Focusing of microscopes and reading of microarrays

Abstract

Microscopes, including viewing and other microscopic systems, employ a hinged, tiltable plate to adjust focus on a flat object such as a microscope slide or biochip by motion, achieved by tilting, which is substantially normal to the focus point on the plane of the object. By employing two such tiltable arrangements, relatively long scan lines of e.g., flying objective, single pixel on-axis scanning can be accommodated. The tilting support plate is specifically constructed to provide tailored locations for different objects in series along the Y axis of the plate. The plate can accommodate heaters and cooled plates and/or the flat object being examined. In a fluorescence scanning microscope, locations are specifically adapted to receive microscope slides and biochip cartridges such as Affymetrix's “Gene Chip®”. A scanning microscope under computer control, employing such a focusing action, enables unattended scanning of biochips with a simple and economical instrument. Also shown are flexure-mounting of a support plate to define the hinge axis, techniques for automatically determining position and focus, and a rotatably oscillating flying micro-objective scanner combined with the tilting plane focus system. Construction and control techniques are shown that realize simple and accurate focusing. Methods of examination of biological materials are disclosed. Simple and efficient focused scanning with a flying micro-objective of ordered arrays of nucleotides and nucleic acid fragments carried upon a microscope slide or other substrate is disclosed. Quantified fluorescence imaging is economically achieved by combined use of the described scanning and focusing arrangement and use of simple and accurate calibration modules respectively for example for Affymetrix's “Gene Chip” microarray modules and for microscope slides.

What is claimed is:  
     
         1 . A microscope having an objective lens with a restricted field of view about an optical axis for examination or treatment of a portion of an object lying at the optical axis, including a tiltable focusing member defining a support plane for the object, the focusing member being mounted to rotate about a pre-established hinge axis to position said portion of the object at the focal plane of the microscope, the hinge axis lying in a plane substantially normal to the optical axis at a distance spaced therefrom sufficient that rotation of the portion of the object at the optical axis sufficiently approximates translation along the optical axis to enable focusing, a drive mechanism for rotating the focusing member about the hinge axis effective to bring into focus said portion of the object and a mechanism for rotating the plane about an axis orthogonal to the optical axis and the hinge axis so that the object can be scanned on a line substantially in a direction parallel to the hinge axis.  
     
     
         2 . The microscope of  claim 1  in which the mechanism for rotating the plane about an axis orthogonal to the optical axis and the hinge axis is a device mounted to rotate about a pre-established hinge extending along an axis that is perpendicular to the hinge axis and the optical axis of the instrument.  
     
     
         3 . The microscope of  claim 1  in which the drive mechanism for the tiltable focusing member is a driver located outwardly along the tiltable member, more distant from its hinge than the position in which the optical axis of the microscope intersects the tiltable member, whereby a lever effect is obtained in which a given motion of the driver results in a finer movement of the portion of the object at the optical axis.  
     
     
         4 . The microscope of  claim 3  in which the distance of the driver from the hinge axis is greater than about twice the distance of the optical axis from the hinge axis.  
     
     
         5 . The microscope of claims  1 ,  2  or  3  in which the position of the drive mechanism is controlled by an automated control system.  
     
     
         6 . The microscope of  claim 5  in which the control system includes a detector that senses the relationship of the object relative to the microscope.  
     
     
         7 . The microscope of  claim 6  in which the detector is an optical, capacitive or inductive position sensor that senses the height of the object.  
     
     
         8 . The microscope of  claim 7  in which the detector comprises a light source and a sensor arranged to determine the height of the object relative to the microscope on the basis of light reflected at an angle from the object.  
     
     
         9 . The microscope of  claim 6  in which the detector is a through-the-lens image analyzer constructed and arranged to enable determination of best focus position.  
     
     
         10 . The microscope of  claim 1  in which the pre-established hinge is defined by a flexible joint.  
     
     
         11 . The microscope of  claim 10  in which the flexible joint is defined by one or more planar springs or the flexible joint is defined by a thinned section of a support member.  
     
     
         12 . The microscope of claims  1 ,  2  or  3  in which a laterally movable carrier is mounted on the tiltable focusing member, the carrier arranged to advance the object, relative to the optical axis.  
     
     
         13 . The microscope of  claim 12  in which the direction of advance includes motion in the direction of the radius of the tiltable focusing member.  
     
