There are currently two practical methods of acquiring very dim images. One is to amplify the image using an image intensifier, the other is to accumulate (integrate) the image over a period of time.

If the image is extremely dim, in the range of 10³ to 10⁵ photons/second/cm², then an intensifier is the clear choice. This is because an intensifier is inherently more sensitive. If the image has a significant movement component, then an intensifier is again the clear choice because it is inherently faster. If the image light level is above 10⁵ photons/second/cm² and the image is stationary, then integration is the clear choice. If the image is moving, then intensification is necessary to prevent blurring from movement during acquisition.

Typically low light level fluorescent images available from a microscope camera port are in the range of 10⁶ to 10⁷ photons/second/cm². The choice of imaging methods then reduces to whether the image has a significant movement component with respect to integration time. At these light levels, typical integration times to accumulate an adequate image can range from a hundred milliseconds to several seconds. The times are influenced by such factors as the susceptibility of the particular dye to photobleaching, the total length of time over which the observations need to be made, the quality of the microscope optics and the sensitivity of the particular camera. Movements of more than several pixels during integration will cause noticeable blurring of the image and result in loss of data from the image. Using a 40x objective and a 2/3 inch integrating CCD camera integrating for 1 second, movements of 10’s of microns can be a problem.

Other factors to consider are:

Intensifiers are expensive, ranging from $10K to $25K. They can be damaged by exposure to bright light. They have shot noise that increases with gain. It is random noise than can be removed by averaging several (typically 4 or 8) successive images together. However averaging can cause blurring of the image if there is significant movement with respect to the averaging time (133ms for 4 averages, 266ms for 8 averages). Intensifiers come in two forms, separate intensifiers which can be mounted to a video camera or a unit combining the intensifier and camera into one unit.

Integration requires a synchronized trigger or gate, usually generated from an imaging program running on a computer. Cooling the CCD array improves the signal-to-noise ratio of integrating cameras. Most CCD cameras are capable of integrating. Many now come with the appropriate controls to do this.

Recommendations:

For intensifiers or intensified cameras, Video Scope is an industry leader.

For integrating cameras there are a number of choices. Cameras using the Sony XC-77 CCD array tend to be quieter and more linear. They are excellent for densitometry. The XC-77 CCD array can be found in cameras from Sony, Video Scope, Xybion and Dage, to name a few.