Excitation and imaging of fluorescent arrays Number:7,154,598 from the United States Patent and Trademark Office (PTO) owispatent

Title: Excitation and imaging of fluorescent arrays

Abstract: A support for an array of fluorescently labeled samples comprises a transparent body defining: (a) an array-support surface and (b) under the support surface, in spaced apart relationship thereto, a field of embedded optical features exposed to be illuminated by a broad light beam of excitation radiation addressed to the support from a predetermined general direction selected to produce a surface wave effect at the support surface, the field of embedded optical features and the support being so constructed that light of the beam incident on the features is launched through the support at an angle to the support surface that produces the surface wave effect of radiation in the manner that it can produce fluorescence from the labeled samples to be imaged beyond the support from a direction different from the direction of the illumination. Fine transmissive and reflective features having surfaces generally normal to radiation substantially at the critical angle, and a grating illuminated at a non-normal surface are shown. A data acquision system employing an elastic rotary motion reducer driven by a stepper motor, under computer control, directs a broad illumination beam through a series of small angular increments, an image is taken at each increment by a CCD camera, and based upon energy references on the array-support surface, a quilt image is formed, based on responses of the energy references associated with localized regions of each image.

Patent Number: 7,154,598 Issued on 12/26/2006 to Montagu,   et al.

---

Inventors:	Montagu; Jean I. (Brookline, MA), Fantone; Stephen D. (Lynnfield, MA)
Assignee:	Decision Biomarkers, Inc. (Waltham, MA)
Appl. No.:	10/618,838
Filed:	July 14, 2003

---

Current U.S. Class:	356/244 ; 356/445
Current International Class:	G01N 21/01 (20060101); G01N 21/55 (20060101)

---

References Cited [Referenced By]

---

U.S. Patent Documents

	4649280		March 1987		Holland et al.
	4857273		August 1989		Stewart
	RE33581		April 1991		Nicoli et al.
	5166515		November 1992		Attridge
	5341215		August 1994		Seher
	5344784		September 1994		Attridge
	5351127		September 1994		King et al.
	5437840		August 1995		King et al.
	5486452		January 1996		Gordon et al.
	5629213		May 1997		Kornguth et al.
	5631170		May 1997		Attridge
	5633724		May 1997		King et al.
	5666197		September 1997		Guerra
	5754514		May 1998		Guerra
	5776785		July 1998		Lin et al.
	5822472		October 1998		Danielzik et al.
	5830766		November 1998		Attridge et al.
	5910940		June 1999		Guerra
	5945334		August 1999		Besemer et al.
	6078705		June 2000		Neuschafer et al.
	6161437		December 2000		Brennan et al.
	6255642		July 2001		Cragg et al.
	6268125		July 2001		Perkins
	6289144		September 2001		Neuschafer et al.
	6356676		March 2002		Herron et al.
	6362004		March 2002		Noblett
	6469755		October 2002		Adachi et al.
	6579721		June 2003		Natan et al.
	6579726		June 2003		Natan et al.
	6692974		February 2004		Perkins
	2002/0018199		February 2002		Blumenfeld et al.
	2002/0021443		February 2002		Venkatasubbarao et al.
	2003/0129654		July 2003		Ravkin et al.
	2003/0151735		August 2003		Blumenfeld et al.
	2003/0223059		December 2003		Li

Foreign Patent Documents

	0 575 132		Dec., 1993		EP
	WO 90/06503		Jun., 1990		WO
	WO 01/34846		May., 2001		WO
	WO 01/59503		Aug., 2001		WO

Other References

Dubendorfer et al., "Reference and Sensing Pads for Integrated optical Immunosensors", SPIE, 2928:90-97(1990). cited by other . Dunphy et al., "New planar waveguide attenuated total reflectance techniques for organic thin film spectroscopy and chemical sensing", SPIE, 3602:140-148 (1999). cited by other . Duveneck et al., "A Novel Generation of Luminescence-based Biosensors: Single-Mode Planar Waveguide Sensors", SPIE, 2928:98-109 (1998). cited by other . Duveneck et al., "Review on Fluorescence-Based Planar Waveguide Biosensors", SPIE, 3858:59-71 (1999). cited by other . Enderlein et al., "Comparison between a conventional epifluorescence microscope and a new highly efficient evanescent wave detector in single molecule spectroscopic applications", SPIE, 3602:94-101 (1999). cited by other . Harrick N. J., "A Thin Film Optical Cavity to Induce Absorption or Thermal Emission", Applied Optics, 9:2111-2114 (1970). cited by other . Harrick N. J., "Multiple Internal Reflection Fluorescence Spectrometry", Analytical Chemistry, 45:687-691 (1973). cited by other . "Modern Techniques in Applied Molecular Spectroscopy", Edited by Francis M. Mirabella, Equistar Chemicals, LP, pp. 146-147 (1998). cited by other . Pinkel et al., "High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays", Nature Genetics, 20:207-211 (1998). cited by other . Silk, Ely, "LED Fluorescence Microscopy in Theory and Practice", Microscope, 50:101-118 (2002). cited by other . Wittrup et al., "Fluorescence Array Detector for Large-Field Quantitative Fluorescence Cytometry", Cytometry, 16:206-213 (1994). cited by other.

