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

2015

Sebastian Karpf, Matthias Eibl, and Robert Huber,
Nanosecond two-photon excitation fluorescence imaging with a multi color fiber MOPA laser, in Advanced Microscopy Techniques IV; and Neurophotonics II , Emmanuel Beaurepaire and Peter T. C. So and Francesco Pavone and Elizabeth M. Hillman, Eds. SPIE, 072015. pp. 953616.
DOI:10.1117/12.2183854
Bibtex: BibTeX
@inproceedings{10.1117/12.2183854,
author = {Sebastian Karpf and Matthias Eibl and Robert Huber},
title = {{Nanosecond two-photon excitation fluorescence imaging with a multi color fiber MOPA laser}},
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 = {953616},
abstract = {A system is presented that uses a fiber based Master Oscillator Power Amplifier (MOPA) with nanosecond-range pulses for two-photon excitation fluorescence (TPEF) imaging. The robust laser in the extended near infrared is based on an actively modulated electro-optical modulator (EOM), enabling free synchronization of the pulses to any other light source or detection unit. Pulses with a freely programmable duration between 0.4 and 10 ns are generated and then amplified to up to kilowatts of peak power with ytterbium doped fiber amplifiers (YDFA). Since we achieve peak power and duty cycles comparable to standard femto- and picosecond setups, the TPEF signal levels are similar, but realized with a robust and inexpensive fiber-based setup. The delivery fiber is further used as an optional, electronically controllable Raman shifter to effectively shift the 1064 nm light to 1122 nm and to 1186 nm. This allows imaging of a manifold of fluorophores, like e.g. TexasRed, mCherry, mRaspberry and many more. We show TPEF imaging of the autofluorescence of plant leaves of moss and algae, acquired in epi-direction. This modular laser unit can be integrated into existing systems as either a fiber-based, alignment free excitation laser or an extension for multi-modal imaging.},
keywords = {multi-photon imaging, TPEF, MOPA, TPA, fiber laser, Raman shifter, non-linear imaging, multi-modal imaging},
year = {2015},
doi = {10.1117/12.2183854},
URL = {https://doi.org/10.1117/12.2183854}
}
Sebastian Karpf, Matthias Eibl, Wolfgang Wieser, Thomas Klein, and Robert Huber,
Time-encoded Raman scattering (TICO-Raman) with Fourier domain mode locked (FDML) lasers, in Optical Coherence Imaging Techniques and Imaging in Scattering Media , Brett E. Bouma and Maciej Wojtkowski, Eds. SPIE, 072015. pp. 95410F.
DOI:10.1117/12.2183859
Bibtex: BibTeX
@inproceedings{10.1117/12.2183859,
author = {Sebastian Karpf and Matthias Eibl and Wolfgang Wieser and Thomas Klein and Robert Huber},
title = {{Time-encoded Raman scattering (TICO-Raman) with Fourier domain mode locked (FDML) lasers}},
volume = {9541},
booktitle = {Optical Coherence Imaging Techniques and Imaging in Scattering Media},
editor = {Brett E. Bouma and Maciej Wojtkowski},
organization = {International Society for Optics and Photonics},
publisher = {SPIE},
pages = {95410F},
abstract = {We present a new concept for performing stimulated Raman spectroscopy and microscopy by employing rapidly wavelength swept Fourier Domain Mode locked (FDML) lasers [1]. FDML lasers are known for fastest imaging in swept-source optical coherence tomography [2, 3]. We employ this continuous and repetitive wavelength sweep to generate broadband, high resolution stimulated Raman spectra with a new, time-encoded (TICO) concept [4]. This allows for encoding and detecting the stimulated Raman gain on the FDML laser intensity directly in time. Therefore we use actively modulated pump lasers, which are electronically synchronized to the FDML laser, in combination with a fast analog-to-digital converter (ADC) at 1.8 GSamples/s. We present hyperspectral Raman images with color-coded, molecular contrast.},
keywords = {swept lasers, FDML, TICO-Raman, fiber lasers, stimulated Raman microscopy, Raman spectroscopy, molecular contrast, multi-modal imaging},
year = {2015},
doi = {10.1117/12.2183859},
URL = {https://doi.org/10.1117/12.2183859}
}
Sebastian Karpf, Matthias Eibl, Wolfgang Wieser, Thomas Klein, and Robert Huber,
A Time-Encoded Technique for fibre-based hyperspectral broadband stimulated Raman microscopy, Nature Communications , vol. 6, no. 1, pp. 6784, 04 2015.
DOI:10.1038/ncomms7784
Bibtex: BibTeX
@Article{HU_2015_Karpf_a,
  Title                    = {A Time-Encoded Technique for fibre-based hyperspectral broadband stimulated Raman microscopy},
  Author                   = {Karpf, Sebastian and Eibl, Matthias and Wieser, Wolfgang and Klein, Thomas and Huber, Robert},
  Journal                  = {Nature Communications},
  Year                     = {2015},
  Volume = {6},
  pages = {6784 1--6},
keywords = {AG-Huber_NL},
  Doi                      = {10.1038/ncomms7784}
}

