User manual LEICA TCS SP5 AOBS BROCHURE

[. . . ] A technique called , , incident light microscopy", therefore, uses a device in the beam path, which decides which light goes where, very much like a three-way valve. For a singly FITC stained sample, blue light has to reach the sample and green light has to reach the detector. Classically, this is achieved by a color-discriminating mirror: in the FITC-case, it is a mirror for blue light, but a window for green light. For other fluorochromes, one has to insert other dichroics, meeting the different color requirements. [. . . ] The sample bleaching results from high numbers of averaging. The transmission of the AOBS® is superior to most dichroic mirrors over the full visible spectrum. Consequently, less averaging is necessary. Bright and crisp images require wide emission bands as provided by the AOBS®. This is important to channel as much photons as possible from the sample to the detector ­ again improving the image quality. Low bleaching during image acquisition is important to protect the sample from fading and to protect living specimen from toxic chemicals that accumulate on photolysis of fluorochromes. The AOBS® has very steep slopes allowing collecting emission very closely to the excitation band. Any visible-range dye can be excited, as the position of the reflection-pins can be tuned individually. Multiparameter fluorescence is solved: up to eight laser lines programmable, leaving still sufficient space for emission collection ­ and the frequencies are tunable!Ratio dyes, like excitation ratio metabolite-probes, e. g. for Ca 2+, membrane potential, pH or chloride expect fast switching in sequential scanning. The AOBS® has switch times of only few microseconds. Reflected light imaging as another option. The very strong suppression of the excitation can be reduced individually, if necessary for reflection imaging. ROI-scanning is improved as well: different excitation patterns are possible for different regions during a single scan. Large 3D volume recording, in sequential mode will benefit as well from fast switching devices, as speed improves dramatically the system efficiency. Fluorescence correlation spectroscopy (FCS) requires very low background and stray-light. Only the AOBS® sufficiently blocks close co-emitted lines, e. g. Spectral recording (lambda scan) supply correct spectra, as the transmission of the AOBS® is "white", which means, that is does not alter the emission spectra ­ a common problem, if spectral scanning is done in a dichroic-mirror system. True confocal optical sectioning requires point-shaped illumination and emission. The AOBS® fits to point-scanning confocal devices. Multiphoton and UV-imaging can be done in parallel without any drawbacks or restrictions. [. . . ] It is a series of tunable elements at very high transmission, allowing selecting any wavelength band for emission collection. Tunable emission bands fit perfectly to tunable dichroics: the combination of these two technologies will not leave open any application requirement. Ulrich: Advances in multispectral confocal imaging. Progress in Biomedical optics and imaging. [. . . ]