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 Ultra-sensitive photon counting camera

To study the dynamics of molecules and interactions with putative binding partners we have established an experimental set up for Fluorescence Lifetime Imaging Microscopy (FLIM), which is based on Time and Space Correlated Single Photon Counting (TSCSPC).

The high temporal resolution of the ultrasensitive camera system allows to follow the spreading of light. To demonstrate this feature we made movies of objects, in which the spreading of light is seen as virtual slow motion with a time resolution in the ps-range.

The specialness of this worldwide fastest non-scanning camera is its recording principle. In contrast to CCD, CMOS or Streak cameras, which acquire pixel or line-based image-informations, the camera developed at LIN continuously records single photons by a special image sensor. In other words: light is measured directly in form of individual quanta. From millions of such individually acquired light quanta images are reconstructed subsequently in the computer by using a special software.

In addition to space coordinates and the absolute arrival time of the single photons, the recording principle of the camera allows to acquire further parameters, e.g. the wavelength, polarisation and travel time and grants a post-recording classification of the photons by certain criteria. Sorting recorded photons with respect to their travel time in 50 ps steps (according to the time resolution of the camera) reveals an image sequence with a moving light gap of 1.5 cm width. The distance of 1.5 cm/50 ps is due to the fact that light spreads with a velocity of approximately cm/s. This is roughly 1 million times faster than a bullet from a small bore rifle.

The overlay of the light wavefront (see movie top right), taken by the single photon counting camera (see image top left), with a brigthfield image of the brain model, taken by a digital photo camera, clearly demonstrates that the light hits the brain model 500 ps before the shadow of the model becomes visible at the wall behind the object.

As illumination source repetitive short laser pulses (10 ps) from a ultra short pulse laser were used. Light reflected by the object was registered photon by photon at the detector and its position and travel time stored in form of a table (i.e. list mode storage). Because a single laser pulse does not provide enough energy to collect sufficient photons by the camera, the exposure continued over several minutes during which the stream of photons was registered sequentially and then disposed into 50 ps time channels. Hence, the image sequence represents the averaged information of many laser pulses.

The time-resolving novel camera system is used by Life Sciences for Fluorescence Lifetime Imaging microscopy (FLIM) to study protein-protein interactions via Förster Resonance Energy Transfer (FRET) in biosensors, local pH or ion alterations or metabolic changes (e.g. NADH autofluorescence) in living specimen under low light conditions. Moreover the camera principle could be used for applications in medical technology, particularly to improve PET and SPECT tomography based imaging, where single particle counting is necessary.

Acquisition system and operational principle

The single photon counting detection technique developed in the Special Laboratory is based on the principle of electron multiplication by microchannel plates (MCP). The time and position sensitive detector is a vacuum-sealed photomultiplier tube which houses a photocathode, a stack of microchannel plates for amplification and a position sensitive anode.

An incident photon interacting with the photocathode results with certain probability in an initial electron that is driven towards the MCP amplification stack and gets multiplied. The resulting electron avalanche carries up to 10 million electrons following an applied electrical field and falls on the position sensitive anode.

Schematic of the detector head

25 mm detector head manufactured by ProxiVision, Bensheim, Germany

The widefield FLIM camera system provides everything required for robust and reliable single photon counting based imaging. Realtime event selection logic processes registered photons to avoid artifacts like multi-photon events, MCP noise and pile-up effect.

In addition to position and picosecond time information absolute arrival time (AAT) is acquired with ns precision. The system features a number of additional digital and analog I/O lines available for the user. The values are read and stored along with every detected photon or in custom protocol basis that allows one to extend the set of acquired parameters.


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