Primary Examiner: Stafira; Michael P.
Attorney, Agent or Firm: Fish & Richardson P.C.

---

Claims

---

What is claimed is:

1. A support for an array of fluorescently labeled samples comprising a transparent body that enables imaging of the array, the transparent body defining: (a) sample-receiving array-support surface disposed to receive an array of the labeled samples, the surface adapted to enable a surface wave effect to travel laterally in the transparent body adjacent the surface and (b) a field of embedded optical features located below and spaced from the array-support surface so that light reaching the optical features must then pass through the transparent body to reach the support surface and the labeled samples, the field of optical features, exposed to be illuminated by a broad light beam of excitation radiation, the field of optical features and the support being constructed and arranged so that a light beam addressed to the optical features can be launched through the transparent body at an acute angle to said sample-receiving array-support surface to produce a surface wave effect that travels laterally in the transparent body adjacent the surface, thereby to excite fluorescence from the array of samples to enable an image of the fluorescing array along an axis independent of the angle of the beam of illumination.

2. The support of claim 1 in which said support surface is an uninterrupted planar surface that bears spots of sample of spot size between about 50 and 500 micron.

3. The support of claim 1 in the form of a microscope slide constructed to enable imaging of the support surface alone an axis normal to the support surface.

4. The support of any of the claims 1 in which said support surface defines a wetted surface of a flow cell, the flow cell having a window for viewing fluorescence from said support surface, and the field of embedded optical features is unwetted.

5. The support of any of the claim 1 in which said embedded optical features are exposed for illumination by a beam directed toward the side of the support opposite from the array-support surface.

6. The support of claim 5 in which said embedded optical features comprise transmissive facets disposed at an angle to the array-support surface and substantially at right angles to the general direction of the selected beam.

7. The support of claim 6 in which said angle of said beam lies between about 30 and 60 degrees to the normal to said array-support surface.

8. The support of claim 7 in which said angle of said beam lies between about 38 to 44 degrees to said normal.

9. The support of the claim 6 in which said optical features are defined by sides of triangular grooves.

10. The support of claim 5 associated with an imager having an axis normal to the support surface and in which said optical features define a diffraction grating arranged to receive an illuminating beam at an angle substantially less than perpendicular to said support surface.

11. The support of claim 1 in which said embedded optical features comprise reflective surfaces exposed for illumination by a beam directed toward said support surface.

12. The support of claim 11 in which said reflective surfaces are defined by sides of triangular grooves.

13. The support of claim 1 in which said optical features are so defined and arranged in a pattern to accomodate variations in the critical angle of said array-support surface.

14. The support of claim 1 wherein the body of said support is substantially comprised of disposable plastic.

15. The support of claim 14 in which said disposable plastic is polystyrene, PMMA or polycarbonate.

16. The support of claim 1 wherein said embedded optical features comprise features of an article cast of molten material in a mold.

17. The support of claim 1 wherein said embedded optical features comprise features that are embossed or press-molded.

18. The support of claim 1 having a plurality of coatings or layers along said array-support surface, the array-support surface comprising a layer of a substance capable of adhering to an adjacent lower substance of the support and to the samples.

19. The support of claim 18 in which said outer layer comprises polystyrene.

20. The support of claim 1 having a plurality of coatings or layers along said support surface that define a wave guide for a wave propogating along that surface.

21. The support of claim 2 having reflective or transmissive embedded optical features, said support surface being adapted to receive samples of a predetermined minimum spot size, the size and periodicity of said features being in the range of about 1/4 th to 1/50th said spot size.

22. The support of claim 21 wherein said size and periodicity is of the order of 1/10th said spot size.

23. The support of claim 1 in which the portion of the array support surface on which said array of samples will reside lies directly opposite to the field of embedded optical features below and spaced from said surface.

24. The support of claim 1 carrying energy references.

25. The support of claim 24 in which the array-receiving area of said array-support surface is defined as a matrix of localized planar regions, at least one energy reference being associated with each of said localized regions.

26. The support of claim 24 in which at least some of said energy references comprise Kapton.

27. The support of claim 24 in which at least some of said energy references comprise labeled biological material.

28. The support of claim 24 in which at least some of said energy references comprise a selected glass or quartz.

29. The support of claim 1 constructed to launch an evanescent wave along said array-support surface.

30. The support of claim 1 constructed to guide one or more wave modes along said support surface.

31. The support of claim 1 constructed to cause the sample spots to function as Fabry Perot cavities during their absorption of energy.

32. The support of claim 1 carrying an array of sample spots.

33. The support of claim 32 wherein the sample spots are fluorescently labeled spots of biological material.

34. The support of claim 32 wherein the sample spots are of size between about 50 and 500 micron.

35. The support of claim 34 in which said embedded optical features are transmissive or reflective and have a size and periodicity of between about 1 and 50 micron.

36. The support of any of the claim 32 in which the spots have shape determined by being deposited as fluid spots from which liquid carrier has evaporated.

37. The support of claim 36 in which said spots are pin-deposited spots.

38. The support of claim 36 in which energy references associated with the sample spots comprise spots the shape of which has been determined by being deposited as fluid spots from which liquid carrier has evaporated.

