Confocal Microscopy

The principle of confocal microscopy is to use lasers, a pinhole and dichroic mirror(s) to get a precise image of your proteins, cells, tissues, or even animals (very tiny) without interference from light outside the narrow plane of focus. 

The confocal uses fluorescence for visualization; non-fluorescent samples cannot be imaged (unless you can use their autofluorescence to see what you are interested in).

The Waisman Center CMN Core has a Nikon C1 laser scanning confocal microscope with three lasers:

  • one argon laser, exciting at 488 nm
  • one HeNe laser, exciting at 546 nm
  • one HeNe laser, exciting at 633 nm

Three PMTs collect the light emission using filters:

  • one at 515/30
  • one at 590/40
  • one 650 long pass

Examples of fluorophores typically used with the confocal are:

  • FITC/GFP/Alexa488/Cy2
  • Rhodamine/Texas Red/Alexa546/propidium iodide/Cy3
  • Cy5/Alexa633

We do have a filter cube for DAPI that would allow you to use DAPI to know where you are in the tissue, but the confocal cannot take images of DAPI fluorescence.  We do NOT have a filter cube for you to visualize the far red channel (Cy5) before using the confocal lasers, because the human eye cannot see that wavelength. 
We should be able to see Q Dots with the confocal, but we have not yet tried to do so; please let me know how it goes if you are planning to try this.

Here are some fluorophore resources and confocal microscopy resources.