ScanImage 2018 includes a new photon counting module, which uses a fast digitizer (>1GHz) to directly sample the output of the transimpedance amplifier. ScanImage analyzes the voltage signal and performs a peak detection on the signal to count photons in real time. By synchronizing the fast digitizer to the sample clock, time correlated photon counting is supported. This allows ScanImage to bin detected photons into virtual output channels depending on their arrival time.
The fast digitizer can be operated in photo current integration mode, while the photon counting is active. This allows to display the photo current integration signal and compare the photon counted signal at the same time to get a direct comparison between the two modes.
Photon counting is most useful for photon limited experiments. This means that less than 1 or less photons are expected to arrive at the PMT per laser pulse. In this regime, photon peaks are clearly separated in the signal. Once the photon rate rises, photon responses start to overlap and the photon discrimination will count multiple photons as a single event. At this point it is advised to switch to photo current integration mode.
Since ScanImage can perform photo current integration and photon counting at the same time, it is possible to use photon integration for bright regions, and photon counting for photon limited regions in the sample. This maximizes the dynamic range of the microscope, and provides an effective way of noise suppression in the low signal regions.
See Photon Counting Controls for more information on the graphical user interface.
The following hardware is required to use ScanImage's photon counting software module:
- National Instruments NI5771 fast digitizer and an NI PXIe-7975 FPGA module
- A fast transimpedance amplifier (bandwidth >80MHz) (such as the Femto DHPCA-100)
- (optional) For time correlated photon counting, a clock multiplier board such as the AD9516-0 is required
Principle of Operation
For each of the two physical input channels, ScanImage implements two processing pipelines: A photon integration pipeline, and a photon counting pipeline. The output of these pipelines can be mapped to ScanImage channels by the user such that the same sample can be recorded both in photo current integration mode and in photon counting mode at the same time.
The photon counting pipeline implements the following stages:
|Pipeline stage||Photo Current|
|1||Offset subtraction and scaler||x||x|
|2||Centered differentiation filter||x|
|3||Programmable FIR filter||x|
|4||Programmable Photon Discriminator||x|
The offset subtraction and scaler applies to the photon integration pipeline and the photon counting pipeline. The remaining stages apply to the photon counting pipeline only. The centered differentiation filter can be used to remove low frequency noise from the signal. The programmable FIR filter can be used to remove high frequency noise from the signal. The photon discriminator uses a sliding window and a debounce filter to discriminate photons (see figure).