39. The support of claim 1 disposed at an illumination and imaging station.

40. The support of claim 39 in which the imaging station includes a wide angle imaging system viewing the support surface along an axis normal to the support surface.

41. The support of claim 40 in which the imaging system is constructed to image an array area of about 15 mm by 15 mm.

42. The support of claim 41 in which the support is constructed of the general size and shape of a microscope slide, and said array-support area defines two areas to be imaged, each area of about 15.times.15 mm, and each associated with its respective field of said embedded optical features.

43. The support of claim 39 in which said imaging station comprises a stationary CCD camera.

44. The support of claim 43 in which said camera has a resolution of about 612.times.612 pixels.

45. The support of any claim 1 in which an imaging system is arranged over the array-support surface of the support and having a viewing axis normal to said surface.

46. The support 39 associated with a mirror constructed to operate with a broad beam light source, the mirror sized to direct the beam from the light source toward said support in a manner that the field of embedded optical features launch the radiation at said angle to said support surface.

47. The support of claim 46 associated with a tilt control mechanism capable of receiving tilt angle signals from a controlling computer and to direct the beam to said support, or move the support relative to the beam, at the commanded angles.

48. The support of claim 47 in which the tilt control mechanism has a range to change the angle of the beam reaching the support surface by about 30 degrees.

49. The support of claim 47 associated with a driver comprising a stepper motor and an elastic motion divider.

50. The support of claim 49 in which the stepper motor is a rotary stepper motor, and the elastic motion divider comprises a weak torsion spring driven by the rotary stepper motor, the weak spring driving a relatively stiff elastic torsional resistance, the mirror or support mounted in the region generally between the weak torsional spring and the torsional resistance so that the mirror is deflected by an angle substantially less than 10% of each step of the stepper motor.

51. The support claim 48 in which the mirror or the support is adapted to advance in steps of the order of 0.1 milliradian.

52. The support of claim 1 associated with an imaging system adapted to acquire a series of images over an angular range of illumination of the support, and to determine the results of imaging by comparing data obtained in each image.

53. The support of claim 52 in which the support carries energy references, and said comparison is based on the imaged results of those references at the various angles of illumination.

54. The support of claim 53 in which the support carries a matrix of energy references distributed over the image area, and localized regions for the final image are selected based upon the imaged results of references associated with localized regions in the respective images of the set, and a final image comprises a quilt formed of localized portions of the images selected from said set of images, or the sum of two or more localized portions.

55. The support of claim 1 in which the light source produces at the support at least a quasi-collimated beam, of not more than 5 degrees convergence or divergence.

56. The support of claim 55 in which the beam has no more than about 2 degrees convergence or divergence.

57. The support of claim 55 in which the light source comprises at least one array of light emitting diodes.

58. The support of claim 1 in which the beam is substantially collimated and the light source comprises at least one laser.

59. The support of claim 1 in which the light source comprises a multiplicity of selectable light source units whose outputs are merged into a single path leading to a mirror that directs the light to the support.

60. The support of any claim 1 having a matrix of energy references associated with the array support surface, and an imaging system adapted to produce image data normalized with respect to sensed results at said energy references.

---

Description

---

TECHNICAL FIELD

This invention relates to the field of microscopic imaging of large fields of view. The invention provides optical systems and methods for high speed imaging of arrays of samples containing fluorescently labeled material, e.g. biologic polymer sequences such as protein, nucleic acid and oligonucleotide arrays, and other fluorescently labeled materials.

The aim of the present invention is to achieve improved performance versus cost of such imaging systems. In general, in low-cost implementations of the invention, it is foreseen that the invention will enable clinical and diagnostic uses that have not previously been regarded as practical economically. Similarly, it is foreseen that lower order educational and investigative uses will be enabled by the invention. The invention is also foreseen to provide higher quality data and better performance in a number of respects than presently possible with available commercial equipment.

According to one aspect of the invention, a versatile, disposable support is provided having fine embedded optical features (microelements) located under the array of fluorescently-labeled samples. By illuminating the field of these features with a broad beam at one or a series of selected angles, the support plays an important role in the lowered cost and accurate functioning of the overall fluorescence excitation and imaging system.

According to another aspect of the invention, novel illumination, imaging and data acquisition techniques are provided that can accommodate variations in the optical characteristics of low-cost disposable supports over their broad surfaces so that data of high accuracy is obtainable despite the low-cost of the system and its disposable components.

Numerous other features of the invention that will be described contribute to achieving these overall goals.

BACKGROUND

Because the conversion efficiency of fluorophores is extremely low, fluorescence microscopy is an extremely inefficient process in which light source-to-detector efficiency may be in the range of parts or a fraction of a part per billion. Another limitation of fluorescence imaging is that the intensity of an illumination source needs to be limited to avoid destruction of the sample or so-called "photo bleaching" in which the capability of the fluorophores to fluoresce is diminished; even before the condition of photo bleaching is reached, the behavior of most fluorophores becomes significantly non-linear or unpredictable, imposing further optical constraints. Numerous non-optical constraints also affect the practicality of the fluorescent microscope design, such as the acceptable duration of a scan of an array, the reliability of the data, the cost of the biochip, the processing complexity and the cost of the scanner.