     
         14 . The microscope of  claim 12  in which a linear guide rail is mounted on the tiltable focusing member, the moveable carrier member movable along the guide rail, the carrier member having a planar surface for supporting a planar object, the planar surface of the carrier member being parallel to the linear guide.  
     
     
         15 . The microscope of  claim 12  including a driver arranged to position the carrier member under computer control.  
     
     
         16 . A scanning microscope having an objective lens with a restricted field of view about an optical axis for examination of a portion of an object lying at the optical axis one picture element at a time, including a tiltable focusing member defining a support plane for the object, the focusing member being mounted to rotate about a pre-established hinge axis to position said portion of the object at the focal plane of the microscope, the hinge axis lying in a plane substantially normal to the optical axis at a distance spaced therefrom sufficient that rotation of the portion of the object sufficiently approximates translation along the optical axis to enable focusing, and a drive mechanism for rotating the member about the hinge axis effective to bring into focus said portion of the object, and a mechanism for rotating the plane about an axis orthogonal to the optical axis and the hinge axis, drive mechanism to produce relative oscillating scanning motion between the object and the objective lens in a direction transverse to the radial direction of the tiltable member and a photosensitive detector for detecting the stream of picture elements produced by the objective lens as it is scanned over the object, while the object is scanned substantially in direction parallel to the hinge axis.  
     
     
         17 . The microscope of  claim 16  in which the scanning microscope comprises a moving objective microscope.  
     
     
         18 . The microscope of  claim 17  in which the moving objective is supported upon an oscillating rotary arm that describes an arc generally centered on a radial axis of the tiltable member.  
     
     
         19 . The microscope of  claim 16  in which the objective has resolution of less than about 10 micron and a depth of field less than 200 micron.  
     
     
         20 . The microscope of  claim 1  or  16  in the form of a scanning microscope having a controller constructed to perform dynamic focus by varying the position of the drive mechanism during scanning.  
     
     
         21 . The microscope of  claim 20  in which the controller responds to through-the-objective image data.  
     
     
         22 . The microscope of  claim 21  including a system constructed to determine best focus data for an array of points during a prescan, to store this data, and to employ this data during microscopic examination of the object.  
     
     
         23 . A scanning microscope having an objective lens with a restricted field of view about an optical axis for examination of an object lying at the optical axis, including a tiltable focusing member defining a support plane for the object, the focusing member being mounted to rotate about a pre-established hinge axis to position said portion of the object at the focal plane of the microscope, the hinge axis lying in a plane substantially normal to the optical axis at a distance spaced therefrom sufficient that rotation of the portion of the object sufficiently approximates translation along the optical axis to enable focusing, and a drive mechanism for rotating the member about the hinge axis, effective to bring into focus said portion of the object, and a mechanism for rotating the plane about an axis orthogonal to the optical axis and the hinge axis to enable the object to be scanned substantially in a line in a direction parallel to the hinge axis, a drive mechanism to produce relative oscillating scanning motion between the object and the objective lens in a direction transverse to the radial direction of the tiltable member, and a photosensitive detector for detecting the stream of single picture elements produced by the objective lens, the microscope constructed and arranged to scan in a direction transverse to the radial direction of the tiltable focusing member, and a laterally movable carrier mounted on the tiltable member, the carrier arranged to advance the object, relative to the optical axis, in motion in the direction of the radius of the tiltable member.  
     
     
         24 . The microscope of  claim 23  in which the scanning microscope comprises a moving objective microscope.  
     
     
         25 . The microscope of  claim 24  in which the microscope includes a flying micro-objective lens.  
     
     
         26 . The microscope of  claim 24  in which the moving objective is supported upon an oscillating rotary arm that describes an arc generally centered on a radial axis of the tiltable member.  
     
     
         27 . The microscope of  claim 1 ,  16  or  23  in which the depth of field of the microscope is between about 30 and 200 micron, and the drive mechanism is a driver located outwardly along the tiltable member, more distant from the hinge than the position in which the optical axis of the microscope intersects the tiltable member.  
     
     
         28 . The microscope of  claim 27  in which the distance of the driver from the hinge axis is greater than about twice the distance of the optical axis from the hinge axis.  
     
