Calibration

Optical microscopy serves as a standard tool in various research fields. For life sciences, it enables selective labelling of specific targets, to track their spatial organization and/or time dependence in a non-destructive manner. Various developments in the field, either in the imaging itself, sample labelling, or image analysis continuously increase the amount and accuracy of the information derived.

Despite their relative simple operation, optical microscopes contain many components, optical, mechanical and electronic. Therefore, the user should consider not only the scientific reliability of the system imaged (by imaging relevant controls, verifying different dyes – See Sample Preparation) but also the technical alignment of these different components. These considerations are of absolute necessity in order to derive conclusions based on the biology of the sample images, rather than ones reflecting the inherent technical variability. Here you can find information that can help you to determine if the phenotypes observed are independent of technical issues: If its verifying localization accuracy, laser intensity or correcting aberrations.

Remember! Always keep in mind that your image is a result of interaction between light (passing through various optical/mechanical/electronic components) and the chosen label for your target- and accordingly requires careful interpretation.

*KNIC offers a range of accessories to provide calibration data! 

Here you can find useful resources to help you determine whether the phenotypes observed are independent of technical issues: 

• A common criteria for illumination stability is <10% variability over long term (hours)/ <3% variability over short (min). you can read more at: Quality Assurance Testing for Modern Optical Imaging Systems. Microscopy and Microanalysis.
• Avoid cross talk! Make sure you image a co-labelled sample with the same excitation intensities of each single fluorophores! Check your microscope efficiency in separating emissions and plan your labelling accordingly. 
  *when using confocal- don’t forget to check the effect of pinhole size on cross talk! 
  *Read more about spectral bleed-through here (Olympus) or here (SVI).
  *How to localize cellular structures: A guided tour into subcellular colocalization analysis in light microscopy.
• Aberrations modify the shape of your imaged target, and you need to find their source and correct for it early in your imaging! Read more about it here (Olympus) and here (Evaluating optical aberrations using fluorescent microspheres: methods, analysis, and corrective actions).
• And if you already have your bead slide for calibration on the microscope stage, why not evaluate the point spread function? Further reading: Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control and ConfocalCheck - A Software Tool for the Automated Monitoring of Confocal Microscope Performance.
• Determination of the Pixel Size.
• Camera performance.