Dr Vipoir has a look at spinning disk technology

It’s been a while since we had any new microscopes in the lab and I’ve been thinking about buying a new confocal microscope. The advantage of all forms of confocal microscopy is the clear image quality, free from out of focus blur. This means that confocal images have an increased resolution compared to conventional, wide field microscopy, which is good for my research.

So just what is the difference between the two techniques? Well, in a wide field microscope, the sample is completely illuminated by the excitation light, so all of the sample is fluorescing. This contributes to a background haze in the resulting image. In a confocal microscope, the excitation light is provided by a laser and the emission light has to pass through a pinhole before it reaches the detector.

Adding a pinhole solves the problem of background haze. Because the focal point of the objective lens of the microscope forms an image where the pinhole is, they are said to be conjugate planes. The pinhole is conjugate to the focal point of the lens, thus it is a confocal pinhole.

However we’re worried about the toxicity of the laser illumination light to our live cells and so wish to minimize this. We also want to increase the speed of our image capture, as this will help maintain the viability of our cells; if we can acquire the images faster the cells will be exposed to the light for less time, and this will also mean that we can image those exciting kinetic events, the details of which have so far eluded us.

So for these reasons we’re looking at getting a spinning disk confocal microscope.

In a laser scanning confocal microscope, the laser scans across the sample using one pinhole in order to build up the image. In contrast, the spinning disk confocal uses a rotating disk with thousands of pinholes arranged in a spiral pattern. A second disk with a corresponding pattern of microlenses increases the efficiency of illumination and therefore the suitability of this technique for fluorescent samples.

As light is projected on to the disk the holes trace concentric arcs of excitation light across the sample. When the fluorescent or reflected light returns through the disk only light from the plane of focus makes it past the holes. Light from above or below the focal plane of the objective comes to the disk at an angle. Only light which comes to the pinholes at right angles is able to penetrate and therefore reach the detector.

Rather clever, don’t you think?

The spinning disk confocal microscope therefore collects multiple points simultaneously rather than scanning a single point at a time, which means that the technique is both faster and hits the sample with a lower dose of laser light. A high sensitivity CCD camera leads to the possibility of blistering fast frame rates. So high speed imaging together with surprisingly little photobleaching makes this tool is ideal for my experiments with live cells.

This is definitely a must have if I’m going to achieve that Nobel Prize this year! Now, where did I put my credit card…

Images acquired with the Volocity Spinning Disk Confocal

Live cell imaging of phagocytosis performed by a mouse macrophage. Hospital for Sick Children, Toronto. Drosophila cells in mitosis. UCSF.

Movie – Drosophila embryo development. UCSF.

Drosophila embryo movie (8.42 MB)