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Tips and tricks to get the most out of your imaging#

Clean your coverslips before putting them on the scope#

Example

Coverslips after mounting
1. Dirty coverslips after mounting

Coverslips after dabbing with H2O
2. After cleaning with water, the coverslip on the right still shows residue

Coverslips after using EtOH and glass cleaner
3. After wiping with EtOH and glass cleaner, coverslips are clean

Keep coverslips centered as much as possible.

  • If coverslips are too close to the edge, your coverslip may be un-imageable.
  • Coverslips close to the edge also increase the risk of the objective hitting a stage insert.

Beware of photobleaching#

Don't expose your sample to any more light than absolutely necessary.

  • Use the least amount of SOLA/laser power needed and close the shutter when not needed.
  • Set z-limits and/or multi-point positions using an OC with low laser power.
  • Doubling the laser power more than doubles the potential for photobleaching.

Avoid mounting media with DAPI/Hoechst#

  • Both DAPI and Hoechst photoconvert with UV light into green-emitting and red-emitting forms.
  • There'll be increased background (noticeable on widefield but not as much on confocal)

Know where your objective is at all times#

  • Have an idea of the rough z-position for your sample and keep an eye on the z-position (preferably using the joystick pad display).
  • Make sure you aren't at risk of hitting the stage (insert) with the objective.

Lower immersion objectives if switching between wells or coverslips#

  • To prevent possible objective lens damage, either use the Escape functionality or lower the objectives manually

Focusing on your sample#

  • Ensure that the stage insert and sample are positioned and secured properly.
  • If using oil immersion objectives, raise the objective while watching for oil contact.
  • Use the eyepieces when finding your sample.

Avoid objective lens damage during focusing

One easy way to damage objectives is to raise them too high. The risk is not only to the objective that you're using--but also for the adjacent ones since they can hit the stage.