2014

Sebastian Karpf, Matthias Eibl, Wolfgang Wieser, Thomas Klein, and Robert Huber,
Time-Encoded Raman: Fiber-based, hyperspectral, broadband stimulated Raman microscopy, ArXiv e-prints , 05 2014.
DOI:10.48550/arXiv.1405.4181
Bibtex: BibTeX
@Article{HU_2014_Karpf_a,
  Title                    = {{Time-Encoded Raman: Fiber-based, hyperspectral, broadband stimulated Raman microscopy}},
  Author                   = {Karpf, Sebastian and Eibl, Matthias and Wieser, Wolfgang and Klein, Thomas and Huber, Robert},
  journal = {ArXiv e-prints},
  Year                     = {2014},
  Archiveprefix            = {arXiv},
  Arxivid                  = {1405.4181},
  Eprint                   = {1405.4181},
keywords = {AG-Huber_NL},
  Url                      = {http://arxiv.org/abs/1405.4181}
}
Matthias Eibl, Sebastian Karpf, Wolfgang Wieser, Thomas Klein, and Robert Huber,
Broadband, High Resolution Stimulated Raman Spectroscopy with Rapidly Wavelength Swept cw-Lasers, in CLEO: 2014 , Optica Publishing Group, 2014. pp. ATu3P.4.
DOI:10.1364/CLEO_AT.2014.ATu3P.4
Bibtex: BibTeX
@inproceedings{Eibl:14,
author = {Matthias Eibl and Sebastian Karpf and Wolfgang Wieser and Thomas Klein and Robert Huber},
booktitle = {CLEO: 2014},
journal = {CLEO: 2014},
keywords = {Lasers, tunable; Scattering, stimulated Raman; Spectroscopy, Raman; Laser light; Laser sources; Master oscillator power amplifiers; Raman spectroscopy; Self phase modulation; Stimulated Raman scattering},
pages = {ATu3P.4},
publisher = {Optica Publishing Group},
title = {Broadband, High Resolution Stimulated Raman Spectroscopy with Rapidly Wavelength Swept cw-Lasers},
year = {2014},
url = {https://opg.optica.org/abstract.cfm?URI=CLEO_AT-2014-ATu3P.4},
doi = {10.1364/CLEO_AT.2014.ATu3P.4},
abstract = {A fast all fiber based setup for stimulated Raman spectroscopy with a rapidly wavelength swept cw-laser is presented. It enables flexible acquisition of broadband (750 cm{\textminus}1 to 3150 cm{\textminus}1) spectra with high resolution (0.5 cm{\textminus}1).},
}
Sebastian Karpf, Matthias Eibl, Wolfgang Wieser, Thomas Klein, and Robert Huber,
Hyperspectral Stimulated Raman Microscopy with Fiber-based, Rapidly Wavelength Swept cw-Lasers, in CLEO: 2014 , Optica Publishing Group, 2014. pp. SM3P.3.
DOI:10.1364/CLEO_SI.2014.SM3P.3
Bibtex: BibTeX
@inproceedings{Karpf:14,
author = {Sebastian Karpf and Matthias Eibl and Wolfgang Wieser and Thomas Klein and Robert Huber},
booktitle = {CLEO: 2014},
journal = {CLEO: 2014},
keywords = {Lasers, tunable; Scattering, stimulated Raman; Raman microscopy; Biological imaging; Medical imaging; Optical coherence tomography; Raman microscopy; Raman scattering; Swept lasers},
pages = {SM3P.3},
publisher = {Optica Publishing Group},
title = {Hyperspectral Stimulated Raman Microscopy with Fiber-based, Rapidly Wavelength Swept cw-Lasers},
year = {2014},
url = {https://opg.optica.org/abstract.cfm?URI=CLEO_SI-2014-SM3P.3},
doi = {10.1364/CLEO_SI.2014.SM3P.3},
abstract = {A hyperspectral stimulated Raman microscopy system using rapidly wavelength swept lasers is presented. Imaging of biological samples with shot noise limited detection is demonstrated with the fiber based setup.},
}

