Biology subjects

McConnell, G.

Publications and source records attributed to McConnell, G..

3 recordsLinked to original sources

bioRxiv2018

Fast Optical Sectioning for Widefield Fluorescence Mesoscopy with the Mesolens based on HiLo Microscopy

We present here a fast optical sectioning method for optical mesoscopy based on HiLo microscopy, which makes possible imaging of specimens of up to 4.4 mm x 3 mm x 3 mm in volume in under 17 hours (estimated for a z-stack comprising 1000 images excluding computation time) with subcellular resolution throughout. Widefield epifluorescence imaging is performed with the Mesolens using a high pixel-number camera capable of sensor-shifting to generate a 259.5 Megapixel image, and we have developed custom software to perform HiLo processing of the very large datasets. Using this method, we obtain comparable sectioning strength to confocal laser scanning microscopy (CLSM), with sections as thin as 6.8{+/-}0.2 m and raw acquisition speed of 1 minute per slice which is up to 30 times faster than CLSM on the full field of view (FOV) of the Mesolens of 4.4 mm with lateral resolution of 0.7 m and axial resolution of 7 m. We have applied this HiLo mesoscopy method to image fixed and fluorescently stained hippocampal neuronal specimens and a 5-day old zebrafish larva.

biophysics↗

bioRxiv2018

Application of the Mesolens for sub-cellular resolution imaging of intact larval and whole adult Drosophila

In a previous paper (McConnell et al., 2016) we showed a new giant lens called the Mesolens and presented performance data and images from whole fixed and intact fluorescently-stained 12.5-day old mouse embryos. Here we show that using the Mesolens we can image an entire Drosophila larva or adult fly in confocal epifluorescence and show sub-cellular detail in all tissues. By taking several hundreds of optical sections through the entire volume of the specimen, we show cells and nuclear details within the gut, brain, salivary glands and reproductive system that normally require dissection for study. Organs are imaged in situ in correct 3D arrangement. Imaginal disks are imaged in mature larvae and it proved possible to image pachytene chromosomes in cells within ovarian follicles in intact female flies. Methods for fixing, staining and clearing are given.

developmental biology↗

bioRxiv2017

A 340/380 nm Light Emitting Diode Illuminator For Fura-2 AM Ratiometric Ca2+ Imaging Of Live Cells With Better Than 5 nM Precision

We report the first demonstration of a fast wavelength-switchable 340/380 nm light emitting diode (LED) illuminator for Fura-2 ratiometric Ca2+ imaging of live cells. The LEDs closely match the excitation peaks of bound and free Fura-2 and enables the precise detection of cytosolic Ca2+ concentrations, which is only limited by the Ca2+ response of Fura-2. Using this illuminator, we have shown that Fura-2 acetoxymethyl ester (AM) concentrations as low as 250 nM can be used to detect induced Ca2+ events in tsA-201 cells and while utilizing the 150 s switching speeds available, it was possible to image spontaneous Ca2+ transients in hippocampal neurons at a rate of 24.39 Hz that were blunted or absent at typical 0.5 Hz acquisition rates. Overall, the sensitivity and acquisition speeds available using this LED illuminator significantly improves the temporal resolution that can be obtained in comparison to current systems and supports optical imaging of fast Ca2+ events using Fura-2.

neuroscience↗