Non-linear bio-molecular imaging

Nonlinear optical microscopy is a powerful technique in bio-molecular science that allows to gain a better understanding of biochemical processes on a cellular level. The main goal is to identify not only morphology but also the chemical composition of a sample.

We are currently working on two contrasting mechanisms:

Time-Encoded (TICO) Raman spectroscopy and microscopy

The TICO-Raman approach uses a wavelength swept Probe laser to record spectral points sequentially in time. With a spectral resolution limited only by the narrowband laser line width and a  broadband coverage, this system has great spectroscopic capabilities but the flexibility also allows for fast imaging applications.

Details of the TICO-Raman concept

The Time-Encoded (TICO)-Raman approach uses rapidly wavelength swept FDML lasers in combination with a narrowband, actively modulated pump laser to encode and detect the Raman spectra directly in time. The FDML probe laser changes its wavelength continuously and repetitively in time. This is indicated in Figure 1A as a rainbow colored line. The blue line shows the single color narrowband pump laser. It is evident that the energy difference between these two light sources changes over time. Therefore, with different timing, different Raman transitions get probed (Figure 1B). When the energy difference matches a Raman transition, pump photons will be converted to probe photons and the FDML laser intensity gets increased at that position. This intensity change can be measured with a fast photodetector (Figure 1C).

To achieve high signal levels, the pump laser is not power on all the time, but modulated to short pulses (Figure 1D). To probe all possible Raman transistions, these pulses get delayed more and more compared to the start of a FDML laser sweep for each new sweep. With the known time-to-wavelenght encoding, the intensity changes can be directly mapped onto a Raman spectrum.

Figure 1: Concept of Time-Encoded (TICO)-Raman
Concept of Time-Encoded (TICO)-Raman

Two-photon excited fluorescence (TPEF) microscopy

a) TPEF imaging setup b) time trace of excitation pulse
TPEF image of a plant leaf

In recent years, multiphoton microscopy evolved as a way of biomedical imaging of scattering tissues with a highly specific selection of layers. Current multiphoton microscopes employ complicated lasers systems. Hence, we investigate different approaches. The excitation laser of our system is an all fiber based, sub-nanosecond pulsed laser. This makes the overall setup very robust, reliable, and maintenance free. One key advantage of the system is the direct synchronization of the excitation pulses to the data acquisition which makes new imaging modalities possible.

Two-Photon Single-Pulse Fluorescence Lifetime Imaging (SP-FLIM)

SP-FLIM images of a convalaria majalis stem.

One of the new imaging modalities that are possible with our new laser source is Single-Pulse Two-photon-excited fluorescence lifetime imaging microscopy (SP-FLIM). Two-Photon FLIM is a chemically specific 3-D sensing modality where the lifetime of a fluorescence decay gives valuable information on the chemical composition of the sample under investigation. Due to our longer pulse length compared to commonly used Ti:Sa lasers, more fluorescence photons are generated per pulse, which allows us to derive the lifetime with a single excitation pulse only. With this methode, we achieve a very rapid acquisition of FLIM images with pixel rates upto 1MHz.

related Publications

2019

  • Kolb, J P;Weng, D;Hakert, H;Eibl, M;Draxinger, W;Meyer, T;Gottschall, T;Brinkmann, R;Birngruber, R and Popp, J: Virtual HE histology by fiber-based picosecond two-photon microscopy. in Multiphoton Microscopy in the Biomedical Sciences XIX Proc. SPIE,10882, pp. 108822F, International Society for Optics and Photonics, 2019
    BibTeX Link
    @inproceedings{Kolb2019/2,
       author = {Kolb, J P;Weng, D;Hakert, H;Eibl, M;Draxinger, W;Meyer, T;Gottschall, T;Brinkmann, R;Birngruber, R and Popp, J},
       title = {Virtual HE histology by fiber-based picosecond two-photon microscopy},
       booktitle = {Multiphoton Microscopy in the Biomedical Sciences XIX} {Proc. SPIE,10882},
       editor = {Ammasi Periasamy; Peter T. C. So; Karsten König},
       publisher = { International Society for Optics and Photonics},
       year = {2019},
       pages = {108822F},
    keywords = {AG-Huber_NL},
       
