Unlike conventional optical microscopes, confocal scanning optical microscopes (CSOMs) image samples one point at a time through pinholes. A primry advantage of CSOMs is that they have a shallower depth of field than other optical microscopes. Thus, a CSOM is able to resolve both height and width information, and to image (independently) areas of a sample which are separated in height with reduced interference.
T. R. Corle (one of the inventors of the present application), G. Q. Xiao, G. S. Kino, and N. S. Levine, in their paper entitled "Characterization of a Real-time Confocal Scanning Optical Microscope," presented at the SPIE Symposium on Microlithography, San Jose, Calif., Feb. 26-Mar. 3, 1989, describe a CSOM in which light from an arc lamp propagates through a spinning Nipkow disk (a perforated disk through which a large number of holes have been drilled or etched in a spiral pattern). Each illuminated hole of the Nipkow disk produces a spot on the sample to be imaged. Light reflected from the sample propagates back through the disk to an eyepiece.
Many points on the sample are simultaneously illuminated by light through the holes of the Nipkow disk. The sample is scanned as the disk spins and the spinning spiral hole pattern sweeps the illuminated point pattern across the sample. As the disk spins, the system generates a real-time confocal image of the sample.
A conventional real-time confocal scanning microscope is shown in FIG. 1. The system of FIG. 1 includes motor 13, for spinning Nipkow disk 1 about its axis 2. Light from source 3 (which may be an arc lamp or other intense source) is focused by condensor 5 and polarized by polarizer 7, and then propagates through iris 9 and is reflected by partially reflective beamsplitter 11 toward disk 1. Some of the light incident on disk 1 from beamsplitter 11 propagates through the holes in disk 1, through tube lens 15, quarter wave plate 17, and objective lens 19 onto sample 21.
Some of the light reflects from sample 21, and propagates back through lens 19, quarter wave plate 17, lens 15, and the same set of holes in disk 1. The reflected light is then transmitted through partially reflective, partially transmissive beamsplitter 11, secondary objective lens 25 in the aperture of aperture plate 23, and analyzer 27. Polarized light from analyzer 27 reflects from mirror 29 and is incident on eyepiece 31. Alternatively, eyepiece 31 may be replaced by an image recording device such as a CCD camera.
By using the same set of pinholes of disk 1 for illumination and imaging, fewer optical components are required, and the procedure for aligning the system components is simplified.
Axis 2 of disk 1 can be tilted at a nonzero angle with respect to the optical axis 22 of objective lens 19 and sample 21. This diverts unwanted reflections from disk 1, preventing them from reaching eyepiece 31.
Polarizer 7, quarter wave plate 17, and analyzer 27 together function to reduce interference from unwanted reflections from the disk. Analyzer 27 is oriented to transmit polarized radiation that has propagated twice through quarter wave plate 17 (once before reaching sample 21, and once after reflecting from sample 21), and to block radiation that has not propagated twice through quarter wave plate 17.
FIG. 2 is a top view of Nipkow disk 1 of the FIG. 1 system. Pinholes 14, which extend through disk 1, are arranged in spiral pattern. Typical Nipkow disks include on the order of one million pinholes, each 10-30 microns in diameter and spaced 50-200 microns from adjacent pinholes. In early confocal microscopes, the Nipkow disk pinholes were drilled through a thin copper sheet. More recently, Nipkow disks have been formed by etching silicon wafers or using a chrome-glass photomask technique.
The major problem with conventional Nipkow disk based confocal microscopes is that the light signal returned to the detector from the specimen is very small. A conventional Nipkow disk with 30 micron holes spaced 200 microns (center to center) will transmit only 2% of the radiation incident thereon. The other 98% of the radiation is not used, and is reflected in undesirable directions (such as toward the eyepiece). Additional optical loss results each time the light propagates through (or reflects from) the other system components (including the separate polarizer and analyzer components).
Until the present invention, it had not been known how to design Nipkow disk based confocal microscopes so as to increase the total amount of light in the image of the sample, while also reducing background noise.