R. Winn Hardin
TRENTON, N.J. -- The new generation of high-resolution, back-illuminated intensified charge-coupled device (CCD) cameras will allow scientists to directly study the processes that regulate individual molecules, expanding the bodies of knowledge in material and analytical chemistry as well as bioresearch.
A main challenge to studies of molecular motion in these fields has been the limitations of CCD camera sensitivity imposed by noise levels generated within image intensifiers. Recent advances by Princeton Instruments should allow scientists to image a single molecule as it emits ultralow light levels (a single photon) during fluorescence.
Designers faced a difficult task improving the cooled, CCD detector systems that use thinned, back-illuminated devices and offer better than 85 percent quantum efficiency. The low-noise electronics of these detectors brings the threshold of a measurable signal level down to tens of photons.
But even these levels are high when one is studying a single, fluorescing molecule, and scientists are forced to take still images instead of viewing the molecule at video frame rates.
Clearly, a new strategy was needed to reduce the minimum detectable signal from many tens of photons to just one. Image intensifiers act as photon multipliers, raising the signal levels to well over the noise threshold level.
However, image intensifiers were a solution with a penalty: While offering optical gains in the range of 10,000 or more, image intensifiers suffer from a low quantum efficiency, typically 10 to 15 percent, meaning that the intensified CCD simply did not sense most photons.
According to physical chemist W.E. Moerner at the University of California at San Diego, Princeton's camera has the advantage over others because its combination of high-quantum efficiency with relatively low read noise means the camera can create images from single-photon emission with a single-stage intensifier.
An effective combination
The new (fourth) generation image intensifiers are based on gallium arsenide photocathode technology and small-diameter microchannel plates, and as such offer quantum efficiencies approaching 40 percent with resolutions comparable to that of CCDs. It is now possible to build a sensitive, high-resolution intensified CCD that offers single-photon sensitivity.
Princeton Instruments' PentaMAX GenIV device can operate at rates up to 5 million pixels per second vs. previous speeds of 150,000 pixels per second. This extra speed results in a full 512 3 512-pixel image readout of 15 frames per second vs. the 1.5 frames per second of the previous systems. The new detector is also 20 times more sensitive than the back-illuminated CCD-based unit, without significant quantum efficiency or resolution penalties.
The improved speed rates in combination with low read noise allow researchers to view molecular motion at video frame rates from 10 to 30 Hz, said Moerner, instead of one shot per 100 ms in previous models.
Moerner was among the first scientists to acquire one of Princeton Instruments intensified CCD cameras and is expected in the near future to release his newest findings on molecular motion and observations on gels used to separate proteins.