    doi = {10.1117/12.2507866},
       type = {Conference Proceedings}
    }
    

2018

  • Eibl ,M; Weng, D; Hakert, H; Kolb, J P; Pfeiffer, T; Hundt, J E; Huber, R and Karpf, S: Wavelength agile multi-photon microscopy with a fiber amplified diode laser. Biomedical Optics Express 9(12), pp. 6273-6282, 2018
    BibTeX Link
    @article{Eibl2018,
    author = { Eibl ,M; Weng, D; Hakert, H; Kolb, J P; Pfeiffer, T; Hundt, J E; Huber, R and Karpf, S},
    title = {Wavelength agile multi-photon microscopy with a fiber amplified diode laser},
    journal = {Biomedical Optics Express} {9(12)},
    keywords = {AG-Huber_NL},
    year = {2018},
    pages = {6273--6282},
    doi = {10.1364/BOE.9.006273},
    }
    

2017

  • Eibl, Matthias and Karpf, Sebastian and Hakert, Hubertus and Weng, Daniel and Huber, Robert: Two-photon-excited fluorescence (TPEF) and fluorescence lifetime imaging (FLIM) with sub-nanosecond pulses and a high analog bandwidth signal detection. in Proc. SPIE, no. 10069, pp. 100691F-6, 2017
    BibTeX Link
    @inproceedings{Eibl2017,
       author = {Eibl, Matthias and Karpf, Sebastian and Hakert, Hubertus and Weng, Daniel and Huber, Robert},
       title = {Two-photon-excited fluorescence (TPEF) and fluorescence lifetime imaging (FLIM) with sub-nanosecond pulses and a high analog bandwidth signal detection},
    booktitle = {Proc. SPIE },
       volume = {10069},
       pages = {100691F-6},
       doi= {10.1117/12.2250831},
      year={2017},
    keywords = {AG-Huber_NL},
       type = {Conference Proceedings}
    }
    
  • Hakert, H; Eibl, M; Karpf, S and Huber, R: Sparse-sampling with time-encoded (TICO) stimulated Raman scattering for fast image acquisition. in Proc. SPIE 10414, pp. 1041408-1041408-7, 2017
    BibTeX Link
    @inproceedings{Hakert2017,
       author = {Hakert, H; Eibl, M; Karpf, S and Huber, R},
       title = {Sparse-sampling with time-encoded (TICO) stimulated Raman scattering for fast image acquisition},
    booktitle = {Proc. SPIE} {10414},
    Year={2017},
       
       pages = {1041408-1041408-7},
       url = {http://dx.doi.org/10.1117/12.2287947},
    keywords = {AG-Huber_NL},
       type = {Conference Proceedings}
    }
    
  • Eibl, M; Karpf, S; Hakert, H; Weng, D; Pfeiffer, T; Kolb, J P and Huber, R: Single pulse two-photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate and an all fiber based setup. in Proc. SPIE 10414, pp. 1041403-1041403-7, 2017
    BibTeX Link
    @inproceedings{Eibl2017,
       author = {Eibl, M; Karpf, S; Hakert, H; Weng, D; Pfeiffer, T; Kolb, J P and Huber, R},
       title = {Single pulse two-photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate and an all fiber based setup},
      booktitle = {Proc. SPIE} {10414},
      
      year = {2017},
    keywords = {AG-Huber_NL},
       pages = {1041403-1041403-7},
       url = {http://dx.doi.org/10.1117/12.2286035},
       type = {Conference Proceedings}
    }
    
  • Matthias Eibl and Sebastian Karpf and Daniel Weng and Hubertus Hakert and Tom Pfeiffer and Jan Philip Kolb and Robert Huber: Single pulse two photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate. Biomedical Optics Express, no. 8, pp. 3132-3142, OSA, Jul, 2017
    BibTeX Link
    @article{HU_2017_Eibla_BOE,
    author = {Matthias Eibl and Sebastian Karpf and Daniel Weng and Hubertus Hakert and Tom Pfeiffer and Jan Philip Kolb and Robert Huber},
    journal = {Biomedical Optics Express},
    number = {7},
    pages = {3132--3142},
    publisher = {OSA},
    title = {Single pulse two photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate},
    volume = {8},
    month = {Jul},
    year = {2017},
    keywords = {AG-Huber_NL},
    doi = {10.1364/BOE.8.003132}
    }
    
  • Karpf, Sebastian and Eibl, Matthias and Wieser, Wolfgang and Klein, Thomas and Huber, Robert: Shot-Noise Limited Time-Encoded Raman Spectroscopy. Journal of Spectroscopy, no. 2017, pp. 1-6, 2017
    BibTeX Link
    @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}
    }
    
