Optical: systems and elements – Compound lens system – Microscope
Reexamination Certificate
1998-05-15
2001-07-17
Spyrou, Cassandra (Department: 2872)
Optical: systems and elements
Compound lens system
Microscope
C359S391000
Reexamination Certificate
active
06262838
ABSTRACT:
BACKGROUND OF THE INVENTION
Focusing mechanisms for microscopes are employed to position the object at the plane of focus of the instrument, to enable the object to be examined, i.e. inspected, illuminated or otherwise acted upon. Typically the object is placed upon a platform that moves laterally relative to the optical axis of the objective, to bring the area of interest of the object into alignment with the optical axis. The platform is then raised or lowered (translated) along the optical axis to achieve best focus. If it is necessary to register the optical axis with regions of the object larger than the field of view of the objective lens, the platform and object are further moved laterally in a sequence of steps to view the entire area. In some cases, microscopes are constructed to translate the objective lens or the entire microscope along the optical axis to reach best focus and in some cases the objective lens is moved laterally to bring the optical axis and areas of interest into alignment.
A known technique for translating the platform and object in the direction of the optical axis employs a precision dovetail mechanism that is activated by a manual rack and pinion or a motorized lead screw. In many cases this mechanism must be constructed with high accuracy to be capable of micron or sub-micron positional resolution, which results in high cost.
In the case of wide field of view microscopes in which lateral motion of the object or microscope objective is required, there is need for reliable, low cost and fast auto-focus mechanisms of high accuracy. This need exists particularly in respect of investigational tools for biology, e.g., for viewing arrays of fluorescently labeled microorganisms and DNA assays as well as for viewing entire biopsy samples, which may be as large as a square centimeter or larger. (“DNA” is used here to designate the full range of nucleic acids of interest to the field of biotechnology.)
According to present biological analytical technology, arrays of fluorescently labeled microorganisms and DNA assays are created in two dimensional fields. The objects to be examined in an array are, for example, DNA fragments that have discriminating sequence information. Biological laboratories have targeted objects for the arrays (e.g., spots of DNA) of diameters of the order of 25 to 250 micron, the spot size depending primarily upon the total number of objects to be represented in the array.
DNA arrays are typically probed with fluorescently labeled fragments of potentially complementary strands. When a match occurs between a fragment in a deposited spot and a fluorescently labeled fragment probe, hybridization occurs, and a positive “score” can be recorded under fluoroscopic examination. Because fluorescence, whether natural or stimulated by illumination, is a weak signal, a “score” is identified for DNA spot by the intensity of the fluorescence from the spot compared to reading(s) for the background that directly surrounds the specific spot. By controlled deposition of spots of a variety of DNA fragments in the array and by observing the matches or “scores” that occur at known spot addresses, important information concerning nucleic acids can be inferred. For this technology to expand widely, economical instruments are required that can rapidly and accurately scan the fluorescently labeled objects over a wide field of view, e.g. a field of view that is approximately 22 mm wide, the width of conventional glass microscope slides.
The large volume of data to be evaluated also calls for unattended operation of such instruments upon a sequence of slides, including automatic focusing of the microscope for each slide.
Microscopes or microscope-like instruments have been developed to inspect, illuminate or otherwise treat wide areas, based on scanning principles. In the case of inspection, the image is constructed electronically from a succession of acquired single picture elements during relative scanning movement between the object and the microscope. Focusing in these instruments is commonly automated, but there are significant economic and operational drawbacks in the systems that have been commercially available.
For a number of reasons, proper focusing is a critical need for automated microscopes where the material to be investigated is disposed over a wide area of a microscope slide. A microscope slide is typically a slab of float glass approximately 25×75 mm in x, y dimensions and about 1+0.1/−0.2 mm thick as defined by industrial standard ISO 8037-1-1986E. It is common for microscope slides to be slightly bowed, as they are not very rigid and can be deformed when clamped. In the normal installation of a slide in a microscope, the slide is caused to rest upon a flat surface and is held in place by gently pushing its edges against stops, a technique which alleviates most deformation. Other types of substrates for microscopic examination, including arrays provided on relatively thin glass cover slips and on plastic substrates, likewise have variation in thickness and are subject to deformation.
The depth of field (focus tolerance) and the resolution of a given microscope are inter-related, being defined by the laws of physics. The better the resolution, the smaller is the depth of focus. Present day biochip examination calls for pixel resolution between 5 and 10 micron which corresponds to a depth of field between approximately 30 and 200 micron, the particular values depending upon the optical configuration and the application. Since the thickness variation of commercial microscope slides is greater than this value, when the slide rests upon its back surface, auto focussing is compulsory.
In cases where the slide or other object is sufficiently uniform for the purpose at hand, auto focus is performed once per slide or object to be microscopically examined, during a setup procedure.
In some cases, automated microscopes employ dynamic focusing features, i.e. features enabling continual adjustment of focus as scanning of a given slide or object proceeds. For this purpose an algorithm is employed to define focus. Commonly, dynamic systems analyze the image acquired through the optical path of the instrument. In response to these readings, the algorithm is employed under computer control, to cause an element of the system to be raised and lowered as scanning proceeds, to translate the object along the optical axis to achieve focus in a dynamic manner. Frequently the pattern of raising and lowering is based upon a prescan of the overall object, from which positional information has been stored for use to control focus during the following examination scan. Typically instruments that enable dynamic focus adjustment with great precision require great cost.
A common attempt to avoid the cost and delays of prior art auto-focus techniques has been to incorporate a mechanism that forces a microscope slide against three buttresses, in an attempt to achieve precise location of the slide. Unfortunately, such techniques have unsatisfactory aspects, in causing the slide to deform, especially with bowing. This frequently results in loss of resolution.
SUMMARY OF THE INVENTION
The present invention provides a novel method and system for focusing a microscope. Though, at its broadest level of generality, it is applicable to all microscopes, it has particular advantage when associated in a system for automatic focusing, and it is presently considered most advantageous when the automatic focusing system is associated with a scanning system in which the object under inspection is translated under either a fixed or moving lens. The invention is especially applicable to instrument systems that operate under computer control such as optical scanners designed for reading biochips. While having a special application in achieving low cost automated scanning, in which focus is established once per slide, it also is advantageous in performing dynamic focus.
The invention provides a simple and low cost technique to bring the relevant surface (typically the top surf
Genetic Microsystems Inc
McGarrigle Philip L.
Sherr Alan B.
Spyrou Cassandra
Treas Jared
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