Spinning disk confocal microscope for visualizing and quantifying host-bacteria interactions

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 527320650

Bacterial pathogens pose a great threat worldwide, and understanding their interaction with human cells is important for developing diagnostics and therapeutics, especially in a post-antibiotic era. Intracellular bacterial pathogens, in particular, present a significant challenge since they can avoid the host immune system and antimicrobial drugs by hiding within host cells to replicate and spread. Host-pathogen interactions have long been studied in static cultures (by placing cells on plastic or glass dishes) that do not effectively recapitulate human in vivo (patho)physiology. Alternatively, animal models rarely recapitulate the infection process in humans and typically do not resolve subcellular detail in a dynamic manner. Recently, organoids and organ-on-chip microfluidic technologies have emerged as experimental systems allowing to modulate one parameter at a time under conditions that more closely mimic in vivo settings. This has added significantly to our understanding of how pathogenesis might unfold in vivo. However, given the inherent three-dimensional (3D) architecture of such samples and the varying time scales of infection-related processes (ranging from seconds to days), specific microscopy modalities are required that enable to resolve biological and infection processes in time and space. The challenges mentioned for the study of infections, in one form or another also apply to the study of host cell responses to sterile insults such as inflammation. Herein, we request the purchase of a spinning disk confocal microscope (SDCM) for fast visualization and quantification of live-cell (infected or sterile) 3D samples over short but also long time scales. The following unique features of SDCM for this specific purpose include, (i) high speed of acquisition, which is critical especially for imaging organ-on-chip devices, live-cell organoids or infected samples in 3D matrices and over time. Laser scanning confocal microscopes (LSCMs) operating with point scanners can take minutes for multi-channel imaging and thick samples, as opposed to seconds with a SDCM. (ii) SDCM reduces out of focus light and aberrations.In thick samples (i.e., >10-15 μm typical thickness of a cell monolayer), wide-field epifluorescence imaging coupled with deconvolution causes aberrations that tend to increase in a non-linear fashion as a function of the specimen thickness. SDCMs are capable of acquiring thin optical sections from specimens in a manner similar to LSCMs, but much faster. (iii)SDCM exhibits lower phototoxicity and photobleaching compared to classical LSCMs and this feature becomes critical for finely resolved live-cell imaging of individual, fast-moving bacteria, motile sensitive immune cells and other cell types expressing live-cell biosensors. Thus, a state-of-the-art SDCM dedicated to an S2 safety level environment would help us advance the study of host-pathogen interactions to an unprecedented level at our campus.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Spinning Disk Konfokalmikroskop: Visualisierung und Quantifizierung von Bakterien-Wirt-Interaktionen
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Eberhard Karls Universität Tübingen
• Leader: Professor Alexander Weber, Ph.D.