, Simon and Kolb, Jan Philip and Kutscher, Tonio and Karpf, Sebastian and Huber, Robert},
  booktitle={2023 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC)}, 
  title={Four-Wave Mixing Fast Wavelength Sweeping FDML Laser with kW Peak Power at 900 nm and 1300 nm}, 
  year={2023},
  volume={},
  number={},
  pages={1-1},
  doi={10.1109/CLEO/Europe-EQEC57999.2023.10232141}}
Philipp Lamminger, Hubertus Hakert, Simon Lotz, Jan Philip Kolb, Tonio Kutscher, Sebastian Karpf, and Robert Huber,
Four-wave mixing seeded by a rapid wavelength-sweeping FDML laser for nonlinear imaging at 900 nm and 1300 nm, Opt. Lett. , vol. 48, no. 14, pp. 3713-3716, 07 2023. Optica Publishing Group.
DOI:10.1364/OL.488181
Bibtex: BibTeX
@article{Lamminger:23,
author = {Philipp Lamminger and Hubertus Hakert and Simon Lotz and Jan Philip Kolb and Tonio Kutscher and Sebastian Karpf and Robert Huber},
journal = {Opt. Lett.},
keywords = {Green fluorescent protein; Laser beam combining; Laser crystals; Laser imaging; Optical amplifiers; Photonic crystal lasers},
number = {14},
pages = {3713--3716},
publisher = {Optica Publishing Group},
title = {Four-wave mixing seeded by a rapid wavelength-sweeping FDML laser for nonlinear imaging at 900 nm and 1300 nm},
volume = {48},
month = {Jul},
year = {2023},
url = {https://opg.optica.org/ol/abstract.cfm?URI=ol-48-14-3713},
doi = {10.1364/OL.488181},
abstract = {Four-wave mixing (FWM) enables the generation and amplification of light in spectral regions where suitable fiber gain media are unavailable. The 1300 nm and 900 nm regions are of especially high interest for time-encoded (TICO) stimulated Raman scattering microscopy and spectro-temporal laser imaging by diffracted excitation (SLIDE) two-photon microscopy. We present a new, to the best of our knowledge, FWM setup where we shift the power of a home-built fully fiber-based master oscillator power amplifier (MOPA) at 1064 nm to the 1300-nm region of a rapidly wavelength-sweeping Fourier domain mode-locked (FDML) laser in a photonic crystal fiber (PCF) creating pulses in the 900-nm region. The resulting 900-nm light can be wavelength swept over 54 nm and has up to 2.5 kW (0.2 {\textmu}J) peak power and a narrow instantaneous spectral linewidth of 70 pm. The arbitrary pulse patterns of the MOPA and the fast wavelength tuning of the FDML laser (419 kHz) allow it to rapidly tune the FWM light enabling new and faster TICO-Raman microscopy, SLIDE imaging, and other applications.},
}
Philipp Lamminger, Hubertus Hakert, Simon Lotz, Jan Philip Kolb, Tonio Kutscher, Sebastian Karpf, and Robert Huber,
