When father-son duo Hans and Zacharias Janssen invented the first so-called compound microscope in the late 16th century, they probably didn’t understand the extent to which it would fuel modern science, or how future scientists would expand upon it. The lens-based magnification of the compound microscope is still very much the basis of the technology, but the depth at which researchers are now able to image—inclusive of live cells—is staggering. No matter the year, or century for that matter, microscopy is a workhorse technique in the laboratory. Let’s review a few microscopy best practices to keep your technique up-to-par.
Tip 1: Keep it Clean
When working with a compound or stereo microscope, always keep the instrument clean. To clean the lenses, first remove any dust and dirt by using a camel hair brush or canned air. Then, moisten the end of a Q-tip with lens cleaning solution and clean the optical surface using a circular motion. With a dry Q-tip, use the same circular motion to dry the surface. For the lens solution, a combination of Windex and vinegar works well. Use a dust cover to store the microscope when not in use.
Tip 2: Ensure Exposure Time is Correct
When the exposure time is appropriate during fluorescence imaging with a microscope camera, you will see separated peaks related to the background area and bright area. An exposure time that is too short will dim the area and merge the two peaks. In contrast, an exposure time that is too long will saturate the signal and create a sharp cliff at the maximum signal level. Some image acquisition software have an automated adjustment feature that defines the length between signal intensity and displayed brightness.
Tip 3: For Live Cell Imaging, Choose the Right Technique
Biological imaging of sensitive samples requires a technique that can capture images while maintaining the material’s physiological integrity. The method should minimize photobleaching, phototoxicity and other potentially harmful outcomes, while also delivering ultra-sharp resolution. Some of these attributes depend on your sample. For example, if you are working with thick dynamic samples, use spinning disk confocal, multi-photon or light sheet microscopy. For thin dynamic samples, turn to fluorescence widefield, total internal reflection or light sheet. If you are working with thick static samples, utilize point scanning confocal, multi-photon or light sheet microscopy. Finally, if your samples are thin and static, use widefield or super-resolution.
Tip 4: Test for Autofluorescence
Grappling with autofluorescence cuts into precious imaging time when working with live cells. It can also lead to bleaching or phototoxicity. Riboflavin, tryptophan and phenol red are well-known culprits. Therefore, to improve signal to noise, you can reduce non-specific background fluorescence by using a phenol-red-free medium, for example. Regardless of the imaging technique you settle on, be sure to test for autofluorescence beforehand.
Tip 5: Establish Favorable Environmental Conditions
Long-term live cell imaging requires a certain level of control over environmental conditions to maintain sample viability, especially temperature and atmosphere. While most mammalian cells grow at 37°C, if bacterial or yeast cells are used, higher or lower temperatures may be required. If you do not already manually pre-set a proper temperature, alter your workflow to include some type of temperature control step. The same goes for atmospheric control. For mammalian cells, the ideal atmosphere of 5% CO2/95% air should be maintained at all times. Establishing and keeping these environmental conditions allows live cell imaging to occur without the risk of sample degradation.