Rapid Refocusing System

The endothelial and smooth muscle cell layers of an artery are intrinsically linked via rapid communication.  However, study of the interplay between these two layers is limited due to the difficulty in imaging the two layers at a high enough frame rate. 
Rapid multi-plane imaging is often required when imaging dynamic processes at high magnification in live samples.  However, movement of the sample or immersion medium necessary to image these planes can blur the images and disturb the sample.  Remote refocussing microscopy provides a simple method of imaging two focal depths of a sample by carrying out the focal displacement elsewhere on the bench.

A simple solution to image two planes in succession rapidly is to move the sample or the imaging objective up and down.

However, these methods are generally quite slow and may cause disturbance of the sample

We use a technique where the refocussing is carried out elsewhere in the optical train


Our system is built around a conventional epi-fluorescence microscope.  We have added:

1. A 4-colour laser (Vortran Laser Technology) to excite different fluorophores in different layers

2. An oscillating stage coupled to a mirror which oscillates between two focal planes of a sample (click to find out more)

3. A camera (Photometrics) synced to the stage to take exposures only at the two focal planes.


Rapid refocussing videos

Results - fixed tissue samples

Via dyeing the vascular smooth muscle and endothelial cell layers with different dyes, we may illuminate each one separately and combine the two images later.

The images below show the smooth muscle layer (false coloured red), where the cells are perpendicular to the endothelial cell layers (false coloured green).  Through combining the two images it is possible to see where the cells overlap in each layer (scalebars are 20 microns).


Fixed vascular smooth muscle tissue, dyed with Propidium Iodide and illuminated at 488nm


Fixed endothelial tissue, dyed with Sytogreen and illuminated at 561nm

Combination of the previous two images


Results - live tissue samples


(Hover over images for brightfield)

Live endothelial tissue, dyed with Sytogreen and illuminated at 561nm

Fixed vascular smooth muscle tissue, displaying autofluorescence


Sequential imaging of the two vascular tissue layers (separated by 15 microns) of a live carotid artery with our system allows for calcium signalling to be quantified in each layer.  Each trace is taken from the circular regions of interest shown in the images on the left.  Solid lines represent signals from the endothelial layer and dotted lines represent signals from the smooth muscle layer.  ACh (acetylcholine) was added at 5.5 seconds and the F/F0 signal plotted over time.