900 nm swept source FDML laser with kW peak power, in Fiber Lasers XX: Technology and Systems , V. R. Supradeepa, Eds. SPIE, 032023. pp. 124001I.
DOI:10.1117/12.2649663
Bibtex: BibTeX
@inproceedings{10.1117/12.2649663,
author = {Philipp Lamminger and Hubertus Hakert and Simon Lotz and Jan Philip Kolb and Tonio Kutscher and Sebastian Karpf and Robert Huber},
title = {{900 nm swept source FDML laser with kW peak power}},
volume = {12400},
booktitle = {Fiber Lasers XX: Technology and Systems},
editor = {V.  R. Supradeepa},
organization = {International Society for Optics and Photonics},
publisher = {SPIE},
pages = {124001I},
abstract = {A wavelength agile 900 nm 2.5 kW peak power fiber laser is created by four-wave mixing (FWM) in a photonic crystal fiber (PCF), while amplifying a 1300 nm Fourier-domain mode-locked (FDML) laser. The FWM process is pumped by a home-built 1064 nm master oscillator power amplifier (MOPA) laser and seeded by a home-built 1300 nm FDML laser, generating high power pulses at wavelengths, where amplification by active fiber media is difficult. The 900 nm pulses have a spectral linewidth of 70 pm, are tunable over 54 nm and have electronic pulse-to-pulse tuning capability. These pulses can be used for nonlinear imaging like two-photon or coherent anti-Stokes Raman microscopy (CARS) microscopy including spectro-temporal laser imaging by diffracted excitation (SLIDE) and time-encoded (Tico) stimulated Raman microscopy.},
keywords = {Fourier domain mode locking,  FDML, Raman, two photon microscopy, SLIDE, 900 nm, fiber laser, photonic crystal fiber, swept source},
year = {2023},
doi = {10.1117/12.2649663},
URL = {https://doi.org/10.1117/12.2649663}
}
Matthias Strauch, Jan Philip Kolb, Christian Rose, Nadine Merg, Jennifer Hundt, Christiane Kümpers, Sven Perner, Sebastian Karpf, and Robert Huber,
Accelerating intraoperative tumor histology with sectioning-free multiphoton microscopy, European Journal of Surgical Oncology , vol. 49, no. 2, pp. e210, 02 2023.
DOI:https://doi.org/10.1016/j.ejso.2022.11.575
Datei: S0748798322013245
Bibtex: BibTeX
@article{STRAUCH2023e210,
title = {Accelerating intraoperative tumor histology with sectioning-free multiphoton microscopy},
journal = {European Journal of Surgical Oncology},
volume = {49},
number = {2},
pages = {e210},
year = {2023},
issn = {0748-7983},
doi = {https://doi.org/10.1016/j.ejso.2022.11.575},
url = {https://www.sciencedirect.com/science/article/pii/S0748798322013245},
author = {Matthias Strauch and Jan Philip Kolb and Christian Rose and Nadine Merg and Jennifer Hundt and Christiane Kümpers and Sven Perner and Sebastian Karpf and Robert Huber}
}