  • Eibl, Matthias and Karpf, Sebastian and Hakert, Hubertus and Weng, Daniel and Blömker, Torben and Huber, Robert: Pulse-to-pulse wavelength switching of diode based fiber laser for multi-color multi-photon imaging. in Proc. SPIE, no. 10083, pp. 100831C-6, 2017
    BibTeX Link
    @inproceedings{Eibl2017,
       author = {Eibl, Matthias and Karpf, Sebastian and Hakert, Hubertus and Weng, Daniel and Blömker, Torben and Huber, Robert},
       title = {Pulse-to-pulse wavelength switching of diode based fiber laser for multi-color multi-photon imaging},
    booktitle = {Proc. SPIE },
       volume = {10083},
       pages = {100831C-6},
       year= {2017},
       doi= {10.1117/12.2251965},
    keywords = {AG-Huber_NL},
       type = {Conference Proceedings}
    }
    
  • Eibl, M; Karpf, S; Hakert, H; Blömker, T; Kolb, J P; Jirauschek, C and Huber, R: Pulse-to-pulse wavelength switching of a nanosecond fiber laser by four-wave mixing seeded stimulated Raman amplification. Opt Lett 42(21), pp. 4406-4409, OSA, Nov, 2017
    BibTeX Link
    @article{2017Eibl,
    author = {Eibl, M; Karpf, S; Hakert, H; Bl\"{o}mker, T; Kolb, J P; Jirauschek, C and Huber, R},
    journal = {Opt Lett} {42(21)},
    keywords = {NLI, TPEF, Multiphoton, AG-Huber_NL},
    
    pages = {4406--4409},
    publisher = {OSA},
    title = {Pulse-to-pulse wavelength switching of a nanosecond fiber laser by four-wave mixing seeded stimulated Raman amplification},
    
    month = {Nov},
    year = {2017},
    doi = {10.1364/OL.42.004406},
    }
    
  • Klein, Thomas and Huber, Robert: High-speed OCT light sources and systems [Invited]. Biomedical Optics Express, no. 8, pp. 828-859, 2017
    BibTeX Link
    @article{Klein2017,
       author = {Klein, Thomas and Huber, Robert},
       title = {High-speed OCT light sources and systems [Invited]},
       journal = {Biomedical Optics Express},
       volume = {8},
       number = {2},
       pages = {828-859},
       abstract = {Imaging speed is one of the most important parameters that define the performance of optical coherence tomography (OCT) systems. During the last two decades, OCT speed has increased by over three orders of magnitude. New developments in wavelength-swept lasers have repeatedly been crucial for this development. In this review, we discuss the historical evolution and current state of the art of high-speed OCT systems, with focus on wavelength swept light sources and swept source OCT systems.},
       keywords = {AG-Huber_FDML, AG-Huber_OCT, AG-Huber_NL},
       DOI = {10.1364/BOE.8.000828},
       year = {2017},
       type = {Journal Article}
    }
    

2016

  • Karpf, Sebastian and Eibl, Matthias and Sauer, Benjamin and Reinholz, Fred and Hüttmann, Gereon and Huber, Robert: Two-photon microscopy using fiber-based nanosecond excitation. Biomedical Optics Express, no. 7, pp. 2432-2440, 2016
    BibTeX Link
    @article{Karpf2016,
       author = {Karpf, Sebastian and Eibl, Matthias and Sauer, Benjamin and Reinholz, Fred and Hüttmann, Gereon and Huber, Robert},
       title = {Two-photon microscopy using fiber-based nanosecond excitation},
       journal = {Biomedical Optics Express},
       volume = {7},
       number = {7},
       pages = {2432-2440},
       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.},
       keywords = {AG-Huber_NL},
       DOI = {10.1364/BOE.7.002432},
       year = {2016},
       type = {Journal Article}
    }
    

