Image analysis – Applications – Biomedical applications
Reexamination Certificate
2000-01-04
2002-02-05
Boudreau, Leo (Department: 2721)
Image analysis
Applications
Biomedical applications
C382S133000, C382S128000, C377S010000
Reexamination Certificate
active
06345115
ABSTRACT:
This is a continuation of international application Serial No. PCT/CA98/00762, filed Aug. 7, 1998, the entire text of which is incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to a system for creating digital images of fluorescent, luminescent, or bright field specimens. The system is flexible, both in its mode of illumination and in that specimens may be arrayed in matrices (e.g. well plates) or randomly arranged (e.g. chemiluminescent colonies, gel media).
BACKGROUND OF THE INVENTION
The present invention is primarily an assay imaging system. An assay may be defined as a measurement of physical properties (chemical, biochemical, physiological or other) within a specimen. Assays are used, typically, in the areas of molecular biology, genomics, and pharmaceutics.
The standard assay specimen container is a plastic or glass plate containing 96 small chambers, termed wells. Detection instruments and robotic plate handling technologies have evolved to make efficient use of the 96 well plate, and to provide as high a throughput as possible with this plate format. Current screening technologies can allow a large screening laboratory using 96 well plates to process some thousands of compounds per day.
Recently, there has been a massive growth in the number of compounds available for testing. In part, this is due to an increased exploitation of biodiversity resources to generate natural compounds. In greater part, the proliferation of new compounds is a result of a new chemical technology, termed combinatorial chemistry. In combinatorial chemistry, large numbers of related compounds are synthesized (using permutations of chemical building blocks), and must then be tested for medicinal value.
With new discoveries in natural compounds, and with the advent of combinatorial chemistry, pharmaceutical companies and biotechnology companies are generating extensive “libraries” of untested compounds. These libraries can easily contain millions of compounds. Standard measurement technologies cannot cope with the volume, and new technologies are needed which will increase the rate at which compounds can undergo initial testing (screening) for medicinal value. To show a major advantage over standard technologies, new technologies should allow throughput to exceed 100,000 compounds/instrument per day. Imaging technologies have the promise to increase throughput to the required levels. They have the additional advantage of flexibility, in that an imaging system can be applied to assays in wells and other formats, and to assays which are static or which change over time.
Most assays are designed so that changes in the absorbance, transmission, or emission of light reflect reactions within the specimen. Therefore, most assay measurement instruments detect alterations in luminance as their operating principle. For detection instruments, bioluminescence or chemiluminescence provides the simplest type of assay, in that there is no need to apply illumination. Absorbance assays involve transilluminating the specimen, usually with monochromatic light. The reaction of interest affects the extent to which the light is absorbed by a fluid, and this absorbance may be measured.
Fluorescence is emitted when a fluorophore interacts with an incident photon (excitation). Absorption of the photon causes an electron in the fluorophore to rise from its ground state to a higher energy level. Then, the electron reverts to its original level, releasing a photon (fluorescence emission) hose wavelength depends upon the amount of energy that is released during reversion. A given fluorophore may emit at single or multiple wavelengths (creating an emission spectrum), as electrons drop from various orbitals to their ground states. The emission spectrum is constant for each species of fluorophore.
Fluorescence assays require the application of an intense monochromatic illumination beam, termed “excitation”. Fluorescence assays are used for the following types of applications:
1. An detector tuned to a specific emission spectrum can be used to localize a fluorophore. For example, wells which contain cells expressing a fluorescently tagged protein can be discriminated from wells which do not.
2. By measuring the intensity of fluorescence, an detector system can estimate the concentration of a fluorescent molecule.
3. Changes in the fluorophore molecule (such as binding of fura-2 to Ca++) will lead to alterations in the emission spectrum. A detector can be used to measure these spectral changes, as an indication of changes in the environment of the fluorophore.
Wells
Each well contains a discrete condition of the experiment, and alterations in light emission are measured to determine whether that condition yields favorable properties. “Well plate” assays are higher in throughput and lower in cost than similar assays in discrete containers.
Reactions within the wells may be of many kinds. In chemistry assays, molecules of different compounds (e.g. a drug candidate and a receptor molecule) are placed into the same well, and the interaction between those compounds is observed. In cell-based assays, each well contains a population of living cells, and effects of compounds on these cells are observed.
Most assays are conducted by making a single measurement from each well. However, it is also possible to record changes over time, by measuring each well repeatedly. The use of repeated observations could be termed a “dynamic” assay.
Standard well plates contain 96 or 384 wells in an area of about 8×12 cm. The trend is towards miniaturization of the wells. Prototypes containing 864 wells or more are under evaluation at many sites. The goal is to develop plates with high density arrays of small “microwells” (e.g. thousands/plate) with small fill volumes. That is, miniaturized wells might contain 1 ul of fluid instead of the 100 ul or more used in a typical 96 or 384 well assay.
Miniaturized assay formats promise to achieve dramatic cost reductions and to simplify disposal procedures, while allowing many more assays to be conducted.
Hybridization Arrays and Genetic Assays
Low throughput methods of genetic analysis use various electrophoretic procedures. Methods for increasing throughput and decreasing costs of genomic assays include arraying DNA clones (cDNAs) or synthetic oligonucleotides onto flat support membranes or slips of treated glass. The arrays of cDNAs or oligonucleotides (termed high density grids) are then hybridized to samples of genomic material to quantify levels of gene expression, or to localize relevant sequences. In the past, most hybridization assays have been conducted using isotopic label and storage phosphor imaging systems for detection. However, nonisotopic methods (particularly fluorescence) are under investigation in many laboratories.
Nonisotopic high density grids provide the potential for very high throughput at low cost, and various detection technologies are being developed for these specimens.
Free Format Assays
Assays which occur within a regularly spaced array (wells, cDNAs within a grid) can be referred to as fixed format assays. Specimens that are irregularly distributed can be termed free format assays. Examples of free format assays include electrophoregrams, bacterial colonies in culture, and various combinatorial assays in which bead-bound compounds are distributed over a tissue culture. The common factor in these free format assays is that areas of altered luminance can occur at any spatial location.
Instruments designed for fixed format assays (fluorescence plate readers, liquid scintillation counters, etc.) only read from defined locations in the specimen. They are not useful with specimens in which effects lie at locations that are not predefined. In contrast, imaging systems are able to detect and quantify reactions at any position within an image, and there is an extensive history of image analysis being applied to free format assays.
Summary of Types of Assays
Pharmaceutical companies are faced with unprecedented numbers of new compounds,
Ramm Peter
Sun Gang
Boudreau Leo
Choobin M B
Imaging Research Inc.
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