Note: This posting is taken directly from a Wikipedia Article I wrote on “Imaging Particle Analysis”, which was initially submitted for review 10/18/13, and published on 1/2/14. The article can be found at: https://en.wikipedia.org/wiki/Imaging_Particle_Analysis
Imaging particle analysis systems can be broadly categorized into two different types based upon the image acquisition technique: static or dynamic. Quite simply, in static image acquisition, the sample is stationary while the images are captured, whereas in dynamic image acquisition the sample is actually moving while being imaged.
Static image acquisition is the most common form. Almost all microscopes can be easily adapted to accept a digital camera via a C mount adapter. The sample is prepared on a microscope slide which is placed on the microscope stage. Once the sample has been focused on, then an image can be acquired in digital format, and image processing algorithms can be used to isolate particles in the field of view and measure them.
In static image acquisition only one field of view image is captured at a time. If the user wishes to image other portions of the same sample on the slide, they can use the X-Y positioning hardware to move to a different area of the slide. Care must be taken to insure that two images do not overlap so as not to count and measure the same particles more than once.
The major drawback to static image acquisition is that it is time consuming, both in sample preparation (getting the sample onto the slide with proper dilution if necessary), and in multiple movements of the stage in order to be able to acquire a statistically significant number of particles to count/measure. Computer-controlled X-Y positioning stages are sometimes used in these systems to speed the process up and to reduce the amount of operator intervention, but it is still a time consuming process, and the motorized stages can be expensive due to the level of precision required when working at high magnification.
The major advantages to static particle imaging systems are the use of standard microscope systems and simplicity of depth of field considerations. Since these systems can be made from any standard optical microscope, they may be a lower cost approach for people who already have microscopes. More important, though, is that microscope-based systems have less depth of field issues generally versus dynamic imaging systems. This is because the sample is placed on a microscope slide, and then usually covered with a cover slip, thus limiting the plane containing the particles relative to the optical axis. This means that more particles will be in acceptable focus at high magnifications.
In dynamic image acquisition, large amounts of sample are imaged by moving the sample past the microscope optics and using high speed flash illumination to effectively “freeze” the motion of the sample. The flash is synchronized with a high shutter speed in the camera to further prevent motion blur. In a dry particle system, the particles are dispensed from a shaker table and fall by gravity past the optical system. In fluid imaging particle analysis systems, the liquid is passed perpendicular past the optical axis by use of a narrow flow cell.
The flow cell is characterized by its depth perpendicular to the optical axis. In order to keep the particles in focus, the flow depth is restricted so that the particles remain in a plane of best focus perpendicular to the optical axis. This is similar in concept to the effect of the microscope slide plus cover slip in a static imaging system. Since depth of field decreases exponentially with increasing magnification, the depth of the flow cell must be narrowed significantly with higher magnifications.
The major drawback to dynamic image acquisition is that the flow cell depth must be limited as described above. This means that, in general, particles larger in size than the flow cell depth can not be allowed in the sample being processed, because they will probably clog the system. So the sample will typically have to be filtered to remove particles larger than the flow cell depth prior to being evaluated. If it is desired to look at a very wide range of particle size, this may mean that the sample would have to be fractionated into smaller size range components, and run with different magnification/flow cell combinations.
The major advantage to dynamic image acquisition is that it enables acquiring and measuring particles at significantly higher rates of speed, typically on the order of 10,000 particles/minute or greater. This means that statistically significant populations can be analyzed in far shorter time periods than previously possible with manual microscopy or even static imaging particle analysis. In this sense, dynamic imaging particle analysis systems combine the speed typical of particle counters with the discriminatory capabilities of microscopy.