Optical: systems and elements – Compound lens system – Microscope
Reissue Patent
1999-09-22
2003-11-11
Nguyen, Thong (Department: 2872)
Optical: systems and elements
Compound lens system
Microscope
C359S368000, C359S370000, C359S383000
Reissue Patent
active
RE038307
ABSTRACT:
CROSS-
REFERENCE TO RELATED APPLICATIONS
Not Applicable
REFERENCE TO A MICROFICHE APPENDIX
Not Applicable
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention pertains generally to three-dimensional optical microscopy, and more particularly to a method and apparatus for three-dimensional optical microscopy which employs dual opposing objective lenses about a sample to obtain a high level of depth resolution.
2. Description of the Background Art
Optical microscopy has experienced a remarkable renaissance in the medical and biological sciences during the last decade. The increased importance of optical microscopy has been due to new developments in fluorescent probe technology, and the availability of quantitative three-dimensional image data obtained through either computational deconvolution or scanning confocal microscopy.
Optical microscopy offers several advantages over non-optical microscopy techniques. Use of optical microscopy allows viewing of living tissue samples in their natural state. Electron microscopy, in comparison, requires microscopy samples which are dried and exposed to vacuum. Additionally, the interior of the sample can be viewed and mapped in three dimensions using optical microscopy, whereas scanning electron microscopy and other scanned probe microscopies map only the surface of the is sample, and thus cannot provide information about the sample interior. Yet another advantage of optical microscopy is that particular cellular components can be recognized and mapped out with great specificity by staining with fluorescent probes. It is now possible to synthesize fluorescent probes with specificity for nearly any given biomolecule.
The only important drawback to optical microscopy is its limited resolution, which is related to the angle over which the objective lens can collect light, and ultimately from the finite wavelength of light. Thus, any technology such as the present invention that significantly increases the resolution of optical microscopy will have important applications in cellular biology, medical imaging, and other biotechnology fields.
Presently there are two primary approaches to three-dimensional optical microscopy: optical sectioning microscopy, which is also known as computational deconvolution, and scanning confocal microscopy.
In optical sectioning microscopy, a series of images of the microscopy sample are acquired, with the focus moved successively through sections of the sample to obtain successive images. Each image contains in-focus information from the parts or sections of the sample which are in the focal plane, and blurred, out-of-focus information from the other parts of the sample. Analysis of the entire data set by computer allows reconstruction of the three-dimensional structure of the sample. The reconstruction process employs computational algorithms and a previously stored reference data set describing the blur caused by a single point source. Optical sectioning microscopy is a “widefield” microscopy in which large area images are recorded, typically by a charge-coupled device array (CCD) camera. Thus, high light throughput and high data acquisition speeds are possible with this technique.
In confocal microscopy, a focused laser beam is used as a light source, and light is detected by a photomultiplier tube through a pinhole which is focused onto the same spot in the sample as the laser. This combined focal point is then scanned in three dimensions through the sample, and the detected intensity as a function of spot position is used to obtain a three-dimensional image of the sample. The pinhole partially suppresses out-of-focus information and improves the resolution, but at the cost of discarding much of the light. This loss of light necessitates long exposure times, which makes operation slow and often causes severe sample bleaching problems. Confocal microscopy operations are further slowed down by the fact that the data pixels are acquired one at a time, as opposed to up to a million in parallel for the large area imaging employed in optical sectioning microscopy.
Both optical sectioning microscopy and confocal microscopy suffer an important drawback in that the depth resolution or Z-direction resolution is several times worse than that in the transverse, or XY, plane. The limitation on Z-direction resolution is caused by fundamental geometrical limitations which are discussed in detail below. The present invention provides a method and apparatus for optical microscopy in which the Z-resolution is not only equal to that of the resolution in the XY plane, but is increased to more than double the resolution in the XY plane obtained heretofore with optical sectioning microscopy. This increase in Z-direction resolution is achieved by the present invention while also maintaining the high light throughput and data acquisition speeds available through optical sectioning microscopy.
There are two previously known optical microscopy methods which employ dual opposing objective lenses. One method, which is known as 4Pi Confocal Microscopy, is a confocal, rather than a widefield, microscopic method. 4Pi Confocal Microscopy can generally be employed in three ways. In a first mode, focused laser light is used to illuminate a sample from both objective lenses and interfere in the sample. In a second mode the emitted light is collected from both directions and combined onto a single pinhole detector. The third mode involves the combination of the first two modes simultaneously. Being a confocal technique, however, all modes of 4Pi Confocal Microscopy have poor light throughput and lengthy data acquisition times due to loss of light caused by the pinhole
photodector
photodetector
and the slowness of the pixel-by-pixel data acquisition.
The second known optical microscopy method which employs two opposing lenses is generally called Standing Wave Fluorescence Microscopy (SWFM). This technique requires a light source with great temporal and spatial coherence, typically in the form of a laser. The spatially and temporally coherent light source results in an interference pattern in sample space which is a sinusoidal standing wave (hence the name) that extends throughout the observed region of the sample.
SWFM could in principle achieve similar Z resolution as one embodiment of the present invention (the I
3
M embodiment described herein) but only by combining several different standing wave patterns in sequence through use of scanning mirrors on similar dynamic devices, or by using multiple individually coherent but mutually incoherent light sources, such as a plurality of lasers. The present invention provides the increased Z-direction resolution without requiring such moving parts, requires only a single, spatially incoherent light source such as an arc lamp or incandescent bulb, and does not require temporal coherence beyond that exhibited by any band-limited light source. The use of a simple incoherent light source allows free choice of wavelength of the illumination light, while lasers are available in only a limited selection of wavelengths. Furthermore, one embodiment of the present invention (the I
5
M embodiment described herein) achieves greater Z resolution than is possible through SWFM alone.
Thus, the present invention differs from, and has advantages compared to, all previously known 3D microscopy techniques. Compared to any mode of microscopy that uses a single objective lens, the present invention offers higher Z resolution. Compared to SWFM, the present invention uses simpler illumination means and offers a greater selection of illumination wavelengths, and in one of its embodiments offers higher Z resolution. Compared to 4Pi Confocal Microscopy, the present invention offers simpler illumination means, a greater selection of illumination wavelengths, greater data acquisition speed, and more efficient use of observed or emitted light, which can lead to less severe sample bleaching.
Thus, there is a need for a method and apparatus for three-dimensional optical microscopy which provides great
Agard David A.
Gustafsson Mats G. L.
Sedat John W.
Nguyen Thong
O'Banion John P.
The Regents of the University of California
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