2015

  • Karpf, Sebastian and Eibl, Matthias and Wieser, Wolfgang and Klein, Thomas and Huber, Robert: Time-encoded Raman scattering (TICO-Raman) with Fourier domain mode locked (FDML) lasers. in Proc. SPIE, no. 9541, pp. 95410F-95410F-6, 2015
    BibTeX Link
    @inproceedings{HU_2015_Karpf_b,
    author = {Karpf, Sebastian and Eibl, Matthias and Wieser, Wolfgang and Klein, Thomas and Huber, Robert},
    title = { Time-encoded Raman scattering (TICO-Raman) with Fourier domain mode locked (FDML) lasers },
    booktitle = {Proc. SPIE },
    volume = {9541},
    type = {Conference Proceedings},
    pages = {95410F-95410F-6},
    year = {2015},
    keywords = {AG-Huber_NL},
    doi = {10.1117/12.2183859}
    }
  • Karpf, Sebastian and Eibl, Matthias and Huber, Robert: Nanosecond two-photon excitation fluorescence imaging with a multi color fiber MOPA laser. in Proc. SPIE, no. 9536, pp. 953616-953616-5, 2015
    BibTeX Link
    @inproceedings{HU_2015_Karpf_c,
    author = {Karpf, Sebastian and Eibl, Matthias and Huber, Robert},
    title = { Nanosecond two-photon excitation fluorescence imaging with a multi color fiber MOPA laser },
    booktitle = {Proc. SPIE },
    type = {Conference Proceedings},
    volume = {9536},
    pages = {953616-953616-5},
    year = {2015},
    keywords = {AG-Huber_NL},
    doi = {10.1117/12.2183854}
    }
  • Eibl, Matthias and Karpf, Sebastian and Wieser, Wolfgang and Klein, Thomas and Huber, Robert: Hyperspectral stimulated Raman microscopy with two fiber laser sources. in Proc. SPIE, no. 9536, pp. 953604-953604-6, 2015
    BibTeX Link
    @inproceedings{HU_2015_Eibl_a,
    author = {Eibl, Matthias and Karpf, Sebastian and Wieser, Wolfgang and Klein, Thomas and Huber, Robert},
    title = {Hyperspectral stimulated Raman microscopy with two fiber laser sources },
    booktitle = {Proc. SPIE },
    type = {Conference Proceedings},
    volume = {9536},
    pages = {953604-953604-6},
    year = {2015},
    keywords = {AG-Huber_NL},
    doi = {10.1117/12.2183822}
    }
  • Karpf, Sebastian and Eibl, Matthias and Wieser, Wolfgang and Klein, Thomas and Huber, Robert: A Time-Encoded Technique for fibre-based hyperspectral broadband stimulated Raman microscopy. Nature Communications, no. 6, pp. 784 1-6;, 2015
    BibTeX Link
    @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

  • Karpf, Sebastian and Eibl, Matthias and Wieser, Wolfgang and Klein, Thomas and Huber, Robert: Time-Encoded Raman: Fiber-based, hyperspectral, broadband stimulated Raman microscopy. ArXiv e-prints, 2014
    BibTeX Link
    @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}
    }
  • Karpf, Sebastian and Eibl, Matthias and Klein, Thomas and Wieser, Wolfgang and Huber, Robert: Hyperspectral Stimulated Raman Microscopy with Fiber-based, Rapidly Wavelength Swept cw-Lasers. in CLEO: 2014, pp. 1-2, OSA, 2014
    BibTeX Link Link
    @inproceedings{HU_2014_Karpf_b,
    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.},
    author = {Karpf, Sebastian and Eibl, Matthias and Klein, Thomas and Wieser, Wolfgang and Huber, Robert},
    booktitle = {CLEO: 2014},
    doi = {10.1364/CLEO_SI.2014.SM3P.3},
    isbn = {978-1-55752-999-2},
    keywords = {Lasers,Raman microscopy,Scattering,stimulated Raman,tunable, AG-Huber_NL},
    pages = {1--2},
    publisher = {OSA},
    title = {{Hyperspectral Stimulated Raman Microscopy with Fiber-based, Rapidly Wavelength Swept cw-Lasers}},
    url = {http://www.opticsinfobase.org/abstract.cfm?URI=CLEO\_SI-2014-SM3P.3},
    year = { 2014}
    }
  • Eibl, Matthias and Karpf, Sebastian and Wieser, Wolfgang and Klein, Thomas and Huber, Robert: Broadband, High Resolution Stimulated Raman Spectroscopy with Rapidly Wavelength Swept cw-Lasers. in CLEO: 2014, pp. ATu3P.4 1-3, OSA, 2014
    BibTeX Link Link
    @inproceedings{HU_2014_Eibl_a,
    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-1 to 3150 cm-1) spectra with high resolution (0.5 cm-1).},
    author = {Eibl, Matthias and Karpf, Sebastian and Wieser, Wolfgang and Klein, Thomas and Huber, Robert},
    booktitle = {CLEO: 2014},
    doi = {10.1364/CLEO_AT.2014.ATu3P.4},
    isbn = {978-1-55752-999-2},
    keywords = {Lasers,Raman,Scattering,Spectroscopy,stimulated Raman,tunable,AG-Huber_NL},
    pages = {ATu3P.4 1--3},
    publisher = {OSA},
    title = {{Broadband, High Resolution Stimulated Raman Spectroscopy with Rapidly Wavelength Swept cw-Lasers}},
    url = {http://www.opticsinfobase.org/abstract.cfm?URI=CLEO\_AT-2014-ATu3P.4},
    year = { 2014}
    }

