Comet assay microscopy hints & tips

Acquiring a good quality source from your microscope will give you consistent and reliable results. When using advanced image analysis software such as Comet Assay IV to score cells, a bright image with a large field of view will help speed up the scoring process.

Choice of Stain

The majority of our customers use one of the following fluorochromes / stains for their comet slides:

• Ethidium bromide
• Propidium iodide
• SYBR Green
• SYBR Gold

Recently there has been an increasing trend towards the use of SYBR Gold which offers very high sensitivity with the added benefit that it does not carry the same level of mutagenic risk as stains such as Ethidium bromide. As far as Comet Assay IV is concerned any stain is acceptable.

Due to the gradual fading of comets, try to standardise the time spent focusing comets for analysis and if necessary consider the use of anti-fading agents such as Vectashield to help prevent or at least reduce excessive fading.

Microscope Setup

Slide preparation is of course key to successful comet assay scoring but it is also important to optimise the image presented to the camera from the microscope.

Illumination

It goes without saying that the correct excitation/barrier filter set must be selected specific for the particular fluorochrome that you are using to stain your slides. Take care to close the shutter to block the light source reaching the filter block when it is not in use otherwise it may cause damage and impair excitation.

It is critically important to ensure that the maximum amount of light is focussed through the objective lens on to the comet slide. Ensure that the working life of the light source has not been exceeded and fine tune the focus and alignment following the instructions given in the microscope’s User manual. It is usually well worth having the microscope regularly serviced and the engineer will be able to optimise the light path for you.

In order to minimise and prevent fluctuations in electronic background noise, ensure that there is a constant, stable power supply to the microscope.

If the image is excessively bright try using the microscope splitter so that perhaps only 50% is directed to the camera, or place a neutral density filter in the light path to the camera.

Some microscopes emit relatively high levels of infra red light from their light source and this can be detected by video cameras which often are capable of detecting light well beyond the visible spectrum, into infra-red. Although undetectable by the naked eye when looking through the microscope’s eyepiece, unless infra-red light is adequately blocked it can lead to a lack of contrast in the video image due to an increase in background brightness. This potential problem is usually prevented by an infra red filter being placed in the light path to the camera.

C-mount adapter

We advocate the use of specialist wide-field C-mount adapters to connect the Comet assay IV camera to the camera port of the microscope, this usually being the trinocular phototube. Wide-field adapters are advantageous in that they focus a greater amount of light to the camera’s sensor and provide a larger field of view than a standard 1x C-mount, allowing multiple comets can be viewed and scored in a single field of view.

We recommend a 0.5x or 0.55x C-mount is used in conjunction with the Comet Assay IV camera which has a 0.5 inch CCD sensor.

The following two tables show how field of view and transmission vary with different magnifications.

Field of View

Transmission

The use of a C-mount adapter can help significantly boost the amount of light reaching the camera sensor. The list below shows how different lens magnifications affect light transmission:

• 0.75x adapter gives 80% transmission increase when compared to a 1x adapter.
• 0.63x adapter gives 150% transmission increase when compared to a 1x adapter.
• 0.55x adapter gives 230% transmission increase when compared to a 1x adapter.
• 0.50x adapter gives 300% transmission increase when compared to a 1x adapter.
• 0.38x adapter gives 615% transmission increase when compared to a 1x adapter.

Choice of objective magnification

The majority of Comet Assay customers use objectives with 20x or 40x magnification and both are well suited to the assay. Ideally you should use high quality objectives specially designed for fluorescence applications. In comparison with a 40x objective, a 20x will provide a larger field of view enabling more comets to be viewed at the same time. Conversely, using a 40x objective will display fewer comets on screen but allows for measurements at higher resolution. The size of cell will also be an important factor in determining which magnification is most suitable.

Another consideration is brightness, in epi-fluorescence microscopy, two factors come into play. Brightness is proportional to the fourth power of the objective numerical aperture but inversely proportional to objective magnification squared. Whatever magnification is chosen, you should choose an objective with sufficiently high numerical aperture to provide a sufficiently bright image.

Useful microscopy links

• Zeiss' Microscopy from the very beginning
• Leica Microsystems fluorescence centre
• Olympus guide to fluorescence microscopy
• Olympus microscopy knowledgebase
• Nikon MicroscopyU: Introduction to fluorescence microscopy
• Nikon general advice on filters