The probe system has been designed for applications where fluorescence measurements are to be made from small areas of the field of view, thus allowing multiple photometry recordings to be captured from a single sample. The size of each region is defined by the light guide probe diameter, with the ability to position up to four probes in the field of view.
The probe mount replaces the standard photomultiplier tube on the single emission coupling box and places the inputs of fibre optic light guides at the primary image plane. These inputs can be moved within the field of view to the point of interest, their precise location in the microscope field of view being identified by means of reverse infra-red illumination of the guides, which is visible on the CCD camera image. The outputs of the fibre optic guides are fed to individual photomultiplier tubes for acquisition of the fluorescence signal. The number of probes connected to the probe mount can be varied from one to four, and for added flexibility each probe can be used to record at a different wavelength.
Set-up for simultaneous photometric measurements from two areas of interest within a microscope field of view:
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The following is an extract from a 1995 Physiological Society Demonstration:
Simultaneous high-speed photometric fluorescence measurements on several independently selectable regions of a microscope image
J. Graham, M.V. Thomas and Peter M. Smith*
Cairn Research Ltd., Brents Shipyard, Faversham, Kent, ME13 7DZ and *Clinical Dental Sciences / Physiology, University of Liverpool, Liverpool L69 3BXPrevious work using ratiometric imaging techniques has demonstrated the importance of resolving both temporal and spatial aspects of the mechanisms by which Ca2+ mobilization regulates secretory (Thorn et al 1993) and other cellular processes. However, CCD camera based imaging systems can only record data for short continuous periods of less than an few minutes, at the relatively poor time resolution of video frame rate. Photometric systems based on a single photomultiplier tube (PMT) have no such limitations and can record data practically indefinitely with much greater time resolution. Unfortunately, conventional PMT based systems cannot provide any information concerning the spatial aspects of [Ca2+]i mobilization.
We demonstrate here a PMT based photometric system which will allow measurement of [Ca2+]i from up to four discrete areas within the field of view with spatial resolution sufficient to discriminate between the apical and basolateral poles of exocrine acinar cells. In this system, light from the real microscope image is refocussed to form a second image, and a number of independently movable fibre optic probes in the second image plane transfer light from discrete regions of the image to individual PMTs. The image and the position of the probes may be observed (and recorded to videotape) simultaneously with fluorescence measurements using infrared illumination and an inexpensive CCD camera. A dichroic mirror is used to split the light from the microscope image into its fluorescence (reflected to the probes) and infrared (transmitted to the camera) components. The positions of the probes in the image can be seen because they are each illuminated from the photomultiplier end by an infrared LED which is reflected by the dichroic back down the path followed by the fluorescence light to form focussed images of the probe openings. A glass coverslip in the microscope image plane reflects back a small proportion of the probe image light to the CCD camera allowing images of the probes to be seen superimposed on the microscope image. The design retains high optical efficiency for the fluorescence signals and automatically gives correct alignment of the probe and microscope images. A variant of this system, which additionally allows fluorescence measurements to be made on the entire image, will also be demonstrated.
References
Thorn P., Lawrie, A.M., Smith, P.M., Gallacher, D.V. & Petersen, O.H. (1993) Cell 74 661-668