2013

  • Karpf, Sebastian and Eibl, Matthias and Wieser, Wolfgang and Klein, Thomas and Huber, Robert: FDML Raman: New High Resolution SRS with ultra broadband spectral coverage. in 2013 Conference on Lasers and Electro-Optics - International Quantum Electronics Conference, pp. PD_A_8 1-3, Optical Society of America, Munich, 2013
    BibTeX Link Link
    @inproceedings{HU_2013_Karpf_a,
    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.},
    address = {Munich},
    author = {Karpf, Sebastian and Eibl, Matthias and Wieser, Wolfgang and Klein, Thomas and Huber, Robert},
    booktitle = {2013 Conference on Lasers and Electro-Optics - International Quantum Electronics Conference},
    pages = {PD_A_8 1--3},
    publisher = {Optical Society of America},
    doi={10.1109/CLEOE-IQEC.2013.6801995},
    title = {{FDML Raman: New High Resolution SRS with ultra broadband spectral coverage}},
    url = {https://doi.org/10.1109/CLEOE-IQEC.2013.6801995},
    keywords = {AG-Huber_NL},
    year = { 2013}
    }
  • Karpf, Sebastian and Eibl, Matthias and Wieser, Wolfgang and Klein, Thomas and Huber, Robert: FDML Raman: High Speed, High Resolution Stimulated Raman Spectroscopy with Rapidly Wavelength Swept Lasers. in CLEO: 2013, pp. ATu3P.4 1-3, OSA, 2013
    BibTeX Link Link
    @inproceedings{HU_2013_Karpf_b,
    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.},
    author = {Karpf, Sebastian and Eibl, Matthias and Wieser, Wolfgang and Klein, Thomas and Huber, Robert},
    booktitle = {CLEO: 2013},
    doi = {10.1364/CLEO_SI.2013.CTu2H.5},
    isbn = {978-1-55752-972-5},
    keywords = {Lasers,Raman,Scattering,Spectroscopy,fiber,stimulated Raman,AG-Huber_NL},
    pages = {ATu3P.4 1--3},
    publisher = {OSA},
    title = {{FDML Raman: High Speed, High Resolution Stimulated Raman Spectroscopy with Rapidly Wavelength Swept Lasers}},
    url = {http://www.opticsinfobase.org/abstract.cfm?URI=CLEO_SI-2013-CTu2H.5},
    year = { 2013}
    }

2009

  • Ploetz, E and Marx, B and Klein, T and Huber, R and Gilch, P: A 75 MHz light source for femtosecond stimulated raman microscopy. Optics express, no. 17, pp. 18612-20, t,, 2009
    BibTeX Link Link
    @Article{HU_2009_Ploetz_a,
      Title                    = {{A 75 MHz light source for femtosecond stimulated raman microscopy}},
      Author                   = {Ploetz, E and Marx, B and Klein, T and Huber, R and Gilch, P},
      Journal                  = {Optics express},
      Year                     = {2009},
    
      Month                    = oct,
      Number                   = {21},
      Pages                    = {18612--20},
      Volume                   = {17},
    
      Doi                      = {10.1364/OE.17.018612},
      Institution              = {Lehrstuhl f\"{u}r BioMolekulare Optik and Center of Integrated Protein Science Munich Fakult\"{a}t f\"{u}r Physik, Ludwig-Maximilians-Universit\"{a}t M\"{u}nchen Oettingenstr 67, D-80538 M\"{u}nchen, Germany.},
      ISSN                     = {1094-4087},
    keywords = {AG-Huber_NL},
      Url                      = {http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-17-21-18612}
    }
    

 

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