Confocal Microscope with Time Correlated Single Photon Counting and Multiphoton Imaging Capabilities

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 565398888

With the present application we seek to replace an existing microscope that is currently used for confocal imaging of fixed and live samples, for two-photon imaging, and second harmonic generation (SHG) microscopy. In addition, it is currently the only available system on campus for time correlated single photon counting (TCSPC) applications such as fluores-cence lifetime imaging (FLIM) microscopy, in particular metabolic FLIM measurements based on NADH fluorescence, and FLIM-based Förster resonant energy transfer (FLIM/FRET) microscopy. The replacement system shall retain these capabilities and up-grade them to the current, technical state of the art. The proposed microscope will be used for confocal imaging of both fixed and live samples. Image scales will begin at low to medium magnifications for overview and tiling images of organoids or small model organisms such as zebrafish. At the high end, resolutions approach-ing the diffraction limit will be required for tracking protein colocalization and trafficking, cyto-skeletal dynamics, or organelle maturation at the subcellular level. Since most of the research groups contributing to this proposal want to extend multimarker approaches also to imaging, the microscope will have to posses a high capability for multi-plexing, and thus needs to offer a wide spectral range of both detectors and available excita-tion lasers. An ability to conveniently separate spectrally overlapping chromophores based on photon arrival times will be essential in this context, and will also help with the suppression of unwanted, background fluorescence in e.g. animal and plant tissues and certain 3D cell cul-ture matrices. For larger samples, primarily organoids, sensitive 3D cell cultures, and small model organ-isms, two-photon excitation will be used to minimize photodamage while increasing penetra-tion depth. Two-photon illumination will also be used by several groups for SHG microscopy for the label-free detection of diffracting structures such as bone or collagen bundles. Beyond regular imaging, the new microscope must also continue to support microscopy-based biophysical modalities. In particular, it must be able to collect TCSPC data for applica-tions such as FLIM microscopy and fluorescence crosscorrelation spectroscopy (FCCS). In the visible range, FLIM-FRET microscopy and FCCS will be used to monitor interactions of fluorescently tagged proteins at the nm scale. A majority of groups will also use FLIM in com-bination with two-photon excitation of unlabelled metabolites such as NADH or FAD to char-acterize the metabolic state of their cells of interest (metabolic FLIM). This investment will allow the Ulm University Core Facility Light Microscopy to continually offer confocal microscopy at the level necessary for nationally and internationally competitive biomedical research, and will ensure critically important redundancy for the confocal imaging needs of the Ulm biomedical community.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Konfokalmikroskop mit zeitkorrelierter Einzelphotonenzählung und Multiphotonenfähigkeit
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Universität Ulm
• Leader: Professor Dr. Gilbert Weidinger