SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
The power of single- and multibeam 2-photon microscopy for
high-resolution and high-speed deep tissue and intravital
imaging
Raluca Aura Niesner 1*, Volker Andresen 2, Jens Neumann 3, Heinrich Spiecker 2 and Matthias Gunzer 4
1 Technical University Braunschweig
2 LaVision Biotec GmbH
3 Institute for Applied Neuroscience, University of Magdeburg
4 Helmholtz Centre for Infection Research
* To whom correspondence should be addressed. E-mail: raluca.niesner{at}tu-bs.de.
Submitted on December 5, 2006
Revised on March 9, 2007
Accepted on 29 May 2007
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Abstract |
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Two-photon microscopy is indispensable for deep tissue- and intravital-imaging. However, current technology based on single-beam point-scanning has reached sensitivity- and speed-limits as higher performance requires higher laser-power leading to sample-degradation. We utilize a multi-focal scanhead splitting a laser-beam into a line of 64 foci allowing sample illumination in real-time at full laser-power. This technology requires CCD-field-detection contrary to conventional detection by photomultipliers (PMT). A comparison of the optical performance of both setups shows functional equivalence in every measurable parameter down to penetration depths of 200µm, where most actual experiments are executed. The advantage of PMT-detection materializes at imaging depths >300µm due to a better signal-to-noise-ratio, while only CCDs allow real-time detection of rapid processes (here: blood-flow). We also find that the point-spread-function (PSF) of both devices strongly depends on tissue-constitution and penetration-depth. However, employment of a depth corrected PSF allows 3D-deconvolution of deep-tissue data up to an image quality resembling surface-detection.
Key Words:
CCD, Charge Coupled Device (synonymous for CCD-camera), OHBS, Organotypic Hippocampal Brain Slices, PMT, Photo Multiplier Tube, PSF, point spread function
