Confocal imaging of a tick

I recently came back from the east coast with an unwanted guest attached to me: a tick, probably a lone star tick, Amblyomma americanum. After removing it, I decided to have some fun with it – I dehydrated it in methanol, cleared it in methyl salicylate, and then imaged it on our spinning disk confocal. The movie below is stitched from four images, and is about 1.8 mm on a side and 1.2 mm thick. Total image size is ~2800 x 2800 x 306. The fluorescence is endogenous autofluorescence excited at 488 and 561 nm. This is probably a nymphal tick, and it looks like the mouthparts are missing.

Nice GCaMP movie

One of the labs we did in this year’s QB3/UCSF Microscopy course was to image cells transfected with a labeled β2-adrenergic receptor and with the calcium reporter GCaMP. Addition of a β2-receptor agonist triggers signaling, leading to calcium influx, reported by GCaMP, and receptor internalization, which can be seen as clustering of the labelled receptors.  This experiment generated some very pretty movies. I’m showing one here, a time lapse acquired on a Spectral Applied Research Diskovery system, operated in spinning disk confocal mode. The first movie is the full field of view, as captured on an Andor Zyla 4.2. The first movie shows the full 2k x 2k field of view, downsampled. The second movie show a full resolution crop from the movie.

Photobleaching and Photoactivation

A few months ago, we purchased a Rapp Optoelectronics galvo-scanning system, along with 405 and 473 nm lasers from Vortran Laser Technology, to provide a photoactivation and photobleaching system for our high speed widefield system. This system is capable of photoactivating or photoconverting any protein that is switched by 405nm light (which is most of them) and photobleaching GFP, while simultaneously acquiring in the GFP, RFP, and Cy5 channels. The entire system is controlled through Micro-Manager.

Today, with help from Nico Stuurman, we took it out for a spin. Nico provided Drosophila S2 cells with either mEos2 or GFP-tubulin as well as help getting everything set up correctly. Here are two videos demonstrating what it can do. Both were acquired on a widefield microscope with a 100x / 1.4 NA objective.

Photoconversion of mEos2-labeled tubulin in the spindle of a Drosophila S2 cell. The video is sped up 20-fold from real time.
Photobleaching of GFP-labeled tubulin in a Drosophila S2 cell. First, a GFP aggregate is bleached, which rapidly recovers. Second, nearby microtubules are bleached, which do not recover over the same time scale. The acquisition doesn't stop during the bleaching, so you can see the bright flash as the bleaching laser is turned on. The video is sped up 1.5x from real time.

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