The present invention relates generally to confocal imaging systems, and more particularly to a confocal macroscope.
Confocal microscopes with coherent optical illumination are capable of producing very thin optical sectioning yielding sharp 3-D image volume data sets and an image of a specimen with much better contrast between fine details than is possible with non-confocal imaging systems such as wide-field instruments known to those skilled in the art. Confocal microscopes are employed to produce images of many types of specimens such as biological materials and semiconductor devices.
A schematic diagram of the essential components of a conventional confocal microscope 100 is shown in FIG. 1. A light source 110, such as a laser in the instance of monochromatic illumination, generates light that is deflected off of a beamsplitter 114, which directs the light toward an objective lens 116. The objective lens 116 focuses the light at a focal point 118 in a specimen 120. The focal point 118 is a small illuminated area in a focal plane, also called an object plane 122, in the specimen 120. In the instance of fluorescent imaging, if the specimen 120 is stained with fluorescent dye that is illuminated with a wavelength near its excitation maximum, then it will emit fluorescent light of a Stokes-shifted wavelength. Fluorescent molecules at the focal point 118 emit Stokes-shifted light rays toward the objective lens 116 which focuses the emitted rays into a confocal pinhole in a conjugate image plane. The confocal pinhole is also called an image pinhole 130 and is located in a plate 132 placed in the conjugate image plane for the focal point 118. In the instance of fluorescent imaging the beamsplitter 114 transmits the fluorescent light to the image pinhole 130, and the fluorescent light passing through the image pinhole 130 is detected by a photodetector 140 such as a photomultiplier tube (PMT). The photodetector 140 generates a signal indicating an intensity of the fluorescent light passing through the image pinhole 130, and the signal is processed by an appropriate data processing system (not shown). An image of the specimen 120 in the object plane 122 is generated by moving the focal point 118 relative to the specimen 120 such that the focal point 118 traverses the object plane 122 in the specimen 120 in a pattern such as a raster pattern. The data processing system assembles the signal from the photodetector 140 to generate the image. Images of different sectional depths of the specimen 120 may be generated by moving the object plane 122 relative to the specimen 120.
If the specimen 120 is reflective then the illumination light is reflected back toward the objective lens 116 and the beamsplitter 114 to be focused on the image pinhole 130 and detected by the photodetector 140. An example of a reflective specimen 120 is an integrated circuit wafer specimen.
Beam-scanning or stage-scanning confocal microscopes differ from wide-field instruments in two major aspects: an illumination spot and an image pinhole. First, in the confocal microscopes rays of light impinging on a specimen from an objective lens are converged along a cone to a single focal point or apex in an object plane in the specimen. This is in contrast to a wide-field instrument where, in each instant, the entire area circumscribed by the field-of-view of the objective lens is illuminated simultaneously. This area includes information from points extending through the entire depth of the specimen, including points above and below the object plane of the objective lens. One advantage of the beam-scanning or stage-scanning confocal microscopes is that all of the light is focused on the focal point in the object plane to produce a much more intense excitation of each scanned point of the specimen, with greater spatial specificity of the area being excited.
A second advantage of the beam-scanning or stage-scanning confocal microscopes is the pinhole in the emission/detection path. Some of the rays of light emanating from the object plane as a result of the illumination light will retrace the path of the impinging path through the objective lens to be collected at a point in the conjugate image plane. The confocal pinhole or image pinhole at the conjugate image plane acts as a spatial-filter to remove out-of-focus rays of light which emanated from points above, below, or to the side of the focal point or apex in the specimen. A single focal point in the specimen is examined at a time. If the focal point of the objective lens is scanned over the specimen at different object planes, then a three-dimensional data set of the specimen may be obtained. The greater intensity of confocal illumination and a segregation of adjacent object planes through which the focal point is scanned allow for the generation of low-distortion images of slices of a thick specimen such as a biological tissue section.
The intensity of illumination in a confocal microscope is enhanced if the excitation light source is a laser such as the laser 110 shown in FIG. 1. Arc-lamps normally used in wide-field instruments have much less optical power at a given excitation wavelength. Arc-lamps are also not as capable as a laser of providing a narrow excitation wavelength while excluding other wavelengths or colors, as arc-lamps emit wavelengths throughout a very broad spectrum. Lasers produce just a few colors or discrete wavelength lines with negligible energy in other spectral regions.