2021

Hubertus Hakert, Matthias Eibl, Marie Tillich, Ralph Pries, Gereon Hüttmann, Ralf Brinkmann, Barbara Wollenberg, Ludwig Bruchhage, Sebastian Karpf, and Robert Huber,
Time-encoded stimulated Raman scattering microscopy of tumorous human pharynx tissue in the fingerprint region from 1500–1800  cm-1, Optics Letters , vol. 46(14), no. 14, pp. 3456-3459, 07 2021.
DOI:10.1364/OL.424726
Bibtex: BibTeX
@article{Hakert2021,
   author = {H. Hakert, M. Eibl, M. Tillich, R.Pries, G. Hüttmann, R. Brinkmann, B. Wollenberg, K-L. Bruchhage, S. Karpf and R. Huber},
   title = {Time-encoded stimulated Raman scattering microscopy of tumorous human pharynx tissue in the fingerprint region from 1500–1800  cm-1},
   journal = {Optics Letters},
   volume = {46(14)},
   number = {14},
   pages = {3456-3459},
keywords = {AG-Huber_NL, Clinical applications, Master oscillator power amplifiers, Optical coherence tomography, Raman scattering, Stimulated Raman scattering, Stimulated scattering},
   DOI = {https://doi.org/10.1364/OL.424726},
   year = {2021},
   type = {Journal Article}
}
Thomas Gottschall, Tobias Meyer-Zedler, Michael Schmitt, Robert Huber, Jürgen Popp, Andreas Tünnermann, and Jens Limpert,
Ultra-compact tunable fiber laser for coherent anti-Stokes Raman imaging, JRS , 06 2021.
DOI:10.1002/jrs.6171
Bibtex: BibTeX
@article{Gottschall2021,
   author = {T. Gottschall, T. Meyer-Zedler, M. Schmitt, R. Huber, J. Popp, A. Tünnermann and J. Limpert},
   title = {Ultra-compact tunable fiber laser for coherent anti-Stokes Raman imaging},
   journal = {JRS},
  keywords = { AG-Huber_NL, coherent anti-Stokes Raman scattering microscopy, four-wave mixing, nonlinear
microscopy, ultrafast laser},
   ISSN = {0377-0486},   
   url = {https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/abs/10.1002/jrs.6171},
   year = {2021},
   type = {Journal Article}
}
Philipp Lamminger, Merle Loop, Julian Klee, Daniel Weng, Jan Philip Kolb, Matthias Strauch, Sebastian Karpf, and Robert Huber,
Combination of two-photon microscopy and optical coherence tomography with fully fiber-based lasers for future endoscopic setups, in Multimodal Biomedical Imaging XVI , SPIE, 032021.
DOI:10.1117/12.2578679
Bibtex: BibTeX
@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},
}
Matthias Strauch, Jan Philip Kolb, Wolfgang Draxinger, Ann-Kathrin Popp, Melanie Wacker, Nadine Merg, Jennifer Hundt, Sebastian Karpf, and Robert Huber,
Sectioning-free virtual H&E histology with fiber-based two-photon microscopy, in SPIE BiOS , SPIE, 032021.
DOI:10.1117/12.2578334
Bibtex: BibTeX
@inproceedings{RN5318,
   author = {Strauch, M;Kolb, J P;Draxinger, W;Popp, A-K;Wacker, M;Merg, N;Hundt, J;Karpf, S and Huber, R},
   title = {Sectioning-free virtual H&E histology with fiber-based two-photon microscopy},
   booktitle = {SPIE BiOS},
   publisher = {SPIE},
   volume = {11648},
Year = {2021},
   DOI = {https://doi.org/10.1117/12.2578334},
   url = {https://doi.org/10.1117/12.2578334},
   type = {Conference Proceedings}
}
Matthias Strauch, Jan Philip Kolb, Christian Rose, Nadine Merg, Christiane Kümpers, Sven Perner, Jennifer Hundt, Sebastian Karpf, and Robert Huber,
Comparison of Sectioning-free Multiphoton Histology to H&E FFPE imaging, in Virtuelle Pathologietage , 2021.
Bibtex: BibTeX
@Conference{Strauch2021b,
  author    = {M. Strauch, J.P. Kolb, C. Rose, N. Merg, J. Hundt, C. Kümpers, S. Perner, S. Karpf and R. Hubert},
  booktitle = {Virtuelle Pathologietage},
  title     = {Comparison of Sectioning-free Multiphoton Histology to H&E FFPE imaging},
  year      = {2021},
  keywords  = {AG-Huber_NL},
}
Matthias Strauch, Jan Philip Kolb, Christian Rose, Nadine Merg, Jennifer Hundt, Christiane Kümpers, Sven Perner, Sebastian Karpf, and Robert Huber,
Quick sectioning-free H&E imaging of bulk tissue using multiphoton microscopy, in 33rd Congress of the ESP , 2021.
DOI:10.1007/s00428-021-03157-8
Bibtex: BibTeX
@Conference{Strauch2021,
  author    = {M. Strauch, J.P. Kolb, C. Rose, N. Merg, J. Hundt, C. Kümpers, S. Perner, S. Karpf and R. Huber},
  booktitle = {33rd Congress of the ESP},
  title     = {Quick sectioning-free H&E imaging of bulk tissue using multiphoton microscopy},
  year      = {2021},
  keywords  = {AG-Huber_NL},
}

