1. Field of the Invention
The present invention relates to a new confocal scanning beam microscope and method for spectrally resolving a confocal image in a significantly short time.
2. Background of the Prior Art
The past decade has seen confocal microscopy emerge as a common tool in many areas of basic and applied science. A confocal microscope sequentially illuminates spots or locations of a sample that are confocal to a pinhole. By scanning the sample, typically in a raster pattern, a complete image is formed. Its main benefit over traditional light microscopy is the capability to resolve a two-dimensional slice (hereinafter referred to as the "sample plane") of a three-dimensional structure without the need to physically section the sample under investigation. A basic point scanning confocal microscope is disclosed by U.S. Pat. No. 3,013,467 of Minsky, which is hereby incorporated herein by reference.
Confocal microscopy has been applied principally in biological and medical sciences. Simultaneous labeling of biological materials by specific fluorescent dyes has become a common tool for locating these materials within tissues and cells. Multiple dyes are often employed, each labeling a distinct molecule or cellular region. This class of application has required fairly sophisticated multichannel detection techniques in which each channel is made sensitive to the presence of each different dye by means of filtering. Detection of fluorescence from these dyes is usually accomplished using as many photomultiplier tubes (PMTs) as dyes. The fluorescence from the sample (the signal) is split and the resulting split signals are filtered and detected. This method frequently suffers from "cross talk": the fluorescence from one dye label overlaps spectrally with that of another, thus reducing the ability to distinguish between separately labeled regions. Furthermore, autofluorescence, the native fluorescence of the sample in the absence of dye labels, often reduces the image contrast.
An alternate approach is to spectrally resolve each pixel of the confocal image, which allows even dyes with very similar spectra to be distinguished from one another and from the autofluorescent background. This approach offers the possibility of detailed exploration of light emitting microscopic environments by analysis of the position and bandwidth of particular materials in a spectrally resolved sample. Besides basic research applications, this detection technique may be applied to medical diagnosis. For example, spectroscopic differences have been observed between healthy and cancerous tissue, and these differences can be detected by spectrally resolved imaging techniques. One significant disadvantage of current confocal microscopes when used to obtain spectrally resolved images of tissue samples, for example, is that the time it takes to produce the spectrally resolved image (referred to hereinafter as "acquisition time") is prohibitively long.
FIG. 1 illustrates the basic components of a typical prior art confocal microscope 20 and having added thereto spectral dispersion device 60. Confocal microscope 20 includes laser 22 that emits a collimated light beam 24 that is expanded by beam expander 23. Light beam 24 is projected onto first scan mirror 30. First scan mirror 30, in this example, is driven to oscillate between two angles by computer control of voltages applied to first scan mirror 30, causing light beam 24 to be scanned vertically, along the y axis. Intermediate placed lenses 34, 36, having focal length f, form a unitary telescope that directs vertically scanned light beam 24 onto second scan mirror 38, which scans beam 24 horizontally to form a light beam moving in a raster pattern. As with first scan mirror 30, second scan mirror 38 is driven to oscillate between two angles by computer control of voltages applied to second scan mirror 38. A raster refers to a scan pattern in which the sample is scanned by the laser beam from side to side in horizontal lines and from top to bottom. In FIG. 1, scan mirror 30 (the Y-axis scan mirror) is set to oscillate much slower than second scan mirror 38 (the X-axis scan mirror). The resulting raster beam moves fast horizontally and slow vertically, and is relayed by a second set of intermediate lenses 40, 42, each having focal length f and forming a second unitary telescope. This arrangement directs the recollimated beam to the entrance aperture 48 of microscope objective 46, such that the angle of light beam hitting aperture 48 varies over time, thereby continuously scanning the sample plane 50 in a tightly focused raster pattern. The light, emitted, reflected, or scattered from the sample (the signal beam) retraces the path of the excitation beam through the microscope objective 46, scan mirrors 38, 30 and intermediate optics 42, 40, 36, and 34. On the retrace path, the signal beam is partially descanned by second scan mirror 38 (horizontal motion is removed) and then fully descanned by first scan mirror 30 (vertical motion is removed). The collimated and fully descanned signal beam from sample plane 50 is then reflected by a dichroic beamsplitter 52 of detection arm 51 of confocal microscope 20, and focused by lens 49 through pinhole 53 that rejects the light emitted, reflected, or scattered from that part of the sample not in the plane of focus of the objective, and passes light to detector 54 only from that part of the sample that is in the plane of focus, i.e. sample plane 50. Detector 54 is typically a CCD camera. Pinhole 53 is critical, because it gives the confocal microscope its sectioning capability. The light that passes through pinhole 53 is detected and recorded as a function of the angles of the scan mirrors over time to create an image.
A spectral dispersion device 60 as shown in FIG. 1, such as a grating, may be placed between pinhole 53 and detector 54. Using the current technology, it is therefore possible to spectrally resolve each pixel, one pixel at a time, along a raster pattern. It is understood in the art that the position of focus on the sample is directly related to the position of both scan mirrors. FIG. 1 illustrates a confocal microscope of a type similar to one manufactured by Kaiser Optical Systems, Inc., Model No. HiRes532.
