Article
Multiphoton imaging of deparaffinized brain tissue sections
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Authors
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Sandra Tamosaityte
- Klinisches Sensoring und Monitoring - Klinik für Anästhesiologie und Intensivtherapie, TU Dresden, Dresden, Deutschland
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Roberta Galli
- Klinisches Sensoring und Monitoring - Klinik für Anästhesiologie und Intensivtherapie, TU Dresden, Dresden, Deutschland
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Ortrud Uckermann
- Neurochirurgie, Universitätsklinikum Carl Gustav Carus, TU Dresden, Dresden, Deutschland
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Elke Leipnitz
- Neurochirurgie, Universitätsklinikum Carl Gustav Carus, TU Dresden, Dresden, Deutschland
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Edmund Koch
- Klinisches Sensoring und Monitoring - Klinik für Anästhesiologie und Intensivtherapie, TU Dresden, Dresden, Deutschland
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Gerald Steiner
- Klinisches Sensoring und Monitoring - Klinik für Anästhesiologie und Intensivtherapie, TU Dresden, Dresden, Deutschland
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Matthias Kirsch
- Neurochirurgie, Universitätsklinikum Carl Gustav Carus, TU Dresden, Dresden, Deutschland
| Published: | June 9, 2017 |
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Outline
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Objective: Label-free multiphoton (MP) microscopy is a powerful tool to study tissue morphochemistry. Most often the experiments are performed on tissue cryosections or on fresh tissues, however the extensive availability of formalin–fixed paraffin-preserved (FFPP) tissues may provide a valuable number of samples for retrospective studies. The purpose of this study was to assess the effects of deparaffinization of FFPP sections and their feasibility for MP imaging.
Methods: 4 μm thick sections of mouse brain and 8 human brain tumors tissues (3 primary tumors and 5 metastases) preserved in FFPP blokes were prepared on glass or CaF2 slides for MP microscopy and Raman micro-spectroscopy, respectively. MP microscopy, simultaneously acquiring Coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) signals, was applied and confirmed by Raman micro-spectroscopy. The FFPP sections were deparaffinized and rehydrated according to standard protocol, using Xylene as dewaxing agent.
Results: The MP images of different tissue samples were acquired providing the following observations: (i) The tissue morphochemical contrast available from CARS signal is based on different protein content, as lipids are washed away during deparaffinization. Depletion of lipids was confirmed by Raman spectroscopy (loss of spectral bands at 1130, 1300, 1440, 1668 cm-1). (ii) TPEF and SHG signals provide additional chemical contrast for autofluorescent structures and collagen, respectively. (iii) The paraffin is never completely removed and gives strong CARS signal. Paraffin deposits can mainly be observed as separate droplets, clusters in and around blood vessels and accumulations in some cells. The insufficient removal of paraffin was confirmed by Raman spectroscopy (paraffin spectral bands in strong CARS active structures at 1062, 1133, 1295, 1439 cm-1). (iv) Tumor borders can be distinguished from normal tissue due to morphological differences provided by MP signals.
Conclusion: Despite the loss of lipids and several paraffin artefacts the FFPP deparaffinized samples provide sufficient morphochemical information about brain tissue structures when investigated by MP microscopy. As long as the paraffin artefacts are corrected, MP microscopy opens a door for label-free imaging of historical FFPP brain tumors for extensive retrospective studies.
