When Should I Use Imaging Instead of Photometry?

The scientist who wishes to measure ion or analyte concentration in a preparation must first decide if the information would best be obtained by photometry or by video imaging, as the equipment appropriate for each of these is significantly different. The main issues are cost and spatiotemporal resolution.

In photometry, the light detector is a photomultiplier tube or photodiode. A pinhole or aperture is positioned so that the emitted fluorescent light from a single Region of Interest (ROI) passes through it and falls on the detector surface. The detector measures the total light intensity over the ROI.

In imaging, usually the entire field of view passes into a camera containing a rectangular array of at least about 500 by 500 sensors; there is no ROI pinhole. The intensity of light over each element of the array is read out at a rate of 30 images per second for video cameras and at rates from 1 to 30 image/s in digital cameras, though greater rates are possible over smaller ROIs.

Photometry has several advantages over imaging. The detector can cost less than a camera, and the sensitivity of a photomultiplier tube can be adjusted over a very wide range of light levels simply by changing the supply voltage to the tube. Photometry also allows a higher rate of measurement; this is because all the light over the ROI is measured by one detector, whereas with a camera the light is spread over a large number of sensor elements, and applications are often light-starved. Therefore photometry has better temporal resolution and typically higher signal to noise ratios at very low light levels. Higher sampling rates can be useful; over 1000 samples/s may be required to monitor fast changes in ion concentration or up to 10,000 samples/s for fast changes in membrane potential.

Imaging has the fundamental advantage over photometry of allowing simultaneous monitoring over a large number of Regions of Interest (ROIs), unlike photometry that can monitor only one ROI at a time. Therefore imaging has better spatial resolution. Imaging requires a camera, which is usually more expensive than a simple photometer. The choice of camera will depend on the expected light levels and the required number of measurements per second. For example, cells that load moderately well with dye and whose [Ca⁺⁺] responses are not too fast can be monitored very effectively using a CCD camera with on-chip image integration that costs under $2,000. At the high end of cost are cameras with scientific grade CCD sensors, image intensification and cooling in the $10,000 to $35,000 range. At higher light levels, the signal to noise ratio of the camera output will exceed that of the photometer due to the typically higher quantum efficiency of most cameras over photometers