Optimizing your imaging parameters

Imaging projects must, by necessity, always be a compromise. To gain better images you must sacrifice time, and vice versa. You must approach this decision at two levels.

Firstly the level of the microscope itself. Consider carefully what it can do and how it will affect your ability to answer your research question. Different microscopes have different advantages and disadvantages and the microscope should never be chosen for a project merely because 'someone you know is using it' or because 'you have used before'.

Secondly, you must consider the choice of parameters. Choice of objective must be considered carefully. Lower magnification objectives have larger fields of view allowing you to acquire data faster. Meanwhile higher magnification objectives allow superior resolution with the cost of having a smaller field of view and shorter working distance. Imaging speed and exposure times must be chosen carefully to ensure that the recorded data remains usable. On confocal microscopes the detector gain must be adjusted according to your sample to avoid overexposure while making sure that the signal to noise ratio remains adequate. Also you should never forget that choices made during sample preparation are part of the parameters you can and should change if the need arises.

The concept of worst usable image

While it is rather easy to determine the 'optimal' imaging settings for a given objective as far as physics and resolution are concerned, there is no guarantee that these 'optimal' settings are actually optimal for you. Because both your time and instrument time have a cost, the actual optimal is usually 'the worst image that still gives you the answers you need' - that is to say, there is a concept of 'worst usable image' that both gives you what you want and can be taken without wasting any time. This optimal is non-trivial to find as it is hard to tell what sort of data you need to get the answers you need. This is why we recommend you spend 2-3 hours on the microscope just to figure out how you will record your data. If and if you have lots of samples, optimizing your imaging parameters to your needs will save both time and money. If you take better images than needed, your data is still useful, but you are wasting time. If you take worse images than what you need, you are getting useless data and still wasting your time. That is to say, it is better to err on the side of caution and take 'bit better' images than strictly necessary. However the image quality should be assessed after the first imaging session - you should always check that the date you have collected is usable before taking more pictures. This is best done by actually running your planned analysis on your data.  Under no  circumstances should you first record the data and then decide how to analyze it. 

Resolution

The resolution of an optical microscope is defined as the shortest distance between two points on a specimen that can still be distinguished by the observer or camera system as separate entities. The better the resolution, the more accurate the microscope can record the morphology of your sample.  It is important to realize that the depth resolution (or z-resolution) is often a lot worse than the x-y resolution and while in optimal case with high magnification the x-y resolution is around 150 nm, the z-resolution is around 600 nm.  This is to say that we can differentiate objects next to each other with much greater precision that objects that lie on top of each other. The optimal resolution and optimal pixel size are connected by Nyquist criteria. Most modern microscopes can calculate this automatically for given field of view. However if and if you plan to deconvolve your image, the actual 'optimal resolution' is smaller than the one that the microscope gives. The optimal resolution for deconvolution should be calculated using Nyquist calculator.  If you are not sure what kind of resolution you need to answer your scientific question, you should take several pictures with different levels of resolution and analyze them to figure out what kind of resolution you actually need to obtain your results.