The state of the art of fluorescence biochip imaging has accordingly been guided by the necessity for a microscope reader to have a very efficient fluorescence-emitted light capture capability. As well, a very shallow depth of field has been important so that only the very thin layer of biological material is imaged, to avoid optical noise perturbations that may be emitted from the support member under the array. This approach has lead to complex and expensive systems: epi-fluorescent scanning confocal microscope readers and cooled CCD camera-based readers that have a small field of view and require moving or tilting with respect to two axes to scan the array.

Some epi-fluorescent confocal or near confocal scanning microscopes employ high precision radiation-directing systems driven by galvanometers or motors and single point detectors such as PMTs or diodes. The text edited by Mark Schena and published by Bio Techniques Books, Natick, Mass. pp. 53 64 carries a summary description of a number of such commercial instruments. Others are also described in U.S. Pat. No. 3,013,467 (Minsky); U.S. Pat. No. 5,459,325 (Hueton); U.S. Pat. No. 5,981,956 (Stern); U.S. Pat. No. 5,895,915 (DeWeerd); U.S. Pat. No. 5,585,639 (Dorsel); U.S. Pat. No. 5,646,411 (Kain); U.S. Pat. No. 5,672,880 (Kain); U.S. Pat. Nos. 6,335,824; 6,201,639 and 6,185,030 (Overbeck).

Examples of fluorescence microscopes that use a CCD array imager as a detector are shown in the Handbook of Biological Confocal Microscopy edited by James Pawley, Plenum Press, 1989 and 1995. Others can be found in U.S. Pat. No. 5,900,949 (Sampas).

In total, the low efficiency of the fluorescent conversion and the other factors mentioned have lead to slow and costly reading of conventional biochips whether by high accuracy scanning of the confocal microscope or by the high cost system of a cooled CCD-based camera associated with a high accuracy scanning mechanism. Such expensive systems have mainly been employed in academic studies and in large efforts directed to drug discovery. No practical way has emerged to enable the technology to be adapted to much lower cost uses such as in medical clinics and diagnostic laboratories, in veterinary medicine, in dealing with agricultural crop diseases and food and water processing, and in lower level educational and investigational laboratories.

An object of the invention is to provide an improved fluorescence imaging approach, and in particular a diagnostic tool that is low-cost and highly effective, useful in direct patient diagnosis and treatment in medicine, as well as for other purposes such as those mentioned.

The invention employs surface light effects to concentrate the illumination in the vicinity of the plane of the sample array. In this way the excitation energy density can be enhanced at the surface of the sample relative to objects at other depths, and imaging can be done without requiring that the imaging system, itself, have a very shallow depth of field. Numerous patents exemplify application of this general technology to experimental microscopy. Examples are U.S. Pat. Nos. 5,633,724; 5,351,127 and 5,437,840 (King) and U.S. Pat. No. 5,341,215 (Seher) and European Patent Application 93304605.4 (EP 0575 132 A1) (King) as well as trade journal articles such as Photonics Spectra, February 2000, pages 24 26. The technique has been described in the Conference on Advances in Fluorescence Sensing Technology IV, 1999, Vol. 3602 pp. 140 148 and pp. 94 101; the Proceedings of SPIE Vol. 4252 pp. 36 46, SPIE Vol. 2928 pp. 90 109 and SPIE Vol. 3858 pp. 59 71, and the book Internal Reflection Spectroscopy by F. M. Mirabella Jr. and N. J. Herrick. See also U.S. Pat. Nos. 5,910,940, 5,754,514 and 5,666,197 (Guerra) and U.S. Pat. No. 6,078,705 (Neuschafer et al.), from different fields.

The potential of surface wave techniques for illuminating and detecting specific binding analytes is also well documented. A number of the techniques proposed use prisms or gratings to induce evanescent fields. Other related techniques such as Surface Plasmon Resonance (SPR) couple evanescent incident radiation into a mode generated between a thin metal layer, such as gold or silver, and a dielectric layer such as silicon or phosphate glasses or silane. Such techniques have been described in U.S. Pat. Nos. 5,830,766 and 5,631,170 and PCT WO90/06503 (Attridge).

A preferred technique to create an evanescence fluorescence-enhancing surface wave described in certain of the above references is to illuminate the substrate at a defined illumination region, via an intermediate support. The light arrives at the surface at a suitable angle to induce an evanescent wave on the surface. To excite the sample, the energy then travels laterally along the surface to a separately defined sample region where the sample is excited. Often a large 90 degree prism member has been employed which is coupled to a separate member carrying the biology. Varying the angle of the incident light permits the accommodation of a range of illumination wavelengths. A conventional microscope has then been used to inspect the fluorescence. By this technique, illumination of the sample has been enhanced without the penalty of incident light being reflected into the objective of the reader. Often a fluid coupling agent between the mated optical parts is required. A variation on this technique has used a grating at an illumination region separate from the imaging region, and the angle of illumination of the grating has been tuned to maximize the signal, see Review on Fluorescence-Based Planar Wave Guide Biosensors, Duveneck et al., Vol. 3858, 1999. In another field a large transparent optical block has been employed to couple light to a sample at various angles for sectioning the sample at various levels, see e.g. U.S. Pat. No. 6,255,642 (Cragg et al.)

