Four simple recommendations that can greatly improve your experience at the microscope:
Use #1.5 coverslips and place them carefully
Most microscope objectives are designed to work with #1.5 (0.17mm thick). Using a coverslip of a different thickness can result in aberrations and loss of signal. This is especially the case for objectives with a numerical aperture (NA) greater than 0.65.
Some objectives, particularly high-performance water, oil, and silicone immersion objectives have correction collars to adjust for different coverslip thicknesses.
If using an inverted microscope, be careful where you place your coverslips, if they are near the edge they could end up tilting your slide when mounted in the stage.
High-precision coverslips can be purchased from a variety of suppliers including ThorLabs and Zeiss.
For more information on coverslip thickness visit Nikon’s MicroscopyU article on coverslips.
Spin down antibodies before use
It's best for all antibodies (primary and secondary) to be spun down before use. If they are not spun down, you may observe bright floating and stationary aggregates in your samples.
Antibodies should be prepared in blocking buffer (e.g. PBS + BSA). This solution should be vortexed, then spun down in microcentrifuge ~10,000 rpm for 10 minutes. This moves aggregates to the bottom of the tube. Use the solution from the top of the tube.
Use the right microscope objective for your sample and experiment
High NA oil immersion objectives are great for applications close to the coverslip, but if you’re working in tissue you may be better off using a water or silicone oil immersion objective to match the refractive index of your sample and avoid loss of signal and resolution from spherical aberration.
Cellular Imaging has a variety of objectives for different applications and we can help match you with the right instrument and objective.
Use mounting media suitable for your application and microscope objective
Hard setting mounting media can be good for long-term storage, but be aware that it can shrink and deform your samples. Aqueous media can be a better choice if preserving the 3D structure of your sample is important.
If you are using an oil objective to image tissue or thick samples, consider mounting media with a refractive index closer to oil. This will allow you to image deeper.
Avoid mounting media that comes with DAPI included.
