Proceedings Article | 10 April 1996
Proc. SPIE. 2655, Three-Dimensional Microscopy: Image Acquisition and Processing III
KEYWORDS: Confocal microscopy, Microscopes, Optical amplifiers, Sensors, Quantum efficiency, Interference (communication), Charge-coupled devices, CCD image sensors, Signal detection, Prototyping
The laser confocal microscope (LCM) is now an established research tool in biology and materials science. In biological applications, it is usually employed to detect the location of fluorescent market molecules and, under these conditions, signal levels from bright areas are often < 20 photons/pixel (from the specimen, assuming a standard 512 X 768, 1 sec. scan). Although this data rate limits the speed at which information can be derived from the specimen, saturation of the fluorophor, photobleaching of the dye, and phototoxicity prevent it being increased. Currently, most LCMs use photomultiplier tubes (PMT, QE equals 1 - 30% 400 - 900 nm). By contrast, rear-illuminated, scientific charge-coupled devices (CCD) now routinely readout the signal from square sensors approximately 30 micrometers on a side with a QE of 80 - 90%, a noise of only +/- 3 e-/pix and with no multiplicative noise. For this reason, in 1989, one of us (JJ) developed a rear-illuminated, single-channel Si sensor, called the Turbodiode, employing some of the sophisticated readout techniques used to measure charge in a scientific CCD. We are now extending this work to a device in which a single 36 X 36 micrometers sensor is read out through a low-noise FET charge amplifier with a reset circuit and then passed to a correlated, double-sampling digitizer. To maintain the desired +/- 3 e noise level at the relatively high data rate of 1 MHz, our new device utilizes 64 separate readout amplifier/digitizer systems, operating in sequence. The resulting detector is more compact, efficient and reliable than the PMT it replaces but as its sensitive area is smaller than that of a PMT, it will require auxiliary optics when used with any LCM having a large (mm) pinhole. As the signal light is parallel, a simple lens mounted axially and with the CCDiode at its focus would suffice. Future versions may use 3 X 3 or 5 X 5 arrays of sensors to `track' the confocal spot as it is deflected by inhomogeneities of the specimen, change its effective size or shape or detect system misalignment.
