Spinning Disk Microscope

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 431398411

The major scientific reason for this instrument application is to move to the next possible level of spatio-temporal resolution in live cell imaging to allow for significantly enhanced visualization of local Ca2+ signals and local 3’,5’-cyclic AMP (cAMP) signals, entitled Ca2+ and cAMP nanodomains. Ca2+ and cAMP nanodomains will be studied in the context of the newly funded SFB 1328 ‘Adenine nucleotides in Immunity and Inflammation’; 1st funding period started July 01, 2018. The following projects will study Ca2+ nanodomains: A01: Ryanodine receptors and NAADP in T cell biology , A02: Dynamic architecture of Ca2+ microdomains in T cells: disentangling contribution of adenine nucleo-tides, Ca2+ release and Ca2+ entry , A03: Adenine nucleotide-modulated T cell differentiation, effector functions, and plasticity, A05: Functional and structural analysis of the nucleotide-activated ion channel TRPM2 , A06: Probing the role of cAMP in T cell-mediated immunosuppression and neuroinflammation , and A10: Role of purinergic receptors in brown adipose tissue. The spinning disc super-resolution system will allow a shift from approx. 370 nm spatial resolution and 40 frames per second (fps) temporal resolution down to 120 nm and 100 fps. Using super-resolution imaging, we plan to describe temporal and spatial components of what currently is termed “Ca2+ microdomain”. These components, here tentatively termed “Ca2+ nanodomains”, likely result from activity of small groups of potentially different ion channels, ion pumps and Ca2+ buffering proteins, but have not been successfully resolved in wild-type cells in live cell imaging. However, the combination of live cell super-resolution imaging, relevant knock-out models, e.g. Ryr1-/- or Orai1-/- T cells, or Ca2+ channel-GECO constructs should allow to characterize “Ca2+ nanodomains” of non-excitable cells for the first time. The expected increase in scientific knowledge is currently only possible with the performance class of spinning disc super-resolution system and thus justifies the selection of this instrument and the high price.Five research groups as well as the University Medical Center Hamburg-Eppendorf Microscopy Imaging Facility are co-applicants. Research topics are all located and funded in SFB1328:1. High-resolution imaging of Ca2+ nano and microdomains in leukocytes, with special emphasis on NAADP forming enzymes and NAADP binding proteins.2. High-resolution imaging of Ca2+ nano- and microdomains in leukocytes, with special emphasis on purinergic receptors/ion channels (P2Y, P2X).3. High-resolution imaging of Ca2+ nano- and microdomains, and induction of fluorescent cytokine reporters in T cells deficient in receptors for adenine nucleotides4. High-resolution imaging of cAMP nano- and microdomains5. Role of adenine nucleotides and purinergic receptors in adipose tissues6. Imaging dynamic processes within intra and extra cellular nano domains by mean of super resolution light microscopy

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Spinning Disk Mikroskop
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Universität Hamburg
• Leader: Professor Dr. Andreas H. Guse