confocal laser scanning microscope

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 539339594

A current confocal laser scanning microscope (CLSM) is requested. In confocal laser scanning microscopes, a laser beam is moved across the object in the XY direction using galvanometer mirrors. To discriminate beams from the different optical slice planes, a CLSM has either a variable diameter pinhole or an array of 32 detectors on which the point spread function (psf) is mapped, thus functioning as an extremely small pinhole. Furthermore, the galvanometer mirrors can be moved at very high frequency to achieve high frame rates (resonant scanning confocal). Other solutions such as Nipkow spinning disk microscopes and ribbon scanning confocal microscopes, are not considered here because of the diversity of applications desired here. The proposed microscope will be used to study protein localizations and protein interactions in living cells and fixed tissues at high resolution and is therefore based on an inverted stand. In addition, neuronal networks will be visualized and physiological studies will be performed using ratiometric sensors. The biological samples include cyanobacteria, green algae, plants, plant and animal/human cell cultures, and immunolabeling in mammalian tissue sections. Thus, interactions within the protein complex of vacuolar H+-ATPase, multiple interactions of the cellular redox network, nuclear import and export as well as the formation of stress granules in plant cells, high-resolution visualization of cell division in Volvox, detection of neuronal networks by tracing in fish, and quantitative detection of tumor markers in human tissue sections will be analyzed. Confocal laser scanning microscopes enable such diverse applications and processing of a wide variety of specimens due to the creation of optical sections, high resolution in the <120 nm range with 488 nm excitation, and the possibility of simultaneous and highly sensitive image acquisition in multiple (at least three) channels. High flexibility of the spectral detection ranges, which is independent of conventional filters, allows reliable separation between autofluorescence and desired fluorescence, especially in pigment-rich plants (cells). This separation is further enhanced based on the different fluorescence lifetimes. Fluorescence lifetime microscopy will also be used for ratiometric sensors and interaction studies. The use of conventional cell nuclear labels and switchable/convertible fluorescent proteins will require a 405 nm diode laser; visualization of fluorescent proteins will require excitation wavelengths of 440 - 560 nm; in addition, long wavelength excitation ranges of up to 730 nm and appropriate detectors will be required for chemical dyes to enable multiple labeling up to the near infrared range. Furthermore, the microscope applied for has controlled temperature control and CO2 gassing for studies on living cells.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Konfokales Laserscanning Mikroskop
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Universität Bielefeld
• Leader: Professor Dr. Christian Kaltschmidt