User manual LEICA TCS SP5 SPECTRAL DETECTOR SP BROCHURE

[. . . ] Immunohistochemical fluorescence tags, fluorescence-insitu hybridization, metabolite probes like Ca2+ or potential sensitive dyes Caenorrhabditis elegans, neurons. 5 channel image, overlay 1) Sensory and interneurons, CFP 2) Excitatory motoneurons, sensory neurons, YFP 3) GABAergic neurons, GFP 4) Glutamatergic interneurons, DsRed 5) Sensory neurons, DiD Courtesy of Dr. Harald Hutter, Max Planck Institute for Medical Research, Heidelberg, Germany and various specific stains for cell compartments and structural proteins fill the huge catalogues of fluorescence probes for current research tasks. And fashionable staining tools like fluorescent proteins and quantum dots, flood the laboratories. [. . . ] Although there might be combinations that "work", they will seldomly lead to optimal results due to loss in efficiency or unacceptable crosstalk ­ usually both. Today: the Leica SP Detector The Leica SP®-detector solves all these problems. The light from the sample, which is a sum of multiple emission spectra, first passes through the pinhole. Then, by means of a prism, the colors are spread into a spectrum. On its way to the first detector, the light passes a slit-photometer device consisting of two motorized barriers, which are controlled via software. The principle is simple but most effective: The device is steplessly adjustable: the barriers can be moved to any wavelength position. By means of this design, this single detector can make up any conventional filter specification in terms of center frequency and bandwidth. Sample protection by high-senstitivity detection. Top: high laser power and rigorous averaging is necessary in conventional detection systems in order to generate noise-free images. Bottom: low illumination intensity and few averages will not damage this extremely bleach-sensitive sample when imaged with SP(R) detector. An ingenious invention However, this is not a multi-parameter fluorimeter. How can we simultaneously collect multiple segments from the spectrum?The clever bits are the barriers: they are not made of low-reflection material as it is the case for standard photometers. So the residing parts of the spectrum on both sides of the band collected at sensor 1 are directed to sensors 2 and 3 with again motorized, reflective barriers ­ ready for the next sequence. Just some high-efficiency coated mirrors. Whether you need to cover the full spectrum for full efficiency or just small specific bands to the detector for unsurpassed crosstalk reduction. (Software for unmixing is, of course, available anyway. ) 3 2 1 2 1 2 3 Prism Sliders Detector Better images in less time Your benefit of Leica's SP detector concept: maximum efficiency and ® G Spectral Detector, five confocal channels True confocal point-scanning ­ real optical sectioning High dynamic sensors Low dark current PMTs High efficiency prism spectrometer Loss-free band selection Free tunable emission bands Any dye adaptable Five bands simultaneously Dye separation: sufficient for five channels simultaneously full tunability which guarantees you best imaging quality and least crosstalk in multiparameter fluorescence. And as a side-effect: the SP detector allows recording of lambda scans ­ spectral image stacks ® G G G G G G G G G to analyze the in-situ emission characteristics of the dyes employed in your sample. There have been several attempts to top the versatility of this concept. But mainly targeting the issue of spectral imaging. Some approaches work with optical grids instead of prisms ­ by far too inefficient for imaging. There are attempts to overcome the grid problem, only by introducing even more optical elements, consequently eating photons. [. . . ] Completely new applications will be possible. More differentiated insights in FRET processes by simultaneous detection of acceptor and donor, and other quenching effects are now available for research. In addition, the SPFLIM has better photon counting efficiency than filter-based concepts. Spectral FLIM ­ a new dimension Fluorescence Lifetime Imaging To further increase the range of applications, it is possible to install two different sources for FLIM excitation in the same system: either a pulsed Titanium-Sapphire laser, which is the standard source for multiphoton excitation, or a pulsed 405 nm laser diode. Lifetime scale (ps) A 1500 2000 2500 Schistocerca gregaria, nervous system, double staining Cy2, Cy3. A) SP-FLIM images left: emission 500-550 nm, right: emission 575-647 nm top: D-FLIM, excitation 405 nm, bottom: MP-FLIM, excitation 780 nm B) Intensity images for comparison B left: excitation 488 nm, emission 500-550 nm right: excitation 561 nm, emission 575-647 nm @ www. confocal-microscopy. com Copyright Leica Microsystems CMS GmbH · Am Friedensplatz 3 · D-68165 Mannheim, Germany · Tel. [. . . ]