LaVision Biotec AsiaPacific

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Cloud Scanner: Flexible Multipoint Scanning

 

Flexible multi point scanning for brighter images and selective excitation below single cell size.

LaVision BioTec's Cloud Scanner improves 2-photon Laser Scanning Microscopy significantly as it allows scanning with eight beams. Eight beams can be arranged to a user defined foci pattern in the object plane of the microscope. The beam arrangement is software controlled and can be optimized during the measurement.

Microscopy of dynamic events always depends on signal to noise ratio and therefore on the fluorescence intensity. In turn the fluorescence intensity depends on the fluorophore, the excitation power and the exited area. If it comes to in vivo Ca2+ imaging of somata the fluorophore is given and the maximum excitation power is limited because of photo bleaching. Exciting the cell body by scanning its entire area increases the fluorescence yield. The Cloud Scanner excites this necessarily larger area at once. It allows defining a pattern of eight diffraction limited foci filling the structure of interest. Therefore the individual soma does not have to be scanned anymore and could be excited at once. Cloud scanning can be applied to all scanning patterns including raster scan, line scan and point measurements. Increasing the fluorescence signal leads to better results and allows faster imaging.

 

 

  • - Higher frame rates
  • - Brighter images
  • - Less photo damage
  • - Fast selective treatment of cell compartments

Adaptation to pixel size

For most applications intravital microscopy has to provide large field of views in combination with high â?? but not diffraction limited â?? resolution. Imaging speed is more important than ultimate resolution. Therefore, low magnification objective lenses and a limited pixel resolution have to be chosen. High NA-objective lenses deliver best 2-photon performance due to their excitation and collection efficiency. One of the most common imaging modalities combines a 20x, NA 1.0 objective lens, 500 by 500 microns FOV and 512 by 512 or even less pixel resolution. The focus size is actually smaller than the pixel size. In other words, only part of the pixel volume will be excited.

 

Scanning and data acquisition remain identical to a standard single beam laser - scanning microscope â?? but it gains brightness. Since speed depends on brightness, the maximum frame rate can be increased substantially.

 

Analysis of the initial decay of the fluorescence

  • - 8 beams in diffraction limited spot  (0 â?? 10 sec) (green) 

  • - 8 beam cloud (11-35 sec) (blue)


Ratio of the decay time >12 for all cells
20% loss is reached 20x later in time

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