Part 1 Principles
Confocal Microscopy tutorial
Part 1 Principles of Confocal microscopy
10. Special features from Leica SP2 confocal microscope
Leica SP2 confocal microscope has two unique hardware innovations. The first one is the emission filter free spectral meter detecting system, introduced in 1999, depicted as below:
Like in picture on the left, the emission is first spread by prism into spectra, this spectra is further selected by a motor-driven slit which can change width or move position laterally as in picture on the right. With this device, the detecting wavelength, detecting band width can be continuously changed within 400 -750 nm to meet emission from any fluorophores without need of add new filter set into the system. a lambda scan within the whole range enable you to determine where is the emission peak of your stain and further optimizing of detecting position and range has more accurate basis.
The beam-splitting-filter free AOBS (acousto optical beam splitter) system is introduce in 2002.
Here the ordinary BSP is replaced by a optical crystal with an ultrasonic field applied. The wavelength and amplitude of the ultrasound can be changed (programmed) to deflect certain band of wavelength and its amount passing through the field (figure on the left). Up to eight excitation bands can be programmed at the same time compared to only three bands in tri-chroic BSP in ordinary filter based system. Besides, the band width of the deflected is very narrow or sharp, around 1 nm only (0.6 - 3 nm proportional to the wavelength concerned), compared to ordinary BSP which has about 10 to 20 nm width of barrier to emission know as "emission hole" (figure on the right).
In short, this function has two major benefits: 1. removal of the limitation on the number of beam splitter available in the system and more accurate control on the reflected bands. 2. Higher efficiency for emission collection.
These two approaches: AOBS and spectra detecting create totally filter-free confocal microscope which provides more versatility and higher efficiency in choosing excitation and collecting emission.
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