Part 1 Principles
Confocal Microscopy tutorial
Part 3 operation, optimization of Leica SP2 LSCM
Average and Accumulation
As discussed above, using Gain and offset to balance the signal intensity and background noise level is often insufficient. And using high settings of some other parameters such as scan speed, scan format, pinhole reduces signal influx and affect SNR and image quality adversely. This side effect can not be compensated by Gain and offset of PMT. In these cased, average is a good option to reduce noise, increase pixel time and compensates the lowered SNR, thus improve image quality.
Average takes the sum of pixel values from the specified number of scan and use arithmetic mean as the final value in the image. Average preserves those persistent pixel values that are mostly specific signal while divide away along times those fluctuated values that are mostly noise; Average also extends the time of scan thus extends the pixel time and improve SNR. So, average reduces noise and improve image quality significantly. Average does not suffer from pixel saturation since the it divides the sum into mean.
Average has two forms, line average and frame
average, both have same effect but are executed differently. Line
average takes specified number of scan first on the same line then go to next
line, repeat for all lines in a frame and finally produces the frame. Frame average
takes the whole frame first then repeat scan the specified number of frames,
each frame is added consecutively, the result is shown immediately after each
addition. Frame average is used more often in frame based acquisition since user
can see the image quality improve progressively after each frame is acquired.
Frame accumulation also takes specified number of scan and adds up the pixel values, but it use the sum as final value in the image. Obviously, accumulation increase signal intensity and extends pixel time thus improve SNR. But accumulation adds both signal and noise thus does not have the effect of preserving persistent signal while canceling signal with varied intensity. Besides, accumulation suffer from saturation on those high intensity value pixels.
In extreme difficult case, line average and frame accumulation can be combined to reduce noise, increase signal intensity, increase SNR and improve image quality. (frame average and accumulation can not be used at the same time because they reject mutually ).
This page was last updated 23.03.2004