CC BY
20LD-I-15
Re-scan Confocal Microscopy of ESCRT-mediated lysosome repair
Stefan G. Stanciu1, Iustin Floroiu1, Radu Hristu1, Efstathios Fiorentis1, Maja Radulovic2, Camilla Raiborg2, Harald A. Stenmark2
[1] Center for Microscopy-Microanalysis and Information Processing, Politehnica University of Bucharest,
Bucharest, 060042 Romania
[2] Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.
E-mail: stefan.g.stanciu@upb.ro
Super-resolution microscopy techniques, capable of circumventing the resolution limit imposed by diffraction, are extremely useful for resolving important properties of eucaryotic and procaryotic cells that are not accessible to conventional microscopies1. While the resolution advantages of some of these techniques come at the cost of high laser beam power, or very specific contrast agents2, Re-scan Confocal Microscopy (RCM)3 has emerged over the past few years as a valuable tool to investigate biological specimens at sub-diffraction resolution using ultra-low beam power, and any type of fluorophores. In addition to a standard Confocal Laser Scanning Microscope (CLSM), where the beam is scanned on the sample by means of a system of mirrors, the RCM incorporates a second system of mirrors whose role is to scan the light emitted by the sample upon laser excitation on the surface of a CCD/s-CMOS image sensor. If the angular amplitude of the camera scanner (re-scanner) is double the angular amplitude of the sample scanner, a resolution advantage of V2 over a conventional CLSM is achieved. With additional deconvolution resolutions down to 120nm under 488nm excitation can be achieved. Conventional RCMs are designed for fluorescence imaging, but recent efforts showed that the Re-scan concept can also be exploited for achieving optical super-resolution based on non-linear effects not requiring contrast-agents, such as Second Harmonic Generation4.
In this work we discuss our results on RCM imaging of the lysosome repair process. Lysosomes are specialized organelles with many important roles, including the degradation of macromolecules, pathogen killing, and cell signaling, but their damage, which can occur due to pathogens, amphiphilic drugs, or other membrane-disrupting agents, impose serious threat to cell viability5. Severely damaged lysosomes are removed by lysophagy, an autophagic pathway that sequesters and degrades damaged lysosomes6. Lysosomes with milder damage are repaired by the endosomal sorting complex required for transport (ESCRT) machinery7. In the current work we place additional attention to this subject, discussing how RCMs resolution advantages are useful to investigate subtle morpho-structural aspects of cells linked to the lysosome repair process.
Acknowledgement: This work was supported by the UEFISCDI Grant RO-NO-2019-0601 MEDYCONAI.
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