Confocal Training Information-User Tips

Confocal Training Information/User Tips

  • If any experiment (such as treatment to tissue culture cells) is necessary for the live imaging, this should be performed by the lab member
  • Bring your own supplies (pipette, tips, and tissue culture media) for the experiment. If confocal supplies (from Ikezu lab) are consumed, it will be charged to the laboratory
  • Strongly recommend following the standard operational procedures(SOPs) for representative experiments, which should contain multiple negative/positive controls. (Experiments without sufficient controls are often a waste of time and effort)
  • For more information, please contact the confocal manager: LeAnn Tiede - 402-559,5509 - ltiede@unmc.edu

Guidelines for Sample Preparation 

  • Glass slides with 170µm-thickness coverglass
  • Cover glass-bottom tissue culture dish for live imaging (MatTek or Willco)
  • Appropriate fluorescent dyes for confocal or fluorescent imaging
  • Appropriate mounting media for fixed samples and dye-free tissue culture media for live imaging
MatTek dishes

 


Limitations

  • If the Signal-to-Noise (S/N) ratio is less than 2, it is difficult to reconstruct acceptable images
  • If the excitation/emission wavelength does not correspond to the laser and filter specifications, it is difficult to obtain maximal intensity from the sample
  • If sample preparation is not adequate (excess mounting media, inappropriate coverglass thickness, inadequate support material), it is difficulty to capture images due to specific focusing depth.

Standard Operational Procedures

  • Basic laser scanning confocal imaging
  • Z-stack for 3D reconstruction and deconvolution
  • Time-course experiment Multiple-positioning experiment
  • Fluo-4 Ca2+ imaging
  • Fura-2 Ca2+ imaging
  • CFP/YFP FRET imaging using Dual/Quadview system

Confocal SOP - pdf file


User Fees* (effective September 1, 2009)

  UNMC**
Consultation/basic training Free
Confocal use (untrained user) $30/hr
Confocal use (trained user) $15/hr
Time lapse studies $10/hr
Time lapse studies (brightfield only) $5/hr
Processing/image analysis $10/hr
Unassisted image analysis Free
   
Supply costs variable

* All fees will be prorated to the nearest half hour
** member of the Department of Pharmacology and Experimental Neuroscience

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