Optical: systems and elements – Compound lens system – Microscope
Reexamination Certificate
2000-04-26
2002-05-14
Spyrou, Cassandra (Department: 2872)
Optical: systems and elements
Compound lens system
Microscope
C359S379000, C359S392000
Reexamination Certificate
active
06388809
ABSTRACT:
FIELD OF THE INVENTION
The field of the present invention is confocal microscopy.
BACKGROUND OF THE INVENTION
Microscopy is used to produce magnified representations of both dynamic and stationary objects or samples; microscopes magnify small things and make them easier to see. There are many different modes of microscopy such as brightfield microscopy, darkfield microscopy, phase contrast microscopy, fluorescence microscopy, reflectance or reflected light microscopy and confocal microscopy. All of these forms of microscopy deliver illumination light in a controlled fashion to the sample and collect as much of the light containing the desired information about the sample as possible. Typically, this is accomplished using Kohler illumination in any of reflectance microscopy, transmission microscopy or epifluorescence microscopy. These methods use appropriately placed diaphragms and lenses to control both the size of the numerical aperture (illumination cone) and the size of the illuminated area of the sample. In Kohler illumination, diaphragms are placed in at least two locations. First, a diaphragm is placed in the conjugate image plane of the sample, a location which permits control of the size of the illuminated area of the sample. Second, a diaphragm is placed in the conjugate image plane of the aperture diaphragm of the objective lens(es) (this location is also a conjugate image plane of the aperture diaphragm of the condenser lens(es)), a location which permits control of the angle(s) of the light illuminating the sample. Typically, any of the diaphragms can be a simple iris (for example, for brightfield microscopy and epillumination fluorescence microscopy), but the diaphragms can also be more complex (for example, in darkfield microscopy, where the diaphragms may comprise cutout rings of different diameters).
An example of a microscope using Kohler illumination is set forth in FIG.
1
. In the figure, microscope
2
comprises a light source
4
that emits a plurality of light rays, which have been divided into first light rays
6
, second light rays
8
and third light rays
10
. The light rays are transmitted along an illumination light path from light source
4
through light source lens
12
, adjustable iris field diaphragm
14
and condensor lenses
16
. An adjustable iris aperture diaphragm (condenser)
18
can be disposed between upstream and downstream condenser lenses
16
. The light then contacts, or impinges upon, sample
20
and then proceeds to pass through objective lenses
22
, which objective lenses can comprise an aperture diaphragm (objective)
24
spaced between the objective lenses
22
, and then the light rays proceed to a light detector
26
. As noted above, the angle of illumination of the sample can be controlled by modulating the light as it passes through conjugate image planes of the aperture diaphragm of the objective lens, which planes can be found, for example, at light source
4
and the upstream aperture diaphragm
18
in
FIG. 1
, while the location and/or area of illumination of the sample can be controlled by modulating light as it passes through a conjugate image plane of the sample, which plane corresponds to the adjustable iris field diaphragm
14
in FIG.
1
.
One preferred form of microscopy is confocal microscopy, in which one or more discreet aperture spots are illuminated in the object plane of the microscope from which transmitted, reflected or fluorescent light is then relayed for observation through conjugate apertures in the image plane. In some embodiments, confocal microscopy can result in spatial resolution about 1.3 times better than the optimum resolution obtainable by conventional light microscopy. See, e.g., U.S. Pat. No. 5,587,832. Additionally, confocal microscopy can reduce the interference of stray, out-of-focus light from an observed specimen above or below the focal plane, and can permit optical sectioning of tissue as well as high-resolution 3-D reconstruction of the tissue. The technique can effectively resolve individual cells and living tissue without staining. Confocal microscopy can be performed using mechanical translation of the specimen with fixed optics, using a fixed specimen and scanning beams manipulated by special rotating aperture disks, or a spatial light modulator (SLM). See U.S. patent application Ser. No. 09/179,185, entitled Apparatus And Methods Relating To Spatially Light Modulated Microscopy; U.S. Pat. Nos. 5,867,251; 4,802,748, 5,067,805, 5,099,363, 5,162,941. The special rotating aperture disks, often called Nipkow disks, typically comprise a plurality of apertures, but only one aperture at a time is used for confocal scanning. Still other known confocal scanning systems have used a laser beam rastered with rotating mirrors to scan a specimen or a laser beam that scans a slit rather than a spot; such slit scanning increases imaging speed relative to rotating aperture disks but slightly degrades resolution. See U.S. Pat. No. 5,587,832. The use of spatial light modulators permits control of either or both of the angle(s) of the light and location of the light, and can provide high speed confocal scanning without the loss of resolution that accompanies slit scanning instead of spot scanning. See U.S. patent application Ser. No. 09/179,185, entitled Apparatus And Methods Relating To Spatially Light Modulated Microscopy; U.S. Pat. No. 5,867,251.
Confocal microscopy, however, does not utilize a significant portion of the light emanating from the spot on the sample that is under investigation, and thus has unnecessarily limited resolution in both the x-y plane (sideways) and in the z-direction (up and down, or depth), and an unnecessarily limited signal to noise ratio. Thus, there has gone unmet a need for improved methods of confocal microscopy that provide enhanced resolution and/or enhanced signal to noise ratio. The present invention provides these and other advantages.
SUMMARY OF THE INVENTION
The present invention provides apparatus and methods that improve the depth resolution of confocal microscopy images. The present invention can be applied to all of reflectance microscopy, transmission microscopy and fluorescence microscopy. The present invention comprises utilizing out-of-focus information from within the focal plane of interest (from the x-y direction) and/or from planes above and below the focal plane of interest (from the z-direction). In general, the present invention takes advantage of the observation that in confocal microscopy the intensity of the light emanating from the illumination spot of the sample falls off or decreases in a regular fashion as the distance from the illumination spot increases. For example, the point spread function (PSF) of the emanating light for a confocal, cylindrically symmetric lens system falls off approximately as sinc{circumflex over ( )}2(z) for the singularly illuminated spot, or central illumination pixel, in the vicinity of the focal plane. This PSF in the x-y plane is a function of depth (i.e., of the z-position). The interaction of (a) a reflective surface or other light-emanating surface, such as a fluorescent surface or transmissive surface, and (b) the PSF formed by a confocal microscope, results in “out-of-focus” information in, above and below the focal plane; this “out-of-focus” information can be measured. By comparing the measurements in the x-y plane, preferably at a plurality of z-positions, one can improve the resolution along each of the x, y, and z-axes. An additional advantage to using the out-of-focus information is that it increases the number of the photons used in the system, thus improving the signal to noise ratio. In addition, incorporation of such information can also improve depth resolution and otherwise help correct for aberrations, such as spherical aberrations or other optical aberrations, in the optical system of a microscope.
Thus, in one aspect the present invention provides confocal microscopes comprising a light detection and analysis system, the system comprising a light detector disposed downstream from a
Digital Optical Imaging Corporation
Graybeal Jackson Haley LLP
Spyrou Cassandra
Treas Jared
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