U.S. Pat. No. 5,192,980 of Dixon et al. discloses a scanning optical microscope for spectrally resolving light reflected, emitted, or scattered from a sample. Dixon et al. recognizes that the diffraction limited spot at the specimen acts like the entrance aperture of an integrated monochromator. The confocal microscope of Dixon et al. therefore acts as the entrance aperture and the first collimating optic of a scanning monochromator. A diffraction grating, lens, and pinhole complete the monochromator. One obvious problem with the Dixon et al. design, and the design of other confocal microscope devices currently used in the art, is that it takes a substantial amount of time to acquire a full spectrally resolved image. For every pixel (smallest image unit at a particular resolution) of the image to be acquired, a full grating scan is required. A small (but not atypical) confocal image is on the order of 200.times.200, or 40,000 pixels. For example, in Dixon et al. and for other confocal microscopes in the art, building a complete spectrally resolved image requires the following summarized steps: (1) position both scan mirrors so that the scan beam focal point is on one spot (corresponding to one image pixel) of sample plane 50; (2) open the shutter attached to the CCD camera; (3) wait long enough to get the spectrum of light corresponding to that pixel; (4) close the shutter; and (5) move the scan mirrors to position the light beam on the point on the sample plane corresponding to the next image pixel and repeat process until all points of the sample plane 50 corresponding to all of the image pixels have been spectrally resolved. Even if a spectral scan of one pixel takes as little time as one second (an unusually short time for a spectral scan with the low light levels available from confocal microscopy), one small spectrally resolved image would take over 10 hours.
Now referring briefly to FIG. 2, which illustrates a confocal microscope arrangement also known in the art, whereby the image is non-descanned when detected. In other words, the scanned image is projected directly onto a two-dimensional detector array 212. To accomplish this, detection arm 202 is placed between microscope objective 204, and scan mirror 206, i.e., dichroic beam splitter 208 reflects fully scanned light beam 201 to lens 210, which focuses light to detector 212. Placing detection arm 202 in a position enabling it to receive the signal beam prior to its descanning has the benefit of extremely rapid image acquisition; however, sectioning capability is not possible with one photon excitation due to the absence of a pinhole to reject light originating from points not in the focal plane. Therefore, in order to retain the sectioning capability of the microscope, multi-photon excitation must be used.
U.S. Pat. No. 5,504,336 of Noguchi is a spectrofluorometric apparatus for obtaining spectral image information from a sample. However, the Noguchi invention is not used in connection with a confocal microscope or other type microscope and appears to be used in connection with laser interferometric detecting and ranging (LIDAR).
An important application of the present invention is as a detection method for DNA sequencing. A typical DNA sequencing technique generates DNA fragment strands of various lengths from a template that is the strand to be sequenced. The polymerization reactions of the fragments are terminated by the incorporation of a dideoxy analog of each of the four bases into each strand fragment thus leading to a mixture of strand fragments of all possible lengths. The strand fragment replica mixture is separated by electrophoresis along a gel microcapillary into discrete bands in accordance with strand replica molecular weight.
One way that detection and analysis of gel bands can be accomplished is by using radioisotope labeled DNA. The radioactive gel slabs containing the separated DNA fragments are exposed overnight to film. After development of the x-ray film, the sequence or size of the DNA separated fragments are read directly from the images on the film. The disadvantage of autoradiographic detection is the time required to expose and develop the film, and the use of hazardous environmentally harmful materials.
An alternative to radioisotope labeling of DNA, is fluorescently labeling the DNA. (L. M. Smith, J. Z. Sanders, R. J. Kaiser, P Hughs, C. Dodd, C. R. Connell, C. Heiner, S. B. H. Kent, and L. E. Hood, Nature vol. 321, pp 674-679) The present detection apparatuses and methods for detection and analysis of fluorescently labeled DNA employ a set of four filters that are selected to pass light emitting from four dyes used to label the four bases of strands of DNA. A desirable set of four dye labels should have very well separated emission spectra, but should have nearly overlapping absorption spectra so that they can all be successfully excited with the same laser line. Such criteria (non-overlapping emission spectra coupled with well-overlapped absorption spectra) are extremely difficult to achieve due to the nature of the relation between absorption and emission. This presents severe limitations on the choice of the set of dye labels. The apparatus and method of the present invention described below broadens the set of dye labels available to researchers by using other spectral characteristics, such as bandwidth and the spectral center of mass to serve as unique identifiers of spectrally overlapping dye sets.
Mathies et al. in U.S. Pat. No. 5,091,652 disclose a confocal microscope and method for detecting and analyzing fluorescently labeled DNA separated using a plurality of gel filled microcapillaries. However, the confocal microscope apparatus of Mathies et al. requires the traditional use of four dyes having well separated emission spectra, and further must read and analyze the fluorescence emitted in bands traveling within each capillary one capillary at a time.
With the foregoing in mind, it becomes a general object of the present invention to provide a scanning beam confocal microscope apparatus and method to reduce the acquisition time to spectrally resolve an entire confocal image.
It is another object of the present invention to provide a scanning beam confocal microscope apparatus and method to project light from a region of the sample plane corresponding to at least two image pixels along one axis of a two dimensional detector array, while using a spectrometer to disperse the spectra of the region's composite pixels along the other axis of the detector array.
It is yet another object of the present invention to provide an apparatus and method to reduce the acquisition time to spectrally resolve an entire confocal image using a partially point-descanned spectral imaging confocal microscope.
It is an object of the present invention to provide an apparatus and method to reduce the acquisition time to spectrally resolve a confocal image using a direct projection line-scan spectral imaging confocal microscope.
It is another object of the present invention to provide an apparatus and method for use in the rapid detection and acquisition of fluorescence emitted from fluorescent labeled samples being separated by microcapillary electrophoresis.