1.5   The Noise Rubric

Obtaining a bright confocal image depends on several factors: 
dye concentration <-> laser power <-> pinhole size <-> PMT voltage <-> signal averaging
Each one of these factors can be optimized and involve practical limitations and compromises.  

• 1.5.1   Dye concentration
  • Dye concentration is determined by the concentration of epitope in the tissue, affinity of the primary and secondary antibodies.  Some fluorophores have better quantum yields than others and are more resistant to photo bleaching.  
• 1.5.2   Laser power
  • More power gives less noise
  • More power increases the rate of photo-bleaching
  • More power increases risk of signal bleed through
• 1.5.3   Pin-hole size
  • Larger pin hole lets more light reach the PMT detector but thickens the optical section.  This gives poorer resolution in the z-axis.  
  • Smaller pin hole sizes down to about 0.5 Airy disk units gives better axial & x-y resolution and thinner optical section, but with less of light sent to the detector.  Pin hole size less than about 0.5 Airy disk units will give smaller optical thickness but will not improve x-y resolution significantly.  Below a certain pinhole size alignment of the pinhole to the optical path axis becomes critical.  Making the pinhole smaller than this value will just decrease light to the PMT without any improvement of  axial or x-y plane resolution.
• 1.5.4   PMT Gain
  • PMT gain (or voltage) will increase the optical signal sent to the video frame grabber (A/D converter).  PMT gain will roughly linearly increase the photonic current but will exponentially increase electronic noise.  With weak fluorescent signals the signal to noise ratio can become very poor above a PMT gain of 800 Volts.  High PMT gain may be avoided by increasing the laser power &/or opening the pin hole. 
• 1.5.5.  Signal Averaging/Integration
  • Signal averaging or integration is done by adding scans of the same view together and dividing the result for each pixel by the number of scans.  Noise being random, will approach zero with increasing number of scans averaged, while the signal being a finite value will approach the finite value.  Signal to noise ratio will decrease in proportion to the square root of the number of scans performed.  e.g. Averaging four scans will yield an image with half the noise compared to a single scan image.

Last updated 07/31/03

					© 1998-2003 Michael Chua, Cell & Molecular Physiology, UNC.  All rights reserved.
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