2013

Sebastian Karpf, Matthias Eibl, Wolfgang Wieser, Thomas Klein, and Robert Huber,
FDML Raman: High Speed, High Resolution Stimulated Raman Spectroscopy with Rapidly Wavelength Swept Lasers, in CLEO: 2013 , Optica Publishing Group, 062013. pp. CTu2H.5.
DOI:10.1364/CLEO_SI.2013.CTu2H.5
Bibtex: BibTeX
@inproceedings{Karpf:13,
author = {Sebastian Karpf and Matthias Eibl and Wolfgang Wieser and Thomas Klein and Robert Huber},
booktitle = {CLEO: 2013},
journal = {CLEO: 2013},
keywords = {Lasers, fiber; Scattering, stimulated Raman; Spectroscopy, Raman; Fourier domain mode locking; Lasers; Optical coherence tomography; Raman lasers; Raman spectroscopy; Swept lasers},
pages = {CTu2H.5},
publisher = {Optica Publishing Group},
title = {FDML Raman: High Speed, High Resolution Stimulated Raman Spectroscopy with Rapidly Wavelength Swept Lasers},
year = {2013},
url = {https://opg.optica.org/abstract.cfm?URI=CLEO_SI-2013-CTu2H.5},
doi = {10.1364/CLEO_SI.2013.CTu2H.5},
abstract = {An all fiber based system for high speed, high resolution Raman sensing is presented. The system is based on a wavelength swept Fourier Domain Mode Locked (FDML) laser for the detection of the Raman signal.},
}
Sebastian Karpf, Matthias Eibl, Wolfgang Wieser, Thomas Klein, and Robert Huber,
FDML Raman: New High Resolution SRS with ultra broadband spectral coverage, in 2013 Conference on Lasers & Electro-Optics Europe & International Quantum Electronics Conference CLEO EUROPE/IQEC , 052013. pp. 1.
DOI:10.1109/CLEOE-IQEC.2013.6801995
Bibtex: BibTeX
@INPROCEEDINGS{6801995,
  author={Karpf, Sebastian and Eibl, Matthias and Wieser, Wolfgang and Klein, Thomas and Huber, Robert},
  booktitle={2013 Conference on Lasers & Electro-Optics Europe & International Quantum Electronics Conference CLEO EUROPE/IQEC}, 
  title={FDML Raman: New high resolution SRS with ultra broadband spectral coverage}, 
  year={2013},
  volume={},
  number={},
  pages={1-1},
  doi={10.1109/CLEOE-IQEC.2013.6801995}}

2009

Evelyn Ploetz, Berit Marx, Thomas Klein, Robert Huber, and Peter Gilch,
A 75 MHz light source for femtosecond stimulated raman microscopy, Opt. Express , vol. 17, no. 21, pp. 18612-18620, Okt. 2009. Optica Publishing Group.
DOI:10.1364/OE.17.018612
Bibtex: BibTeX
@article{Ploetz:09,
author = {E. Ploetz and B. Marx and T. Klein and R. Huber and P. Gilch},
journal = {Opt. Express},
keywords = {Laser amplifiers; Ultrafast lasers; Nonlinear microscopy; Raman microscopy; Laser sources; Raman microscopy; Raman scattering; Raman spectroscopy; Stimulated Raman scattering; Time resolved spectroscopy},
number = {21},
pages = {18612--18620},
publisher = {Optica Publishing Group},
title = {A 75 MHz Light Source for Femtosecond Stimulated Raman Microscopy},
volume = {17},
month = {Oct},
year = {2009},
url = {https://opg.optica.org/oe/abstract.cfm?URI=oe-17-21-18612},
doi = {10.1364/OE.17.018612},
abstract = {In femtosecond stimulated Raman microscopy (FSRM) a spectrally broad pulse (Raman probe) and a spectrally narrow pulse (Raman pump) interact in a sample and thereby generate a Raman spectrum of the focal volume. Here a novel light source for FSRM is presented. It consists of an 8-fs laser (repetition rate of 75 MHz) operating as Raman probe. A Yb3$+$ based fiber amplifier generates the Raman pump light at 980 nm. The amplifier is seeded by the spectral wing of the 8-fs laser output which ensures synchronisation of pump and probe pulses. Spectral and temporal characteristics of these pulses are reported and simultaneous recording of broadband Raman spectra relying on these pulses is demonstrated.},
}