Photon Imaging System
Description

The figure below shows the instrumentation gap filled by single optical photon imaging equipment, which can effectively acquire images from samples producing only a few photons per second.



This system is designed to acquire photon images from bioluminescent and chemiluminescent samples and correlate them with images acquired using standard microscopy techniques such as brightfield, DIC, epifluorescence, etc. A major advantage of this system over photon imaging systems offered by other vendors is that this system records the time and position of each photon event detected during an experiment. This allows the user to rapidly evaluate the results using any number of image integration times chosen after the experiment.



Block diagram of an example system configuration for intracellular calcium imaging with bioluminescent samples labeled with aequorin. In this example, an IPD-425 resistive anode imaging photon detector from Photek, Limited is mounted on a Zeiss Axiovert 200M microscope. The computer controls the illumination shutters and sliding mirror on the microscope. Photon detection is performed by an IPD Controller with DSP technology from Photek, Limited. A Pentium computer manages data acquisition and review through IPDWin2k software written by Science Wares, Inc. System integration and installation was performed by Science Wares, Inc.





Representative axial view of the changes in intracellular free calcium during first cell division of a zebrafish embryo which has been injected with recombinant f-aequorin (a calcium-specific bioluminescent reporter), and observed using an Imaging Photon Detector. The photon images (Panels 1-21) represent 30 seconds of accumulated light with a 30 second gap between each image. Corresponding brightfield images (Panels 0'-18') were grabbed every 3 minutes. It is possible to see a distinct localized rise in intracellular calcium that accompanies the first appearance of the furrow arc at the surface of the cell (Panel 2). This is followed by two sub-surface slow calcium waves which appear to accompany the outward progression of the leading edges of the furrowing arc (Panels 3-7). As these wave fronts approach the margins of the blastodisc, a more intense calcium signal appears in the central region (Panel 8); again it moves out to the margins of the blastodisc (Panels 9 and 10) but it also moves downwards accompanying the deepening of the furrow as the blastodisc is split in two (Panels 11-15). Eventually, at the end of the first cell division, the levels of calcium return to around pre-cleavage levels (Panels 18-21).

This image and caption are provided courtesy of Dr. Andrew L. Miller, HKUST Calcium-Aequorin Imaging Laboratory, Hong Kong University of Science & Technology, Clearwater Bay, Kowloon,Hong Kong, phone: (852) 2358-7324, fax: (852) 2335-1477, web site: http://home.ust.hk/~aequorin/calcium.htm



The movies below show calcium signalling reported by aequorin during later stage gastrulation in a zebrafish embryo. This data is discussed in Proc. Natl. Acad. Sci. USA 96(1), 157-161:







This animation depicts an Aequorin-Calcium pulse in a 100 micron microvial. The integration time for each image is 2 seconds, with a total of 60 frames comprising the animation. The start time for each image is shifted 0.25 seconds and the total event is seen 3 times faster than it actually occured.
This animation demonstrates the usefulness of the Photon Imaging System for quantifying the response of low light bioluminescent assays

System Components:

Automated inverted or upright microscope
  • Computer controlled stage, focus, shutters and mirrors
  • Two imaging ports - one for the photon detector and one for a CCD camera
  • Color, RGB or monochrome CCD camera, NTSC, PAL, or Direct Digital
  • Resistive Anode Imaging Photon Detector
  • 25mm diameter bialkali or S-20 photocathode, quantum efficiencies over 15%
  • Dynamic range up to 30,000 photons per second
  • Noise down to 20 photons per second (~2x10-5 photons/sec/pixel)

Digital Signal Processing and Direct Memory Access hardware
  • Records time and position of every photon event
  • Spatial resolution 256x256 (typical) up to 1024x1024
  • Temporal resolution down to 1 microsecond
  • Digital pulse height distribution filter
  • Digital photon image scale and offset adjustments

Windows 95/98/Me/NT/2000/XP Software
  • Automated, scheduled acquisition of photon images and CCD camera images
  • Programmable fluorescence and brightfield acquisition parameters
  • Programmable scans controlling stage position and focus
  • Replay of photon images at high or low speed with variable image integration times
  • Synchronized display of photon images and CCD camera images, side-by-side or overlay

Other Equipment
  • Custom floor standing dark box - anodized aluminum with a sliding front door
  • Fiber optic system for transmitted light and epi-illumination sources
  • Electronic shutter controls and mounts



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Maintained by Anastasia Karplus
email: sia@sciencewares.com
last updated 1/18/2005
Copyright © 2005 Science Wares, Inc., all rights reserved.