2020

Matthias Strauch, Jan Philip Kolb, Daniel Weng, Melanie Wacker, Wolfgang Draxinger, Nadine Merg, Jennifer Hundt, Sebastian Karpf, and Robert Huber,
Two-photon microscopy for sectioning-free virtual H&E imaging, in 104. Jahrestagung der Deutschen Gesellschaft fuer Pathologie , 062020.
Weblink: https://www.pathologie-dgp.de/media/Dgp/user_upload/Verhandlungsband_2020_final__kompr._.pdf
Bibtex: BibTeX
@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},
}
Matthias Strauch, Jan Philip Kolb, Nadine Merg, Jennifer Hundt, Sebastian Karpf, and Robert Huber,
Evaluation of two-photon fluorescence microscopy for sectioning-free H&E imaging of different tissues, in 32nd Congress of the ESP and XXXIII International Congress of the IAP , 2020.
DOI:10.1007/s00428-020-02938-x
Bibtex: BibTeX
@InProceedings{Strauch2020a,
  author    = {M. Strauch, J.P. Kolb, N. Merg, J. Hundt, S. Karpf and R. Huber},
  booktitle = {32nd Congress of the ESP and XXXIII International Congress of the IAP},
  title     = {Evaluation of two-photon fluorescence microscopy for sectioning-free {H&E} imaging of different tissues},
  year      = {2020},
  keywords  = {AG-Huber_NL},
}

2019

Daniel Weng, Hubertus Hakert, Torben Blömker, Jan Philip Kolb, Matthias Strauch, Matthias Eibl, Philipp Lamminger, Sebastian Karpf, and Robert Huber,
Sub-Nanosecond Pulsed Fiber Laser for 532nm Two-Photon Excitation Fluorescence (TPEF) Microscopy of UV Transitions, in 2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) , IEEE, 062019. pp. 1-1.
DOI:10.1109/CLEOE-EQEC.2019.8872571
Bibtex: BibTeX
@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}}
Jan Philip Kolb, Daniel Weng, Hubertus Hakert, Matthias Eibl, Wolfgang Draxinger, Tobias Meyer-Zedler, Thomas Gottschall, Ralf Brinkmann, Reginald Birngruber, Jürgen Popp, Jens Limpert, Sebastian Karpf, and Robert Huber,
Virtual HE histology by fiber-based picosecond two-photon microscopy, in Multiphoton Microscopy in the Biomedical Sciences XIX , Ammasi Periasamy; Peter T. C. So; Karsten König, Eds. International Society for Optics and Photonics, 022019. pp. 108822F.
DOI:10.1117/12.2507866
Bibtex: BibTeX
@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}
}
Matthias Strauch, Jan Philip Kolb, Daniel Weng, Melanie Wacker, Wolfgang Draxinger, Sebastian Karpf, and Robert Huber,
Sectioning-Free Virtual H&E Imaging of Tissue Samples with Two-Photon Microscopy, in 31st Congress of the ESP , 2019.
DOI:10.1007/s00428-019-02631-8
Bibtex: BibTeX
@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},
}

2018

Matthias Eibl, Daniel Weng, Hubertus Hakert, Jan Philip Kolb, Tom Pfeiffer, Jennifer Hundt, Robert Huber, and Sebastian Karpf,
Wavelength agile multi-photon microscopy with a fiber amplified diode laser, Biomedical Optics Express , vol. 9, no. 12, pp. 6273-6282, Nov. 2018. The Optical Society.
DOI:10.1364/BOE.9.006273
Bibtex: BibTeX
@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}
}

