Flexible multi point scanning
for brighter images and
selective excitation below single cell size.
LaVision BioTec’s Cloud Scanner improves 2-
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
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 – but not diffraction limited – 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-
The Cloud Scanner accounts for this situation allowing the user to define a line of eight foci shorter than the pixel width. 200 ps delays between two foci eliminate cross talk and allow to increase the laser power without damaging the sample.
Time delay between laser pulses while using a utilizing a 80 MHz Ti:Sa laser.
Scanning and data acquisition remain identical to a standard single beam laser -
Example PSF for voxel filling
PSF integrated over the dwelltime (crosses mark the edges of the pixel)
800 nm excitation
Upper row of images showing conventional scanning path at different pixel sizes. In the row below images display the voxel filling scanning path of the Cloud Scanner.
Analysis of the initial decay of the fluorescence
8 beams in diffraction limited spot (0 – 10 sec) (green)
8 beam cloud (11-
Ratio of the decay time >12 for all cells
20% loss is reached 20x later in time