Rolf Borlinghaus

Closing the spectral gap : the transition from fixed-parameter fluorescence to tunable devices in confocal microscopy

Modern microscopy in life sciences is ruled by development and exploration of new dyes and stains (probes for histochemical staining, quantum dots, fluorescent proteins etc.) on one side, and technological improvements and innovations for fluorescence microscopy-especially high resolution and optical sectioning microscopy-on the other side. Concerning the technical innovations, several ingenious inventions have been made available for confocal microscopy. First, the acousto optical tunable filter, which allows switching and dimming of laser lines. Second the spectral detector, employing mirror sliders in front of the detectors which allow continuous tuning of the spectral emission band detected by the sensor. Third, the most challenging task: a substitute to the classical beam splitter-the device which is restricting fluorescence microscopy most. This was solved by introduction of the acousto optical beam splitter. The very last device which is still lacking flexibility is the laser source, operating only at non-equidistant frequencies and requiring a set of quite different laser sources as gas lasers, solid state lasers or diode lasers. A new approach by supercontinuum light sources is presented and discussed, which significantly enhances flexibility and coverage of the excitation spectra of typical, rare and natural fluorochromes.

The White Confocal: Continuous Spectral Tuning in Excitation and Emission

“White,” when describing a physical parameter, means constant distribution over the observed range. The white confocal refers to a concept that allows tuning any wavelength or light pattern from a white laser source for illumination of the sample, simultaneously transmitting a white spectrum to the detector and finally collecting at any selection of tunable bands within the visible spectrum. In this chapter, such a technology is described. It is composed of lasers based on photonic crystal fibers and acousto-optical devices for selection (AOTF) of small bandlets and guiding them onto the sample. Simultaneously, a second acousto-optical device (AOBS) performs white transmission of the emission into a tunable multiband detection system, based on a spectrophotometer concept. This concept is the ultimate solution for efficient recording and optimal separation of multiparameter fluorescence in confocal microscopy.

Benefits and applications of programmable spectral devices in confocal microscopes

Recent developement in confocal microscope systems allow to tune the spectral characteristics of those elements which separate excitation and emission. The new devices replace the classical filter-devices, which have been used in fluorescence microscopy for many years. Excitation filters have been replaced by acousto optical filters, which work as multichannel dimmer devices and allow fine tuning of excitation energy as well as tuning the excitation ratio of multiple excitations. Emission filters have been replaced by tunable multiband detectors, which allow tuning of emission band width for higher efficiency and better band separation. The multiband detector also allows spectral scanning modes. Dichroitic beam splitters have been replaced by acousto optical beam splitters (AOBS) giving significantly increased signal efficiency and allow fast switching of excitation modes without moving mechanical parts. All three devices together lead to much better primary images, either giving highly improved signal to noise ratio or much less photobleaching (which is identically with viability of life samples).