2017

Matthias Eibl, Sebastian Karpf, Hubertus Hakert, Torben Blömker, Jan Philip Kolb, Christian Jirauschek, and Robert Huber,
Pulse-to-pulse wavelength switching of a nanosecond fiber laser by four-wave mixing seeded stimulated Raman amplification, Opt. Lett. , vol. 42, no. 21, pp. 4406-4409, Nov. 2017. Optica Publishing Group.
DOI:10.1364/OL.42.004406
Bibtex: BibTeX
@article{Eibl:17,
author = {Matthias Eibl and Sebastian Karpf and Hubertus Hakert and Torben Bl\"{o}mker and Jan Philip Kolb and Christian Jirauschek and Robert Huber},
journal = {Opt. Lett.},
keywords = {Lasers, fiber; Lasers, Raman; Nonlinear optics, four-wave mixing; Scattering, stimulated Raman; Lasers, ytterbium ; Fiber lasers; Master oscillator power amplifiers; Nanosecond pulses; Raman scattering; Stimulated Brillouin scattering; Wavelength conversion},
number = {21},
pages = {4406--4409},
publisher = {Optica Publishing Group},
title = {Pulse-to-pulse wavelength switching of a nanosecond fiber laser by four-wave mixing seeded stimulated Raman amplification},
volume = {42},
month = {Nov},
year = {2017},
url = {https://opg.optica.org/ol/abstract.cfm?URI=ol-42-21-4406},
doi = {10.1364/OL.42.004406},
abstract = {We report on a multi-color fiber laser based on four-wave mixing (FWM) and stimulated Raman scattering (SRS), delivering rapidly wavelength switchable narrowband output at 1064, 1122, and 1186\&\#x00A0;nm. High-power pulses from a nanosecond pulsed fiber master oscillator power amplifier at 1064\&\#x00A0;nm are combined with 1122\&\#x00A0;nm of seed light for Raman amplification at the first Stokes order in a standard single-mode fiber. With increasing power, we observe a narrowband spectral component at 1186\&\#x00A0;nm, without any additional seed or resonator at this wavelength. We analyze this occurrence of a narrowband second Stokes order both experimentally and theoretically and suggest it is a result of FWM seeding of the SRS amplification in the fiber. We demonstrate that the wavelength shifting can be controlled electronically within microseconds for very rapid and even pulse-to-pulse wavelength changes. This wavelength conversion method can extend the spectral coverage of single-wavelength fiber lasers for biomedical imaging.},
}
Matthias Eibl, Sebastian Karpf, Hubertus Hakert, Daniel Weng, Tom Pfeiffer, Jan Philip Kolb, and Robert Huber,
Single pulse two-photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate and an all fiber based setup, in Advances in Microscopic Imaging , Emmanuel Beaurepaire and Francesco Saverio Pavone and Peter T. C. So, Eds. SPIE, 072017. pp. 1041403.
DOI:10.1117/12.2286035
Bibtex: BibTeX
@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}
}
Hubertus Hakert, Matthias Eibl, Sebastian Karpf, and Robert Huber,
Sparse-sampling with time-encoded (TICO) stimulated Raman scattering for fast image acquisition, in Advances in Microscopic Imaging , Emmanuel Beaurepaire and Francesco Saverio Pavone and Peter T. C. So, Eds. SPIE, 072017. pp. 1041408.
DOI:10.1117/12.2287947
Bibtex: BibTeX
@inproceedings{10.1117/12.2287947,
author = {Hubertus Hakert and Matthias Eibl and Sebastian Karpf and Robert Huber},
title = {{Sparse-sampling with time-encoded (TICO) stimulated Raman scattering for fast image acquisition}},
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 = {1041408},
abstract = {Modern biomedical imaging modalities aim to provide researchers a multimodal contrast for a deeper insight into a
specimen under investigation. A very promising technique is stimulated Raman scattering (SRS) microscopy, which can
unveil the chemical composition of a sample with a very high specificity. Although the signal intensities are enhanced
manifold to achieve a faster acquisition of images if compared to standard Raman microscopy, there is a trade-off between