     
         29 . A method of microscopic examination comprising providing a microscope having an objective lens with a restricted field of view about an optical axis for examination of a portion of an object lying at the optical axis, the microscope including a tiltable focusing member defining a support plane for the object, the focusing member being mounted to rotate about a pre-established hinge axis to position said portion of the object at the focal plane of the microscope, the hinge axis lying in a plane substantially normal to the optical axis at a distance spaced therefrom sufficient that rotation of the portion of the object sufficiently approximates translation along the optical axis to enable focusing, a drive mechanism for rotating the member about the hinge axis effective to bring into focus said portion of the object, and a mechanism for rotating the plane about an axis orthogonal to the optical axis and the hinge axis, enabling the object to be scanned substantially in a direction parallel to the hinge axis, and under control of an automated control system, moving the movable member to bring the portion of the object into the plane of focus of the microscope.  
     
     
         30 . The method of  claim 29  in which the object comprises biological material.  
     
     
         31 . The method of  claim 30  in which the object fluoresces and the microscope is constructed to detect such fluorescence.  
     
     
         32 . The method of  claim 31  in which the object comprises an ordered array of nucleotides that may fluoresce.  
     
     
         33 . The method of  claim 31  in which the object comprises an ordered array of oligonucleotides.  
     
     
         34 . The method of  claim 31  in which the object comprises an ordered array of deposits of nucleic acid fragments.  
     
     
         35 . The scanning microscope of  claim 23  or  25  or the method of  claim 29  in which the scanning drive mechanism is a limited rotation oscillating motor operating at a substantial frequency.  
     
     
         36 . The scanning mechanism of  claim 35  in the form of a fluorescence detection microscope.  
     
     
         37 . The method of  claim 29  in which the microscope is a scanning microscope and includes a drive mechanism for rotating the focusing member about the hinge axis, effective to bring into focus said portion of the object, drive mechanism to produce relative oscillating scanning motion between the object and the objective lens in a direction transverse to the radial direction of the tiltable member and a photosensitive detector for detecting the stream of picture elements produced by the objective lens.  
     
     
         38 . The method of  claim 37  in which the microscope is constructed and arranged to repeatedly scan in a direction transverse to the radial direction of the tiltable member, and a laterally movable carrier mounted on the tiltable member, the carrier arranged to advance the object, relative to the optical axis, in motion in the direction of the radius of the tiltable member.  
     
     
         39 . The method of performing quantified fluorescence microscopy comprising providing a microscope according to any of the claims  1 ,  16  or  23 , calibrating the microscope with a calibrating tool having a surface layer of effective fluorophores, and subsequently scanning a slide or biochip having an array or micro array of specimens.  
     
     
         40 . A microscope having an objective lens with a restricted field of view about an optical axis for examination or treatment of a portion of an object lying at the optical axis, including a tiltable focusing member defining a support plane for the object, the focusing member being mounted to rotate about a pre-established hinge axis to position said portion of the object at the focal plane of the microscope, the hinge axis lying in a plane substantially normal to the optical axis at a distance spaced therefrom sufficient that rotation of the portion of the object at the optical axis sufficiently approximates translation along the optical axis to enable focusing, a drive mechanism for rotating the focusing member about the hinge axis effective to bring into focus said portion of the object, and a mechanism for rotating the plane about an axis orthogonal to the optical axis and the hinge axis, so that the object can be scanned on a line substantially in a direction parallel to the hinge axis, the lens that rotates about an axis being mounted on an oscillating arm and the hinge axis being located outwardly beyond the lens-carrying end of the arm, further from the axis of rotation of the arm than the location of the lens.  
     
     
         41 . A microscope having an objective lens with a restricted field of view about an optical axis for examination or treatment of a portion of an object lying at the optical axis, including a tiltable focusing member defining a support plane for the object, the focusing member being mounted to rotate about a pre-established hinge axis to position said portion of the object at the focal plane of the microscope, the hinge axis lying in a plane substantially normal to the optical axis at a distance spaced there from sufficient that rotation of the portion of the object at the optical axis sufficiently approximates translation along the optical axis to enable focusing, a drive mechanism for rotating the focusing member about the hinge axis effective to bring into focus said portion of the object, and a mechanism for rotating the plane about an axis orthogonal to the optical axis and the hinge axis, so that the object can be scanned on a line substantially in a direction parallel to the hinge axis, the support plane being adapted to define at least two positions in sequence along the Y axis to receive objects or modules of differing dimension.