Densitometry and other light amount measurements
Quantitation of light is used to determine the chemical make up of the specimen. We can measure the amount of stain by measuring the amount of light that it absorbs. By using reflected light we can determine the components of coal. Measuring fluorescent light output can tell us the amount of DNA in a plant cell.
Whether a system is used to measure the absorption of transmitted light, the absorption of reflected light or the emission of fluorescence the system is the same. There is a detector that converts light to voltage and a system to convert voltage to a digital number so that a computer can use the data.
The two kinds of detectors are tv. cameras and photo multiplier tubes (PMT). PMT's are a type of vacuum tube that converts photons into voltages and is very efficient at doing it. Tv cameras are in more versatile and easier to use but are not as sensitive as a PMT. Typically a tv system will be used if wide areas of the specimen need to be examined, PMT's are used if the light amount is very small. Both tv. systems and PMT systems attach to the camera port of a microscope.
The main problem with a PMT system is selecting the area to be read. A PMT system must have a diaphragm visible to the user to isolate the area to be read from the surrounding area. If the area to be read is an irregular area then this can be quite difficult. However if you are trying to quantitate fluorescence a tv camera may not be sensitive enough, only a PMT will work.
For ransmitted light use all systems start by taking a reading of a blank area of the slide, this is the maximum amount. Then the specimen is inserted and the reduced light intensity is read. This lets us know the amount of light that the specimen absorbed. This can be an absolute number or a relative one depending on the application.
Reflected light is used by industry and geology. A reflective standard is used to provide the baseline amount and then the specimen is read. A typical use for a reflected light system is to determine the composition and grade of coal.
Fluorescent systems are usually used for relative measurements. Most fluorescent systems use PMT's since the light output is so low. The specimen is lined up optically under the diaphragm. The diaphragm is adjusted to exclude background light and the reading is taken. A typical use is fluorescent probe quantitation and DNA studies.
If a tv. based system sounds like an automatic image analyser you are right. Most image analyzers can do light amount quantitation. Some typical applications include DNA studies using transmitted light and mineral studies using reflected light.
These systems can provide a lot of interesting data. They can also provide reams of junk. If this area interests you read Inouye's book and find a local guru. There are issues of specimen preparation and measurement accuracy that can only be addressed by an experience user.
The only out of the box system that has found acceptance is the medical area is the CAS system for DNA analysis. This system provides the numbers that clinical users need. I wish they would use a better microscope in the system but overall it works.
What this means to you
Systems are manufactured for microscopes that can measure light amount. They can be very valuable
but there are difficulties in operating one in the real world.