The software saves .tif stacks with format int16. In our experiments, the minimum value of these images is typically around -100 to -150 (with the odd outlier a bit lower).

For reference, max image values for these scans were ~10,000. The dynamic range of the data appears to be limited to a fraction of what is available with a 16bit integer.

Is this correct behavior?


The pixel values are determined by the resolution of the digitizer. The data format used is int16 (signed).

  • The NI 5732 is a 2-Channel 14-bit digitizer, producing pixel values within the interval [-2^13, 2^13-1]
  • The NI 5734 is a 4-channel 16-bit digitizer, producing pixel values within the interval [-2^15, 2^15-1]

The reason a signed integer is used, is because the voltage values range from -1 to +1. The digitizer produces integer values that ultimately map to some voltage value within that range.

The Digitizers mentioned above spew out raw information; thus, the digitizer to voltage mappings are not calibrated by the hardware vendor.

This means that when your digitizer registers zero, it may not exactly map to a zero voltage.

In addition, the PMT may exhibit dark noise. In this case, dark noise relates to how much light comes in when no signal is being received (unless
you completely block the lens, there is always some measure of light present). This would cause a value to register even when no photons are present.

This is where the ScanImage channel offsets can be used to perform a raw calibration of your equipment.

  1. On the Channels Page, make sure the 'Auto Read' checkbox is checked.
  2. On the Channels Page, make sure the 'Subtract Offset' checkbox for each Channel is checked.
  3. On the Main Controls Page, press the FOCUS button. This will automatically set the Offset.

Keep in mind that the Offset value can slightly vary for each iteration of FOCUS (or 'Read Offsets' on the Channels Page). This is
because the digitizer always has some noise. To account for this, ScanImage reads in and averages the first 100 values.


Useful ScanImage Links:

  1. Main Controls Page
  2. Channels Page
  3. Supported Microscope Hardware