Equipment
 

The Photon Imaging Microscope systems

Each of our Photon Imaging Microscope systems rely on an ultra-sensitive light detector (called an imaging photon detector or IPD, Photek Ltd.), which has been attached to a Zeiss Axiovert 100TV microscope so that the photons of light emitted from cells injected with aequorin can be visualised (Miller et al., 1994).  We use a water-cooled thermoelectric system that maintains the detector at approximately –12°C, and dry air (with a –100 F dew point), is continually blown over its detection surface.  In addition, the systems are mounted in dark boxes to protect the detectors from the ambient light during photon collection.  The microscopes’ stage and focus are both under computer control.  This enables remote control of the sample position and microscope focus while the dark box remains closed.  Aequorin luminescence can be imaged continually; however, it may be interrupted periodically to capture bright-field and fluorescent images, thus allowing the correlation of luminescence with the changing morphology of the embryo.
Microscope automation and data acquisition are controlled by a custom-built application called Ipd for Windows 2k (IPDWIN2k; Science Wares Inc.).  The photon image data for an entire experiment consists of 2 files: a list of photon coordinate (x,y) pairs and a list that links each coordinate pair with a time tag.  The bright-field images are collected by a DAGE MTI CCD-300 camera and the video signal output is digitised by a frame grabber into 768 x 576 pixel 8-bit greyscale images, and stored as .TIFF files.
On completion of data acquisition, the IPDWIN2k software is also used to review the acquired photon image data with any chosen integration periods and time steps, and also to review the photon, bright-field and fluorescent image data simultaneously.  The photon events can be correlated with morphological features by superimposing the photon images on the captured bright-field/fluorescent images.
General information about imaging [Ca2+]i with aequorin using a Photon Imaging Detector, and a more detailed description about our system can be found in Miller et al., 1994 and Webb et al., 1997, respectively.