The confocal microscope has undergone many exciting and ingenious changes since its conception by M. Minsky, described in U.S. Pat. No. 3,013,467, with its defining characteristic being a detector pinhole. Minsky used arc-lamp illumination and a pair of orthogonally oriented, electromechanically oscillated tuning forks to translate a specimen. Advances in confocal designs are disclosed in Sheppard et al. xe2x80x9cA Scanning Optical Microscope For The Inspection Of Electrical Devicesxe2x80x9d Microcircuit Eng., Cambridge, 1980, p.447-454 and in Marsman et al. xe2x80x9cMechanical Scan System for Microscopic Applicationsxe2x80x9d; Rev. Sci. Instrument, 1047-1052, 54(8). These confocal microscopes use resonant galvanometers to oscillate the specimen, incorporate laser illumination and PMT detection, scan in real-time, and are used for observing the functional processes of living cells. They are limited to scanning areas of only about 1 mm on a side. The confocal microscopes described so far are categorized as xe2x80x9cstage-scannersxe2x80x9d, because they move the specimen on a support stage with respect to a fixed optical beam.
Laser beam scanning confocal microscopes, also called beam scanners, are described in xc3x85slund et al., xe2x80x9cPHOIBOS, A Microscope Scanner Designed For Micro-Fluorometric Applications, Using Laser Induced Fluorescence, Proceedings of the Third Scandinavian Conference on Image Analysis, Copenhagen, Denmark (1983). Beam scanners angularly deflect an illumination and detection beam with respect to a central axis of an objective lens, using tilting mirrors or acousto-optic devices. Beam scanners have an advantage in that a specimen is not jostled during scanning, and the specimen position may be adjusted without disturbing the scanner. However, spherical and chromatic aberrations in the objective lens are accentuated as the beam is deflected towards the periphery of the field. The field of view is both delimited and restricted by the diameter of the exit pupil of the objective lens which is typically less than half a millimeter. This produces an image that is bowl-shaped which may extend out of the specimen, and is not a flat-field scan. The introduction of beam scanners was contemporaneously accompanied by the implementation of digital storage.
Beam scanners and early stage scanner designs have drawbacks due to an angular scan, nonlinear velocity, and a curved scan path. These result in images that are irregular in shape, flawed quantitatively, and limited in field-of-view. This is partly attributed to the fact that with a fixed time-clock, both the spatial size of samples and the strength of the detected optical signal relative to a concentration of fluorescent dye molecules in a specimen are inversely related to the instantaneous velocity of the beam within the specimen.
Most of the commercially available confocal microscopes sold today are beam scanners. They are more than sufficient for viewing circumscribed specimens which fit neatly within the objective lens"" field of view. They are also more than sufficient for glimpsing isolated fields of a larger specimen. They also are well suited for monitoring live processes, where small scan spaces can translate into short frame times. Their edge distortion makes them problematic for producing images of extended areas. Attempts to seamlessly align image tiles produced by a beam-scanner to create a montage from scans of adjacent fields of view is cumbersome, due to the tiles"" distorted edges and the need for a separate mechanism, independent of the scanner, to move a specimen stage between tiles.
Recently, large field-of-view stage scanners have been patented in U.S. Pat. No. 5,184,021 to Smith and U.S. Pat. No. 5,091,652 to Mathies et al. Each describe flat-bed x-y scanners. These models allow seamless scanning, but at a limited resolution and speed. Such large-specimen stage scanners also place great stresses on a specimen at high scan rates, which are necessary in fluorescent imaging to avoid bleaching fluorescent dye in the specimen, and to collect copious data in a timely manner. Not jostling the specimen is critical in applications in which biological tissue cannot be dry-mounted. Further, even dry-mounted biological specimens are susceptible to impact damage.
U.K. Patent Application Number 2,184,321 to White describes a spiral configuration stage scanner having a scan motion without wasted time or as much jostling due to rectilinear motion reversal. However, the data set it generates is spatially irregular, due to uneven dwell time and a curved scan path.
There remains a need for a confocal imaging system capable of efficiently, accurately, and without jarring a specimen, generating a clear image of the specimen having a large cross-sectional area.
According to one embodiment of the present invention an imaging system comprises a specimen stage, a source of a collimated light beam centered on a beam axis, and a scan-head movably positioned to focus the collimated light beam on a focal point in an object plane above the specimen stage and to receive light emitted or reflected from the object plane. The collimated light beam is comprised of parallel light rays, and infinity space is the region in which the light beam is collimated. The scan-head is translated coaxial to the beam axis and takes advantage of the capacity of infinity space to stretch. According to another embodiment of the present invention a method comprises generating a collimated light beam centered on a beam axis and defining a region of infinity space, focusing the collimated light beam on a focal point in an object plane in a specimen on a specimen stage with an objective lens, and detecting light reflected or emitted from the specimen at the focal point to generate image data.