Teledyne Princeton Instruments PI-MTE, System Manual

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Chapter 5 Operation 55
5.5.2.2 Software Binning
One limitation of hardware binning is that the shift register pixels and the output node
are typically only 2-3 times the size of imaging pixels. Consequently, if the total charge
binned together exceeds the capacity of the shift register or output node, the data will
be lost.
This restriction strongly limits the number of pixels that may be binned in cases where
there is a small signal superimposed on a large background, such as signals with a large
fluorescence. Ideally, one would like to bin many pixels to increase the S/N ratio of the
weak peaks but this cannot be done because the fluorescence would quickly saturate
the CCD.
The solution is to perform the binning in software. Limited hardware binning may be
used when reading out the CCD. Additional binning is accomplished in software,
producing a result that represents many more photons than was possible using
hardware binning.
Software averaging can improve the S/N ratio by as much as the square root of the
number of scans. Unfortunately, with a high number of scans, i.e., above 100, camera
1/f noise may reduce the actual S/N ratio to slightly below this theoretical value. Also, if
the light source used is photon-flicker limited rather than photon shot-noise limited,
this theoretical signal improvement cannot be fully realized. Again, background
subtraction from the raw data is necessary.
This technique is also useful in high light level experiments, where the camera is again
photon shot-noise limited. Summing multiple pixels in software corresponds to
collecting more photons, and results in a better S/N ratio in the measurement.
5.5.3 Background Subtraction
Each CCD has its own dark charge pattern or background that can be subtracted from
the total acquired signal. By subtracting this background, you can eliminate the dark
charge, which might otherwise hide low-intensity signal.
To set up for background subtraction, set up the experiment conditions for acquiring
the actual image (detector temperature, exposure time, region of interest, timing mode,
etc.) and then, while blocking the incoming signal from the array, acquire a dark charge
“background image” under those conditions. Once the background image is acquired,
save it to disk.
After storing the background to disk, you have two choices for background subtraction:
automatic or post-processing.
• Automatic: This approach requires that you activate Background and specify the
background filename on the Acquisition
►
Experiment Setup
►
Data
Corrections tab page before acquiring an image. When you acquire an image,
the specified background file data will automatically be subtracted from the raw
image data before the corrected data is displayed and is available for storage to
disk.
• Post-Processing: If you prefer to acquire and preserve the raw image data, make
sure that Background is not active on the Acquisition
►
Experiment
Setup
►
Data Corrections tab page. Then, acquire the image, save the raw
image data to disk, and, via the Image Math function, subtract the background
file data from the raw image data. The subsequent data can then be saved to a
separate file.