Multiphotonenmikroskopie: Institut für Biomedizinische Optik

Multiphotonenmikroskopie

Publikationen

2017

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

@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},
}

2011

Steven, Philipp and Bock, Felix and Hüttmann, Gereon and Cursiefen, Claus: Intravital Two-Photon Microscopy of Immune Cell Dynamics in Corneal Lymphatic Vessels. PLoS One, no. 6, pp. e26253, 2011

@article{Steven2011,
   author = {Steven, Philipp and Bock, Felix and Hüttmann, Gereon and Cursiefen, Claus},
   title = {Intravital Two-Photon Microscopy of Immune Cell Dynamics in Corneal Lymphatic Vessels},
   journal = {PLoS One},
   volume = {6},
   number = {10},
   pages = {e26253},
   abstract = {<sec> <title>Background</title> <p>The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years.</p> </sec> <sec> <title>Methodology/Principal Findings</title> <p>We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization.</p> <p>Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology.</p> <p>Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1–5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces.</p> </sec> <sec> <title>Conclusions</title> <p>To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions.</p> </sec>},
   year = {2011}
}

2007

Kantelhardt, S. R. and Leppert, J. and Krajewski, J. and Petkus, N. and Reusche, E. and Tronnier, V. M. and Huttmann, G. and Giese, A.: Imaging of brain and brain tumor specimens by time-resolved multiphoton excitation microscopy ex vivo. Neuro Oncol, no. 9, pp. 103-12, 2007

@article{Kantelhardt,
author = {Kantelhardt, S. R. and Leppert, J. and Krajewski, J. and Petkus, N. and Reusche, E. and Tronnier, V. M. and Huttmann, G. and Giese, A.},
title = {Imaging of brain and brain tumor specimens by time-resolved multiphoton excitation microscopy ex vivo},
journal = {Neuro Oncol},
volume = {9},
number = {2},
pages = {103-12},
note = {Kantelhardt, Sven R
Leppert, Jan
Krajewski, Jochen
Petkus, Nadine
Reusche, Erich
Tronnier, Volker M
Huttmann, Gereon
Giese, Alf
United States
Neuro Oncol. 2007 Apr;9(2):103-12. Epub 2007 Feb 26.},
abstract = {Multiphoton excitation fluorescent microscopy is a laser-based technology that allows subcellular resolution of native tissues in situ. We have recently applied this technology to the structural and photochemical imaging of cultured glioma cells and experimental gliomas ex vivo. We demonstrated that high microanatomical definition of the tumor, invasion zone, and normal adjacent brain can be obtained down to single-cell resolution in unprocessed tissue blocks. In this study, we used multiphoton excitation and four-dimensional microscopy to generate fluorescence lifetime maps of the murine brain anatomy, experimental glioma tissue, and biopsy specimens of human glial tumors. In murine brain, cellular and noncellular elements of the normal anatomy were identified. Distinct excitation profiles and lifetimes of endogenous fluorophores were identified for specific brain regions. Intracranial grafts of human glioma cell lines in mouse brain were used to study the excitation profiles and fluorescence lifetimes of tumor cells and adjacent host brain. These studies demonstrated that normal brain and tumor could be distinguished on the basis of fluorescence intensity and fluorescence lifetime profiles. Human brain specimens and brain tumor biopsies were also analyzed by multiphoton microscopy, which demonstrated distinct excitation and lifetime profiles in glioma specimens and tumor-adjacent brain. This study demonstrates that multiphoton excitation of autofluorescence can distinguish tumor tissue and normal brain based on the intensity and lifetime of fluorescence. Further technical developments in this technology may provide a means for in situ tissue analysis, which might be used to detect residual tumor at the resection edge.},
keywords = {Animals
Brain/anatomy & histology/ pathology
Brain Neoplasms/ pathology
Disease Models, Animal
Glioma/pathology
Mice
Mice, Inbred Strains
Microscopy, Fluorescence, Multiphoton/instrumentation/ methods
Sensitivity and Specificity},
year = {2007}
}

Orzekowsky-Schroeder, Regina and Klinger, Antje and Schuth, Anna and Freidank, Sebastian and Huttmann, Gereon and Gebert, Andreas and Vogel, Alfred: Intravital real-time study of tissue response to controlled laser-induced cavitation using 500-ps UV laser pulses focused in murine gut mucosa under online dosimetry and spectrally resolved 2-photon microscopy. no. 7568, pp. 756815, SPIE,

@inproceedings{Orzekowsky2010,
   author = {Orzekowsky-Schroeder, Regina and Klinger, Antje and Schuth, Anna and Freidank, Sebastian and Huttmann, Gereon and Gebert, Andreas and Vogel, Alfred},
   title = {Intravital real-time study of tissue response to controlled laser-induced cavitation using 500-ps UV laser pulses focused in murine gut mucosa under online dosimetry and spectrally resolved 2-photon microscopy},
   editor = {Daniel, L. Farkas and Dan, V. Nicolau and Robert, C. Leif},
   publisher = {SPIE},
   volume = {7568},
   pages = {756815},

}

Hüttmann, G. and Orzekowsky-Schröder, R.: Imaging of immune cell dynamics in small intestine and the eye by 2-photon microcopy. in 7th Workshop on Advanced Multiphoton and Fluorescence Lifetime Imaging Techniques (FLIM 2012),

@inproceedings{Hüttmann2012,
author = {Hüttmann, G. and Orzekowsky-Schröder, R.},
title = {Imaging of immune cell dynamics in small intestine and the eye by 2-photon microcopy},
booktitle = {7th Workshop on Advanced Multiphoton and Fluorescence Lifetime Imaging Techniques (FLIM 2012)},
abstract = {Autofluorescence based 2-photon microscopy was investigated as a
tool for studying the dynamics of immunological processes in vivo. Nearly
all tissue components were simultaneously visible by autofluorescence and a
nearly complete visualization of tissue architecture was possible. Within
the tissue, immune competent cells like lymphocytes, macrophages and
dendritic cells were visualized in their dynamic interaction with other
cells or surrounding tissues.
Immunological processes were studied in the small intestine and at the
surface of the eye. Excitation and emission spectra of the different mucosal
tissue components were quantitatively determined and a compared to the
respective spectra of endogenous chromophores. It was shown, that by using
only two excitation wavelengths within the tuning range of a Ti:Saphire
laser enterocytes, antigen presenting cells and lysosomes of the small
intestine could be discriminated based on the excitation and emission
properties. By additionally using an intravital nuclear stain, motion of
lymphocytes in the lamina propria and the epithelium of small intestine
villi was quantitatively analyzed.

2-photon microscopy was also a powerful tool for studying the conjunctiva
and cornea of the eye. Lymphocyte dynamics and uptake of microspheres or
fluorescing heat-inactivated E. coli was followed over time in the
conjunctiva-associated lymphoid tissue of mice. In a different mouse model
of suture induced corneal hem- and lymphangiogenesis, immune cell migration
into lymphatic vessels and luminal transport of individual cells was
observed in vivo.

Autofluorescence based 2-photon intravital microscopy is a valuable tool to
study dynamic immunological processes. While tissue autofluorescence gives a
good overview over all relevant structures and allows for discriminating
different cell types by spectral analysis, the additional combination of
specific staining with external dyes or fluorescent proteins is easily
possible and enhances the potential of the technique further. },

}