specificity and acquisition speed. Commonly used SRS concepts either probe only very few Raman transitions as the
tuning of the applied laser sources is complicated or record whole spectra with a spectrometer based setup. While the first
approach is fast, it reduces the specificity and the spectrometer approach records whole spectra -with energy differences
where no Raman information is present-, which limits the acquisition speed. Therefore, we present a new approach based
on the TICO-Raman concept, which we call sparse-sampling. The TICO-sparse-sampling setup is fully electronically
controllable and allows probing of only the characteristic peaks of a Raman spectrum instead of always acquiring a whole
spectrum. By reducing the spectral points to the relevant peaks, the acquisition time can be greatly reduced compared to a
uniformly, equidistantly sampled Raman spectrum while the specificity and the signal to noise ratio (SNR) are maintained.
Furthermore, all laser sources are completely fiber based. The synchronized detection enables a full resolution of the
Raman signal, whereas the analogue and digital balancing allows shot noise limited detection. First imaging results with
polystyrene (PS) and polymethylmethacrylate (PMMA) beads confirm the advantages of TICO sparse-sampling. We
achieved a pixel dwell time as low as 35 μs for an image differentiating both species. The mechanical properties of the
applied voice coil stage for scanning the sample currently limits even faster acquisition.},
keywords = {nonlinear microscopy, fiber optics imaging, stimulated raman scattering microscopy, time encoded, sparse sampling, Raman spectroscopy , Fourier Domain Mode Locked Laser, FDML, Lasers, fiber},
year = {2017},
doi = {10.1117/12.2287947},
URL = {https://doi.org/10.1117/12.2287947}
}
Sebastian Karpf, Matthias Eibl, Wolfgang Wieser, Thomas Klein, and Robert Huber,
Shot-Noise Limited Time-Encoded Raman Spectroscopy, Journal of Spectroscopy , vol. 2017, pp. 1-6, 03 2017. Hindawi.
DOI:10.1155/2017/9253475
Bibtex: BibTeX
@article{Karpf2017,
   author = {Karpf, Sebastian and Eibl, Matthias and Wieser, Wolfgang and Klein, Thomas and Huber, Robert},
   title = {Shot-Noise Limited Time-Encoded Raman Spectroscopy},
   journal = {Journal of Spectroscopy},
   volume = {2017},
   pages = {1-6},
   url = {https://doi.org/10.1155/2017/9253475},
   year = {2017},
keywords = {AG-Huber_NL},
   type = {Journal Article}
}
Matthias Eibl, Sebastian Karpf, Hubertus Hakert, Daniel Weng, Torben Blömker, and Robert Huber,
Pulse-to-pulse wavelength switching of diode based fiber laser for multi-color multi-photon imaging, in Fiber Lasers XIV: Technology and Systems , Craig A. Robin and Ingmar Hartl, Eds. SPIE, 032017. pp. 100831C.
DOI:10.1117/12.2251965
Bibtex: BibTeX
@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}
}
Matthias Eibl, Sebastian Karpf, Hubertus Hakert, Daniel Weng, and Robert Huber,
Two-photon-excited fluorescence (TPEF) and fluorescence lifetime imaging (FLIM) with sub-nanosecond pulses and a high analog bandwidth signal detection, in Multiphoton Microscopy in the Biomedical Sciences XVII , Ammasi Periasamy and Peter T. C. So and Karsten König and Xiaoliang S. Xie, Eds. SPIE, 022017. pp. 100691F.
DOI:10.1117/12.2250831
Bibtex: BibTeX
@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}
}
Matthias Eibl, Sebastian Karpf, Daniel Weng, Hubertus Hakert, Tom Pfeiffer, Jan Philip Kolb, and Robert Huber,
Single pulse two photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate, Biomed. Opt. Express , vol. 8, no. 7, pp. 3132-3142, 2017. Optica Publishing Group.
DOI:10.1364/BOE.8.003132
Bibtex: BibTeX
@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.},
}

