Light Delivery in Microscopy
Light delivery in Microscopy:
Along with cold LED illumination, Bluebox Optics recommends the use of a good camera along quality filters to achieve the best signal to noise ratio for your image. In many cases for direct illumination fluorescence microscopy, especially live cell imaging, the most effective illumination is to use the lowest optical power that allows you to get a good pictures using the camera you have available, ideally combined with Camera triggering to only illuminate when capturing pictures. Over illumination will lead to photo bleaching and phototoxicity. The money you save buying the niji LED source can be put towards buying a better quality camera or filters! Please contact us for more information.
Photo bleaching occurs when intense light is delivered onto a fluorophore molecule. The light destroys the chemistry of the fluorophore and can quickly lead to fading of the image. The time taken to photobleach varies depending on the specific molecule and can vary from just a “few” photons through to extensive illumination over many hours. Many common dyes like GFP and FITC can photobleach over seconds.
Minimising light exposure
The easiest way to minimise photobleaching is to minimise the samples’ exposure to light. This can be achieved in a number of ways, ideally exposure time should be kept to a minimum using a more sensitive camera if necessary. The niji also has an optional low light aperture to allow low light delivery whilst running the LEDs at a high enough power for them to be stable in their light delivery. This aperture allows roughly 10% of light to be transmitted.
In some circumstances, it may be worth considering accessories such as autofocus stages to allow minimise the time required for focussing or alternatively focussing and checking exposure on a neighbouring part of the slide than that you wish to capture.
The amount of light delivered in fluorescence imaging is often toxic to the cells being imaged. In live cell imaging if very beneficial to keep the total light delivered onto the cell to an absolute minimum. More details can be found
More details can be found at: https://www.thermofisher.com/uk/en/home/life-science/cell-analysis/cell-analysis-learning-center/molecular-probes-school-of-fluorescence/imaging-basics/protocols-troubleshooting/troubleshooting/photoxicity.html