Multi-plane remote refocusing epifluorescence microscopy to image dynamic Ca<i/>²⁺events

Abstract

Rapid imaging of multiple focal planes without sample movement may be achieved through remote refocusing, where imaging is carried out in a plane conjugate to the sample plane. The technique is ideally suited to studying the endothelial and smooth muscle cell layers of blood vessels. These are intrinsically linked through rapid communication and must be separately imaged at a sufficiently high frame rate in order to understand this biologically crucial interaction. We have designed and implemented an epifluoresence-based remote refocussing imaging system that can image each layer at up to 20fps using different dyes and excitation light for each layer, without the requirement for optically sectioning microscopy. A novel triggering system is used to activate the appropriate laser and image acquisition at each plane of interest. Using this method, we are able to achieve axial plane separations down to 15 μm, with a mean lateral stability of ≤ 0.32 μm displacement using a 60x, 1.4NA imaging objective and a 60x, 0.7NA reimaging objective. The system allows us to image and quantify endothelial cell activity and smooth muscle cell activity at a high framerate with excellent lateral and good axial resolution without requiring complex beam scanning confocal microscopes, delivering a cost effective solution for imaging two planes rapidly. We have successfully imaged and analysed Ca2+ activity of the endothelial cell layer independently of the smooth muscle layer for several minutes.

Myography of isolated blood vessels: Considerations for experimental design and combination with supplementary techniques

“…For the investigation of the spatial organization of changes in the intracellular calcium concentration confocal microscopy has been employed on vessel preparations. Using this method, changes in the intracellular calcium concentration can be detected in either single smooth muscle cells or single endothelial cells ( Simpson, 1999 ; Paredes et al, 2008 ; McCarron et al, 2012 ; Dora and Hill, 2013 ; Wilson et al, 2015 ; Lawton et al, 2019 ) or even in both together when a special arrangement of the vessel is used ( Rahman et al, 2007 ). However, since the dyes used in confocal microscopy are usually non-ratiometric, artefacts induced by movements of the preparation, dye loss, photobleaching, etc.…”

Fast and light efficient remote focusing for volumetric voltage imaging

“…This is especially harmful in ultra-high-speed recordings such as voltage imaging, where integration times are in the sub-millisecond range and the signal-to-noise ratio (SNR) is severely limited by the amount of photons that can be collected in each frame. Remote focusing has therefore mainly been used to achieve fast axial scanning in two-photon microscopy [9,10] and is not widely adopted as a method on the emission light path to refocus the detected image [11][12][13].…”