2016

Sebastian Karpf, Matthias Eibl, Benjamin Sauer, Fred Reinholz, Gereon Hüttmann, and Robert Huber,
Two-photon microscopy using fiber-based nanosecond excitation, Biomed. Opt. Express , vol. 7, no. 7, pp. 2432-2440, 07 2016. Optica Publishing Group.
DOI:10.1364/BOE.7.002432
Bibtex: BibTeX
@article{Karpf:16,
author = {Sebastian Karpf and Matthias Eibl and Benjamin Sauer and Fred Reinholz and Gereon H\"{u}ttmann and Robert Huber},
journal = {Biomed. Opt. Express},
keywords = {Fiber optics imaging; Nonlinear optics, fibers; Lasers, fiber; Fluorescence microscopy; Nonlinear microscopy; Femtosecond pulses; In vivo imaging; Laser sources; Nanosecond pulses; Optical systems; Ultrafast lasers},
number = {7},
pages = {2432--2440},
publisher = {Optica Publishing Group},
title = {Two-photon microscopy using fiber-based nanosecond excitation},
volume = {7},
month = {Jul},
year = {2016},
url = {https://opg.optica.org/boe/abstract.cfm?URI=boe-7-7-2432},
doi = {10.1364/BOE.7.002432},
abstract = {Two-photon excitation fluorescence (TPEF) microscopy is a powerful technique for sensitive tissue imaging at depths of up to 1000 micrometers. However, due to the shallow penetration, for in vivo imaging of internal organs in patients beam delivery by an endoscope is crucial. Until today, this is hindered by linear and non-linear pulse broadening of the femtosecond pulses in the optical fibers of the endoscopes. Here we present an endoscope-ready, fiber-based TPEF microscope, using nanosecond pulses at low repetition rates instead of femtosecond pulses. These nanosecond pulses lack most of the problems connected with femtosecond pulses but are equally suited for TPEF imaging. We derive and demonstrate that at given cw-power the TPEF signal only depends on the duty cycle of the laser source. Due to the higher pulse energy at the same peak power we can also demonstrate single shot two-photon fluorescence lifetime measurements.},
}

2015

Matthias Eibl, Sebastian Karpf, Wolfgang Wieser, Thomas Klein, and Robert Huber,
Hyperspectral stimulated Raman microscopy with two fiber laser sources, in Advanced Microscopy Techniques IV; and Neurophotonics II , SPIE, 072015. pp. 953604.
DOI:10.1117/12.2183822
Bibtex: BibTeX
@inproceedings{10.1117/12.2183822,
author = {Matthias Eibl and Sebastian Karpf and Wolfgang Wieser and Thomas Klein and Robert Huber},
title = {{Hyperspectral stimulated Raman microscopy with two fiber laser sources}},
volume = {9536},
booktitle = {Advanced Microscopy Techniques IV; and Neurophotonics II},
editor = {Emmanuel Beaurepaire and Peter T. C. So and Francesco Pavone and Elizabeth M. Hillman},
organization = {International Society for Optics and Photonics},
publisher = {SPIE},
pages = {953604},
abstract = {A fast all fiber based setup for stimulated Raman microscopy based on a rapidly wavelength swept cw-laser is presented. The applied Fourier domain mode locked (FDML) laser is a fiber ring laser, providing a continuously changing wavelength output over time. This fast swept source allows us to rapidly change the wavelength and, thereby the energy difference with respect to a single color pump laser. The pump laser is a master oscillator power amplifier based on a fiber amplified laser diode and a Raman shifter. By controlled variation of the relative timing between probe and pump laser with an arbitrary waveform generator, the Raman signals are encoded in time and they are directly acquired with a synchronized, fast analog-to-digital converter. This setup is capable of acquiring rapidly high resolution spectra (up to 0.5 cm<sup>-1</sup>) with shot noise limited sensitivity over a broadband (750 cm<sup>-1</sup> to 3150 cm<sup>-1</sup>) spectral region. Here, we show the performance of this system for imaging in the CH-stretch region around 3000 cm<sup>-1</sup> and in the fingerprint region around 1600 cm<sup>-1</sup>. We present hyperspectral images of a plant stem slice with molecular contrast of lignin and a lipid representative as well as images of PS (polystyrene) and PMMA (poly(methyl methacrylate) beads with an acquisition speed of 18 &mu;s per spectral point.},
keywords = {stimulated Raman, multiphoton, microscopy, coherent Raman, fiber laser, FDML, TICO, hyperspectral},
year = {2015},
doi = {10.1117/12.2183822},
URL = {https://doi.org/10.1117/12.2183822}
}