Applications exist where such previously designed surface wave systems may be justified, but these designs have not proven suitable for low-cost clinical usage and the like.

One previously proposed substrate for imaging a fluorescing array has been a microscope slide having an interference grating buried under a thin layer of high index glass. In that example the grating was arranged to reflect normal incident light that has not been absorbed by the sample (a very large fraction) at a suitable angle to induce an evanescent wave at the sample. The intensity of the evanescent wave can be more than an order of magnitude greater than that of the original incident light beam, but, because gratings reflect normal incidence beams at different angles for different wavelengths, to operate beneficially, each slide was generally restricted to its design wavelength.

U.S. patents disclosing other use of gratings include U.S. Pat. No. 5,822,472 (Danieizik et al.) and U.S. Pat. Nos. 6,078,705 and 6,289,144 (Neuschafer et al.) In these and in other cases the array to be imaged has been incorporated in a flow cell arrangement, see also for example U.S. Pat. Nos. 5,166,515; 5,344,784; 5,631,170 and 5,830,766 (Attridge); and U.S. Pat. No. 4,857,273 (Stewart).

Prior art CCD-based, fluorescent, conventional or confocal scanning microscope systems can provide high quality images of material located on the top surface of a support. But in their compromise between depth of field, energy collection efficiency, laser power, damaging of the sample by photo bleaching, capture time requirements and cost and the precision and complexity associated with establishing evanescence light concentration, high cost of the support, uncertainties caused by operating in a non-linear region of the fluorophores, etc., they have not been altogether satisfactory.

It is well known that evanescent illumination of a biochip has had the potential to offer a much higher signal than conventional illumination, such that a CCD-based imaging system can be used to acquire the image information on the biochip without loss of data. The apparent requirements and cost of prior proposals to reliably induce evanescence, however, has apparently impeded commercialization of the techniques.

The present invention provides low-cost, robust and wave-length versatile systems and techniques incorporating surface wave technology that are foreseen to enable a breakthrough in the technology.

SUMMARY

According to the invention, illumination of a broad surface of a biochip substrate is employed to illuminate a large array of spots of the biological material. A pattern of fine embedded optical features (microelements) disposed over a broad surface of the transparent, array-carrying substrate, beneath the array, intercepts the incoming broad beam of illumination traveling at a selectable angle and enables the illumination to travel through the substrate at an appropriate angle to the surface carrying the array of samples to establish an evanescent wave or other surface wave illuminating effect that concentrates the illumination energy substantially at the plane of the array of biological samples.

The present invention also provides novel systems, methods and apparatus for accepting and employing the novel disposable biochip for high accuracy imaging of large regions on the biochip. In particular, the invention provides for obtaining an image at a selectable wave length of thin material of organic or inorganic nature on the support surface at high resolution, high sensitivity and high speed.

The function of the fine embedded optical features of the array support is to efficiently assist the launching of the excitation beam at the suitable angle so as to stimulate the excitation of the fluorophores on the broad surface of the support that lies opposite the embedded features, without causing equivalent emission in the base support material that would create optical noise perturbation. Since they are embedded in the supporting substrate, the embedded optical features do not necessitate coupling of one member to a discretely separate biochip or substrate member. A simple wide field of view CCD-based camera can be used with this system to obtain equivalent information to that which has been obtainable from more expensive commercial systems; or, by employing a more complex CCD camera or scanning microscope principles, superior image information can be obtained, according to the invention.

In one aspect of the invention, embedded optical features of the sample support are simple, shallow transmissive or reflective formations that enable the illumination of the sample and reduce performance requirements of the microscope.

Another aspect of the invention is the provision of a cassette or flow cell that incorporates the described array support within a reaction chamber suitable for further processing in an automated or semi-automated protected environment.

Important features of the design include computer-controlled variation of the angle of approach of the illumination beam to the biochip, and dynamic varying of that angle by steps over a range, taking a full wide image at each step, and processing that data in manner that optimizes the signal obtained for localized portions of the biological array being imaged, to provide a composite or quilted image of localized best results.

Another aspect of the invention is a system which controls incident illumination to optimize surface stimulation and fluorescence emission, that involves both sequential illumination of the array in steps over a range of incident angles and novel protocols for selecting the results from the optimum angle for localized regions of the array based on energy references strategically located with respective localized regions of the array on the disposable biochip or substrate.

In the present invention, because of the high level of excitation at the plane of the sample, a camera with a large depth of field is employed, e.g. a CCD camera, and because the time required to take a complete image of the wide area may be of the order of one second, the very small fraction of a second required to index from one angle of illumination to the next for another full image is negligible; likewise the overall time to take full images in steps over a range of 10 or even 20 adjacent angular increments and to process the information for optimizing the reading for each local portion of the imaged array, according to the invention, is readily affordable.

A particular aspect of the invention is a fluorescence reader, preferably a CCD-based imager or camera, capable of broadly illuminating and acquiring a wide field of view image of the sample. One feature of the reader is a system that provides a variable angle of incidence, broad illuminating light beam constructed with suitable range and resolution that compensates for the variation of the critical angle encountered with a wide range of different biological products deposited on the biochip as well as variations due to manufacturing tolerances in the geometry of the disposable biochip and variations of the index of refraction of its base material.

Another aspect of the invention is the use of quasi-collimated light, i.e. light that is slightly divergent or convergent, i.e. divergent or convergent over an angular range of no more than about 5 degrees, preferably less than about 2 degrees. At the cost of somewhat less efficient use of the light, the angular spread helps to ensure that at least some of the light reaches the array surface at the angle required for producing a desired surface effect in the presence of localized imperfections or misalignments. In conjunction with use of quasi-collimated light, it is advantageous to employ a low-cost L.E.D. chip having an X-Y array of light-emitting diodes as an inexpensive, slightly divergent light source for a low-cost version of the instrument, in lieu of use of a laser light source.

In such ways as described above, a simple, low-cost sensor platform, i.e. a broadly illuminated biochip substrate itself, is able, at various selectable wave lengths, to induce luminous excitation for fluorescently exciting a broad array of biological material deposited, spotted or otherwise provided on the substrate. This disposable biochip and its microscope reader cooperate to optimize consistent and reproducible imaged information within practical commercial manufacturing tolerances.

In preferred embodiments, for producing stepped motion of a broad beam-reflecting mirror for taking a succession of images of the fluorescing array at angular increments, an elastic motion divider is employed in which a motor, preferably a stepper motor, deforms a weak spring attached to a stronger spring of a similar nature anchored rigidly at the other end. The motion at the interface between the weak and stronger springs is approximately proportional to the ratio of the rigidity of the two springs, and is the location where the mirror for stepping the angle of the beam is mounted. Preferably the springs are torsion springs driven by a rotary stepper motor. In certain embodiments two such motion reducers are mounted in cascade to achieve a two axis mirror motion. Damping of settling motions is advantageously employable to increase the operating speed of the system. While this elastic motion reducer is presently preferred for its simplicity and characteristcs, the tilt mirror (or the tilting of the support) can for instance be controlled by other known precision mechanisms, for instance galvanometers, gear-reduced stepper motors DC motors with encoders, and any other suitable motors with motion reduction mechanisms.

Selection of best localized regions from a set of images taken at adjacent angles based on energy references, for forming a quilt or tapestry composite image is preferred. In certain cases, two or more images, or two or more localized images from respective regions, may be added to obtain an image, or localized region image for a tapestry, having improved signal to noise ratio.

While the image acquisition methods proposed here are preferably applicable to wide field of view, two-dimensional CCD-based microscope systems, the unique disposable biochip substrate can also be used with a one dimensional CCD-based microscope with single direction scanning or with conventional fluorescence microscopes, confocal microscopes or flying spot scanning microscope systems.

The fine embedded optical features that assist in launching the light to the top surface are defined such as to cooperate with the angle of the generally collimated, wide beam incident on the support. The periodic pattern of the embedded optical features as well as the array-receiving area of the substrate can extend over the dimension of the field of view of the reading instrument and is selected in accordance with the dimension of the spots or other features of interest of the material to be inspected, as well as in accordance with the angle of incidence of the excitation beam. By suitably fine dimensions of the embedded optical features, the obscuration associated with the edges of the optical features is caused to limit artifacts to those that are small in effect on the response with respect to the response over the full dimension of the spots of the inspected sample. Preferably, to avoid detrimental or non-uniform artifact effects by the edges of the optical features, the period of the features is selected to be in the range between about 1/4 to 1/50 of the size of the sample spots, preferably of the order of 1/10 the dimension of the smallest sample spot size to be employed on the respective substrate.

For the range of biological spot sizes between 50 and 500 micron diameter, a periodicity of the embedded optical features is preferably between about 1 and 50 micron.

For providing the embedded optical features the invention in particular includes the support formed with triangular shaped grooves at a suitable angle in transmissive or reflective geometry such that the light moves from the facets of the grooves through the transparent body of the support at the critical or other appropriate angle to the top surface of the support. In certain preferred embodiments the features are at the bottom surface of the support and are protected, for handling purposes, with a layer of organic or inorganic material.

According to a further aspect of the invention, the sample support with the embedded optical features is constructed as a substrate similar in size and shape to a conventional microscope slide. This provides a disposable substrate that, in size and shape, is entirely familiar to clinical laboratory personnel and the like, and suitable to be handled and spotted by robotic equipment that already exists in laboratories or clinics. In some preferred instances, the top surface of the sample support receives the sample to be imaged and the bottom surface is shaped to transmit light to the top surface such as to cause an evanescent wave or to induce another surface-concentrating effect to the light energy at that surface, to concentrate illumination energy at the plane of the array. For producing such effect, the excitation light enters the bottom surface, e.g. via facets of the embedded optical features, at an angle approximately normal to the critical angle which is defined by the top surface of the substrate, the material of the biochip and the biological medium/air interface. By approaching with approximately normal incidence, detrimental refraction effect by the body of the support is avoided.

According to another aspect of the invention, the sample support having the embedded optical features is likewise built in the form a substrate similar in size and shape to a conventional microscope slide, the top surface which receives the sample to be imaged is coated with single or multiple layers in manner to create wave guide conditions adjacent the top surface, and the bottom surface is shaped to transmit light to the top surface such as to cause light energy to concentrate in that wave guide along the top surface. The excitation light again may enter the bottom surface, e.g. via facets of the embedded optical features, approximately normal to the appropriate angle for entering the wave guide, defined by the top surface, the material of the biochip and the biological medium/air interface on it.

For such transmissive embedded optical features, the invention in particular includes the bottom of the support formed as triangular shaped grooves at a suitable angle such that light incident upon the features from outside is transmitted through the transparent body of the support at the critical angle to the top surface of the support. In certain preferred embodiments that surface is protected, for handling purposes, with a layer of organic or inorganic material.

In another aspect of the invention, the sample support having the embedded optical features is also preferably built in the form of a transparent substrate similar in size and shape to that of a conventional microscope slide, and the top surface receives the sample to be imaged. In this case, the broad top surface bearing the sample array is adapted to receive the excitation light from above e.g. at an approximately 45-degree angle of incidence to the top surface. The bottom surface is shaped to define fine reflective features and is coated to reflect that light which enters the substrate from the top and is not absorbed or deflected by the sample being inspected. Due to the orientation of such fine reflective features, the reflected light is directed back toward the top surface at the desired angle, e.g. the critical angle that creates a surface wave effect, such as an evanescent wave along the top surface. By suitable orientation of the fine reflective surfaces relative to the predetermined location of the illuminating source, substantially no light is directed back into the source, nor is it directed into the imager.

For such reflective embedded optical features, the invention includes the bottom of the support formed as triangular shaped grooves having walls disposed at suitable angles and coated with a reflective material such as aluminum, silver or gold, for suitably internally reflecting incoming light from the top, to be redirected at the critical angle to the top surface. In certain preferred embodiments that reflective surface is protected for handling purposes with a layer of organic or inorganic material.

In order to create a surface wave at the top of a substrate, the light must travel through the substrate to the surface at an angle (the critical angle) defined by the index of refraction of the substrate. If the substrate is polycarbonate or polystyrene with an index of about 1.59 and the sample on the top surface has an index of refraction of 1 (equal to that of air) the critical angle is approximately 38.9 degrees to the normal. The choice of substrate material (e.g. polystyrene) and the index of refraction of the sample defines the angle of incidence with the normal to the surface. For most common biological sample materials the critical angle is in the range of approximately 30 to 60 degrees, a range of about 30 degrees or 0.5 radian, considering the use of materials having conventional indices of refraction as well as those materials having more extreme indices of refraction, some of which are commercially available while others are to be expected. In many cases the preferred range is from 38 to 44 degrees to the normal, a range of about 6 degrees or 0.1 radian, using materials such as those employed in the preferred embodiments described herein.

In some embodiments of the invention, the top surface of the support is coated with a material of lower index of refraction than that of the support, and in other embodiments it is coated with alternate layers of high and low indices of refraction to create a wave guide condition in which the light is confined as it moves along the surface bearing the array of biological material until it is absorbed by the biological material. In such cases, advantageously according to the invention, the top-most layer over such coating layers is a layer of biology-binding material deposited in solution with a solvent that evaporates such as chloroform or other manner of coating such as by vapor deposition. Polystyrene is a suitable such material to which biological material binds, and there are other known coatings that have the characteristic of both adhering to the selected final coated layer of the support and to the biological material to be deposited.

In still other embodiments of the invention the deposited and dried sample spots themselves are sized and adapted to define Fabry Perot-like resonant cavities for the illuminating radiation that proceeds along the plane of the array, the excited fluorescent radiation being able to escape from the sample because of its differing wavelength. In some cases, to properly adjust the spacer thickness of the Fabry Perot cavity, a coating on the support of special thickness and refractive index matched to the sample cooperates with the thickness of the sample spot to define the thickness of the spacer of the Fabry Perot-like cavity.

In other embodiments of the invention, the bottom surface of the biochip support beneath the area of the support that carries the array of biological sample is formed as a diffraction grating having characteristics such that a broad excitation beam of light with non-normal incidence (e.g., offset at 10 to 15.degree. from normal) is diffracted and directed to the top surface of the support at the critical angle, the camera or other microscopic viewing instrument being arranged with viewing axis normal to the array surface, not being in line with the direction of the angled incident radiation so it does not collect any radiation that may continue along its original path.

The invention also provides unique methods of manufacturing low-cost, versatile, disposable array supports having embedded optical features for evanescently exciting, or otherwise employing surface wave effects, to produce luminescence in proteins, antibodies, antigens or nucleic acids labeled with luminescent dyes, as well as luminescence from other labeled materials.

In various embodiments, the embedded optical features are formed as grooves of shape chosen to optimize the uniformity of the surface wave over the entire region of interest. These optical features range between sub-micrometer dimension and periodicity to as large as a fraction of a millimeter, dependent upon other characteristics of the system, especially the size of the spots in the array as previously mentioned. In some embodiments, according to the invention, to accommodate a range of wavelengths, the index of refraction of the support itself is varied according to location of the region of incidence of the light and the location and dimensions of the spots and array pattern. Also in certain embodiments, with some deviation of the angle of incidence of the illumination, adjacent grooves with slightly different reflection angles are provided in the support or, in these and other cases, a slight curvature may be provided in the optical surfaces, or the light is provided as only quasi-collimated light as mentioned above.

It is characteristic of the invention that the source light is reflected to induce a surface wave at the top surface at a location slightly offset from its precise point of incidence on the substrate, the deviation being approximately equal to the thickness of the substrate. In certain embodiments of the invention, this deviation is minimized by use of a thin substrate mounted on a rigid surrounding support.

An alternate manufacturing method according to the invention is to form desirable reflective embedded optical features on the top surface of a suitably rigid base and deposit a coating of suitable thickness and index of refraction onto that, the upper surface of this coating defining the surface on which the sample to be inspected is placed. The index of refraction of such coating is selected to accommodate the geometry of the reflective features, the index of refraction of the inspected sample and the wavelength of interest. Such coating, and others mentioned herein, can be silicon dioxide, titanium dioxide or other material having an index of refraction suitably higher than that of the inspected sample.

In operation, fluorescently labeled biological material is deposited on the top surface of the support as an array opposite to the field of fine embedded optical features; the excitation beam of broad size preferably arrives at the substrate to illuminate an area larger than the wide-field of view of the reading instrument. The light is directed by the beam deflection mirror in cooperation with the field of fine embedded optical features to produce a surface wave effect by any of the techniques described above. The fluorophores are excited and emit light at their specific emission wavelength, and the emitted energy is collected by the objective of the imager. In preferred implementations of the invention, the excitation light beam is approximately collimated and the inclination of the redirecting mirror is defined to accommodate the incidence of the excitation, and constructed to be adjusted to accommodate possible variations in the instrument and biochip geometric features as well as variations or uniformity variations in the indices of refraction of the material of the support are the biology inspected.

In an alternate implementation, the biochip is designed to accommodate an excitation beam aimed axially with the axis of the reading instrument, and, for instance, the angle of the biochip rather than the angle of the incident illumination may be varied, or both may be varied.

The material of the disposable support may be virgin polystyrene with an index of approximately 1.59 or polymethylmethacrylate (PMMA, known as Plexiglas,.TM.) or polycarbonate or similar plastic having an index of refraction between about 1.49 and 1.59, respectively. The fine optical features may be created in the substrate by forced embossing at proper temperature of the substrate, by being cast or press-formed against a suitably formed negative master in a manner akin to the techniques commonly used to create CD and DVD discs or cast of molten material in a mold. The features may be as small as a fraction of a micron but possibly as large as tens of microns or hundreds of microns, depending upon sample spot size, as noted above. The selected geometry of the embedded optical features, e.g. the angle of the reflecting surface or the periodicity of grating lines is determined by the index of the material of the support and any coating as well as the angle of incidence of the excitation source such as to induce a surface wave, these being dimensions which may be chosen to optimize the manufacturing process selected.

Thus, another aspect of this invention is that microscope slides of standard dimension having the broad field of embedded optical features are made employing technology presently used to manufacture CDs and DVDs, at comparable cost.

Another aspect of the invention is a cassette or flow cell for hybridization incorporating the novel support that has been described. The support with its field of embedded optical features is preferably nested in a cassette having a protective cover, for instance a cover of rigid material having a deformable seal rim or bonded membrane that protects the biological material.

Another aspect of the invention is a system, as described, that achieves an optical efficiency that is more than one order of magnitude greater than that which has been achieved with conventional microscope slides, using, according to the present invention, an imager the complexity of which is greatly reduced from that presently available commercially.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagrammatic view of an image acquisition microscope according to the invention, having its broad excitation light beam inclined at a controllable angle to illuminate the broad area of a biochip carrying a field of fine embedded optical features under the array support. The arrangement enables transmission illumination such as to create a surface wave to induce fluorescent emission of tagged biological material located on the top surface of the biochip opposite the embedded optical features.

FIG. 1A is a diagrammatic view of the CCD camera of FIG. 1 with its associated lenses and filters.

FIG. 1B is a perspective view illustrating a beam-directing mirror mounted on a flexure motion reducer having magnetic damping.

FIG. 1C is a partially broken-away side view and FIG. 1D an end view of a preferred rotary motion reducer that moves a beam-directing mirror.

FIG. 2 is a diagrammatic view of an image acquisition microscope similar to that of FIG. 1, but in which the illumination is directed to the top surface of the biochip substrate, and employing internal reflecting features at the bottom of the substrate, opposite the array, to create a surface wave to induce fluorescent emission of an array of tagged biological material samples located on the top surface of the biochip.

FIG. 3 is a perspective view, including, as indicated, a blow-up of a small portion, of a biochip constructed in the form of a microscope slide, according to the invention.

FIG. 3A is a schematic presentation showing, in representative form, the light path striking a biochip built for transmission illumination, employing embedded refractive optical features which stimulate a surface wave to induce fluorescent emission of a tagged area of an array on a biochip.

FIG. 3B is a schematic presentation similar to that of FIG. 3A of the same arrangement specific to inducing fluorescent emission of a tagged area employing a substrate of polystyrene having an index of refraction 1.59 and a sample having an index of refraction of 1.0.

FIG. 3C is a schematic presentation, similar to FIG. 3B, of an embodiment employing a wave guide formed by coating on the top surface.

FIG. 3D is a schematic presentat