Individual colloidal particles larger than roughly 200 nm can be resolved with a conventional light microscope. We use an Olympus IMT2 inverted microscope with a 100 N.A. 1.2 oil immersion objective. The objective lens' 400 nm depth of focus is comparable to a typical sphere diameter, so that only a single layer of spheres is in focus at any time. This is convenient for distinguishing particles in dense suspensions. Three-dimensional views can be reconstructed from a series of images at different focal planes, although multiple light scattering rapidly reduces contrast with increasing depth. The limited working distance of high-powered objective lenses (typically around 200 m including the thickness of the cover glass) poses the ultimate constraint to viewing in depth. Scanning laser confocal microscopes offer true three-dimensional microscopy but are at least an order of magnitude more expensive than conventional microscopes.
Conventional photomicrographs of colloidal suspensions such as that in Fig. 1(a) can capture thousands of particles with spatial resolution limited by the wavelength of light. An optical scanner then can convert these photographs to digital arrays with a degree of detail limited only by the amount of computer memory available. While the image processing methods discussed below can be applied to photographic data, our main interest is in the quantitative analysis of video images which record both spatial and temporal information.
Standard commercial video cameras produce 30 complete images per second but with poorer spatial resolution than is offered by photographic film. The convenience, flexibility, and economy of video technology, however, encourage its use. The usable portion of a single video image typically consists of 480 horizontal lines of 640 pixels, where each pixel records the average brightness of a discrete area in the original scene. Of the many cameras available, those based on charge-coupled device (CCD) technology are superior to older vidicon tube models which suffer from geometric distortions, nonuniform sensitivity, and various nonlinearities which vary with time. Tube cameras also are quite bulky. Monochrome CCD cameras may be preferable to color models not only because they are less expensive but also because they tend to have superior noise figures and greater sensitivity to subtle brightness variations. Color information, furthermore, is not used in the techniques we describe below. We use an NEC TI-324A CCD camera attached to our microscope's video port. The addition of a video eyepiece provides a total system magnification of M = 85 nm/pixel on the CCD. The choice of system magnification is a trade-off among several figures of merit including size of field of view, degree of image contrast, desired tracking precision, and speed of image processing. In our studies of phase transitions and dynamics in suspensions of monodisperse latex microspheres, such compromises typically dictate selecting the magnification to produce images with apparent radii of pixels.
Video images must be converted into digital format before they can be analyzed. Digitizing video frames requires a dedicated frame grabber which typically takes the form of an add-on board for a computer. The frame grabber used in this study is a Data Translation DT-3851A installed in a 486-class personal computer. Frame grabbers such as the DT-3851A convert the analog video stream to digital images in real time, a process which requires more than 12 million analog to digital (A/D) conversions per second. Such high-speed digitization limits most frame grabbers to 8 bits of dynamic range, or 256 gradations of gray scale per pixel. While photographs capture much more subtle gradations, 8 bit resolution suffices if the video signal is adjusted to fill the grabber's dynamic range.
Although frame grabbers can digitize and display images in real time, storing a digital image is time consuming. A single uncompressed gray-scale image takes up about one third of a megabyte. Typical hard disks can store such an image in a quarter second, which is not fast enough to acquire full motion video in real time. Disk arrays and high speed video drives can archive full-screen full-motion digital video in real time and probably will become cost-effective solutions to the video storage problem in the near future. Storing images to fast memory before transferring to disk also is becoming increasingly feasible with the advent of local bus frame grabbers. Real-time hardware compression schemes such as MPEG generally are not appropriate since they achieve their results by throwing out the subtle gradations which we hope to study. Recordable video disks offer another hardware solution, but are prohibitively expensive for routine recording, particularly when only a small percentage of the recorded video is likely to be analyzed. Real-time digital video recording, however, is not the only practical approach for time-resolved digital microscopy.
Video images can be recorded in analog form with commercial video tape recorders. The more advanced formats such as S-VHS or Hi8 faithfully preserve most of the information from the original video signal. Video tape decks with computer interfaces such as the SONY EVO-9650 can be controlled by the same computer which hosts the frame grabber card. A fairly straightforward program then can direct the tape deck to seek out and pause at a particular video frame, have the frame grabber digitize the paused image, and store the result to disk. Repeating this process permits digitizing any sequence of video frames. It should be noted that some low cost frame grabbers have difficulty digitizing pause-mode video signals. While such problems are becoming less common as video technology progresses, they should still be considered when designing a video acquisition system.