Unlike the Confocal microscope the Light Sheet microscope illuminates a single, thin plane at one time thus producing an optical section without the need for a confocal pinhole, but increasing SNR significantly. The detection lens is set at 90 degrees to the plane of illumination, and this perpendicular light path and focal plane illumination significantly reduces photo toxicity of delicate samples.
Application and advantages
Light sheet fluorescence microscopy is the way to observe small animal models, such as Zebrafish and Drosophila in a more gentle and physiological manner, but is also applicable to Arabidopsis, marine organisms, excised tissue and biopsies.
The sample is usually embedded in agarose and mounted vertically in an imaging chamber via glass a capillary. This allows the sample to be rotated in 360O for optimisation of the light sheet angle, but also to obtain a “Multiview” of a sample for whole organism reconstruction.
The system does not image pixel by pixel but will grab full frame using two sCMOS cameras allowing high resolution time lapse recordings for efficient analysis of dynamics within the sample.
Further background reading including downloads on sample preparation and other protocols can be found here;
- Twin sCMOS PCO Edge 16bit cameras for simultaneous 2 channel acquisition
- Twin illumination objectives for single or dual sided sample illumination with dual side fusion of the live image
- Pivot scanning to reduce shadowing
- Time lapse
- Chamber incubation with CO2 available
- System configured for imaging cleared tissue
- Objective lenses, 10x 20x and 40x
- Laser lines, 405,445, 488,514,561 and 638nm
- 32TB 3 RAID drive processing workstation networked to RDS
Software and Network Drives
- Zen SP1 Lightsheet, allows for multidimensional experiment design in 3D, and 4D
- PC 1, HPZ840 4TB acquisition workstation
- PC 2, 32TB 3 RAID drive image processing workstation memory.
Imaging Suite, Room WXG89
Accounts / access / training
For internal users, all requests via the Stratocore MISBU page; https://ppms.eu/bham/login/?MISBU
Dr Robert K. Shaw
Technology Hub Imaging Section
The Imaging Suite – Ground floor IBR West Extension
College of Medical and Dental Sciences
University of Birmingham B15 2TT