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Rapid Imaging of Signaling between the Endothelium and Smooth Muscle; Development of a Rapid Remote Refocusing Epifluorescence Microscope
Author(s) -
Buckley Charlotte,
Lawton Penny,
Wilson Calum,
Girkin John,
McCarron John
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.525.5
Subject(s) - magnification , image quality , microscope , shutter , fluorescence lifetime imaging microscopy , biomedical engineering , lens (geology) , materials science , optics , computer vision , computer science , fluorescence , physics , image (mathematics) , medicine
Rapid multiplane imaging is often required when imaging dynamic processes at high magnification in live samples. However, during rapid focus switching, image quality is compromised by the repeated, rapid agitation of the sample itself and movement of the immersion medium used for the high NA lens. Remote refocusing has been successful in minimising the degraded image quality in rapid multiplane imaging, allowing rapid acquisition of multiple focal planes without sample movement. Remote refocusing lends itself very well to imaging the endothelial and smooth muscle cell layers of an intact artery. These cell layers are intrinsically linked through a rapid communication that is critical for blood vessel function. However, understanding the interplay between the two cell types is hampered by the difficulty in imaging the two layers at a sufficiently high frame rate to capture signal transmission. The cell layers are only ~10 μm apart, therefore we designed and implemented an epifluorescence‐based remote refocusing imaging system. The system can image at over 20fps in two colours, using optical switches to provide TTL pulses to trigger appropriate laser shutter opening and image acquisition at each plane. With 60X objectives, we achieved a lateral resolution of 0.96 μm and an axial resolution of 6.40 μm. We have shown, using appropriate calibration slides, that the magnification remains constant over 100 μm depth and have verified the stability of the image acquisition. Using this method, we are able to image and analyse Ca 2+ activity of the endothelial cell layer independently of the smooth muscle layer and the rapid communication between the two cell types. Support or Funding Information Wellcome Trust, British Heart Foundation This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .