Optics: measuring and testing – By particle light scattering – With photocell detection
Reexamination Certificate
2000-02-19
2003-11-11
Stafira, Michael P. (Department: 2877)
Optics: measuring and testing
By particle light scattering
With photocell detection
C356S343000
Reexamination Certificate
active
06646742
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to devices and methods for the discrimination of particulate elements using flow analysis in walled conduits, and more particularly, to devices and to methods for optically discriminating particulate elements on the basis of different light scattering effects and different light emission behaviors stemming from structures (or sub-structures) of such elements as well as from differences in general compositional element characteristics.
BACKGROUND OF THE INVENTION
Since establishment of the Cell Theory in the 1840's clinicians have found it informative to categorize, analyze and monitor the diverse blood cell populations of man and beast. In the blood of any typical mammal these cells are the erythrocytes, leukocytes and thrombocytes. Their numerical concentration can be estimated with precision; provided however, sufficient cells are counted.
In health, mammals have five subpopulations of circulating leukocytes: neutrophil granulocytes, lymphocytes, monocytes, eosinophil granulocytes and basophil granulocytes. Since perfection of the light microscope around 1880 and the introduction of Ehrlich's dyes for differentiating the granulocyte's granules shortly thereafter, these leukocyte subsets can also be differentiated with precision; provided however, sufficient cells are examined. Additionally there is a need for rapid exact analysis of countless other suspendable complex micro-particles and cell types.
During last century it became clear from optic scattering effects and from other evidence that, even though proteins could not be resolved with classic microscopes and were in nanometer size ranges, these too must be considered micro-particles.
Whilst counting chambers and microscopes could be used for cell evaluations, investigators performing these tasks were prone to fatigue and subjective vagaries. Therefore, in relation to known statistical sampling errors, an inadequate number of micro-particles could be analyzed by microscopy and only extreme pathological deviations were intercepted reliably. On the other hand, by 1930 R. A. Fisher had taught that, if a sufficient number of well-chosen, different, robust measurements were made on complex organic entities such as cells during an investigation, then, it would be straightforward to objectively assign the different tested elements to appropriate natural sets and subsets.
From the 1930's onwards attempts were made to automate the time-consuming number-accumulation chore and classification challenge of cell and micro-particle analysis. For clinical cell analysis, these endeavors evolved along two main paths: (i) image analysis microscopy, which examines all microscopic objects, and (ii) flow analysis, or generalized flow cytometry of micron-sized and of sub-micron particles down to macromolecules.
In classical microscopy a large specimen is placed into an object plane which is orthogonal to the optic axis of the microscope. That classical plane is occupied by a vast expanse of sample material which is illuminated over a wide field, i.e., a field much larger in area (and frequently also in depth) than the arbitrarily definable voxel (or volume element or “cell of space”) occupied, for example, by a small single tissue cell or by an ensemble of granules within, perhaps, an eosinophil cell or a suspension or solution in a spectrophotometric cuvette. In whatever manner the voxel (or “cell”) is defined, there is a very vast number of these illuminated elements in the classical microscopic object. Each such illuminated voxel of such a sample preparation causes the scattering (or, in certain settings fluorescence) of light. In relation to the signal light from a specific voxel of interest, the light from all the other structures in the microscopic field represents signal-degrading background noise. That background noise light can easily exceed 99.9% of the total light received in the microscope's image location of the specific voxel. Hence, significant detail relating to such classically viewed voxels is likely lost to the viewer.
“Confocal” microscopy is an automated technique for avoiding the degrading effects of that frequently overwhelming background noise discussed above. The background is avoided by contemporaneously having both the illumination lens system(s) and the interrogation lens system(s) focus essentially on one voxel at a time. Additionally, computer techniques are used to synthesize three-dimensional high-signal images whose rich information content could not otherwise be captured. Confocal microscopy was first patented in the United States in 1952 by Marvin Minsky. Today, such technique is practiced via instruments which matured in the 1980's (see, for example, S. W. Paddock “Confocal Laser Scanning Microscopy” BioTechniques 27 992-1004 1999).
Thus, in the centuries-old field of microscopy the relatively recent introduction of the optic background-noise-reducing confocal microscopy technique has advanced the classic approach beyond illumination of the entire surrounding tissue to specific illumination and interrogation of optically-isolated elements of space, which can be made smaller than individual biologic cells. This lowers the limit of detection by increasing the signal-to-background ratio that can be differentiated as a separate element in the colloquial signal
oise function for any analyte or measurand. Without the confocal microscopy approach the sample signal is lost in the “sea” of system noise.
Returning now to the evolution of particle flow analysis, by the mid-century it has been said that considerable work had been done to produce instruments capable of counting blood cells. Interestingly, such instruments varied considerably in principle, mode of operation, complexity and cost.
Prior to the work of Crosland-Taylor in this area, optic flow cytometry evolved via the upright optic axis of the microscope with an object plane that was orthogonal to the optic axis. Crosland-Taylor made significant progress in overcoming the optic tissue background noise interference problem of microscopy by exploiting hydrodynamic forces within an optically clear suspension fluid to present biologic cells singly to an optic interrogation area in a so-called hydrodynamically focused cell monofile.
Throughout micro-particle analysis, one broad approach had been to suspend particulates in an appropriate fluid (e.g., as aerosol or hydrosol) in order to isolate a “sample” away from other interfering objects so that ensembles (down to single elements) could be interrogated in physically isolated elements of space. Specific sensors could then respond quantitatively to differing properties of the isolated elements. In this way multi-angle light scatter (MALS) studies had already allowed the interrogation of particle ensembles in the middle of cylindrical cuvettes (i.e., interrogation cells) far from the surrounding walls. The Crosland-Taylor work implemented a general approach to isolating, thin, stable filaments of particulate-containing fluid for interrogation in flow-through cells rather than merely in static cells or cuvettes.
When lasers were introduced, MALS could more appropriately be termed MALLS (multi-angle laser light scatter); and in the 1960's many scientists incorporated lasers into the earlier Crosland-Taylor flow cytometry systems. Thereby, additional optic sensing axes could be placed into the traditional orthogonal microscopic object plane which had been co-opted by flow cytometry for the particle monofile; and the illuminating laser pencil for flow cytometry could also reside in that plane. Indeed, many in the art perceived that an interrogation apparatus could be arranged with laser and particle filament intersecting at the optic scattering and fluorescence origin of a spherical radiation domain. Crosland-Taylor had already introduced bubble-free degassed carrying liquids. Therefore, except for the thin sample fluid filament carrying the test particulates through a finite illuminated radiation centroid, this spherical scatter
Boyd James R.
Gangstead Mervin L.
Pina Jean-Charles
von Behrens Wieland E.
West Jerry B.
MWI, Inc.
Sidley Austin Brown & Wood LLP
Stafira Michael P.
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