System for high throughput analysis

Radiant energy – Luminophor irradiation

Reexamination Certificate

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C250S461100

Reexamination Certificate

active

06750457

ABSTRACT:

TECHNICAL FIELD
This invention relates to optical detection instruments more particularly optical detection systems that may illuminate, collect and detect light of a broad spectral range of wavelengths.
BACKGROUND OF THE INVENTION
In chemical, biochemical and cellular analytical systems, imaging provides a powerful tool. The imaging may use fluorescence, luminescence (e.g. chemiluminescence) or radio labels in a large number of analytical procedures. This imaging allows very sensitive detection. The use of such labels is well characterized, for example as detection agents conjugated to probes or binding agents. Alternatively the label may be directly incorporated into a target of interest. The sensitivity of these labels allows detection of rare events and rapid sample analysis. Optical analysts may be applied to a number of different analytical applications including identification of compounds, assay of array of biomolecules (e.g. biopolymers) or assay of binding events (e.g. competitive binding).
The benefits of imaging technology have led to widespread application of this technology to research needs. One benefit of optical screening has been in increased throughput. The speed and sensitivity of optical scanning is adaptable to automated high throughput assays that are required for screening large numbers of samples against numerous interaction agents.
Developments in the fields of genomics, cytology and chemistry have produced challenges for the evaluation and analysis of large numbers of unique samples, compounds or biological isolates.
For example, in the field of genetics, the human genome project and other genome sequencing efforts have identified tens of thousands of unique oligonucleotide sequences. Screening of gene expression against these known sequences would provide information on gene expression and regulation. This information could be correlated with disease states or applied to evaluate response of cells to therapeutic treatment (e.g. for clinical evaluation of potential pharmaceutical evaluation, monitoring of response to therapy, etc.). Labeled mRNA or cDNA isolated from cells could be used in expression evaluations. Alternatively, genomic fragments may be screened against known sequences to determine homology between the screened DNA and known genes.
Clinical evaluation of DNA expression could require analysis of DNA from hundreds of thousands of individuals. This analysis could involve screening of nucleic acid isolations from hundreds of different cell types against tens of thousands of unique oligonucleotide sequences. The present need for throughput has motivated the development of multiplexed, automated, high throughput analysis of nucleic acid homology.
There is no single, standardized technology for nucleic acid analysis. In part this is a result of the differing throughput needs. The density of analytical arrays or the scale of separation analysis of DNA will depend on the amount of samples that are being analyzed. Gene expression analysis may be performed in wells on a multiwell plate, on a separation gel, on a chip substrate containing an array of reaction spots or using other formats. If a separation gel or chip substrate is used, the separation substrate may either be read directly or transferred to another substrate, (e.g. a transfer [blotting] membrane). Alternatively, an emitted signal from an assay of samples (e.g. chemiluminescent signal, radio signal) may be recorded on a storage device (e.g. a storage phosphor screen), which is subsequently analyzed. The samples of interest may be detected by detecting a signal associated with the sample.
In addition to the label and substrate variability, the sample size may vary. This may range from the relatively large target from a separation gel to a spot on a chip array, which may be orders of magnitude smaller. Presently, most automated analytical systems are limited in types of substrates and target densities that the system is able to analyze. The data from different analytical systems often must be subsequently combined to make a finalized evaluation of a biological sample or compound.
High throughput analysis ability is required to analyze sample nucleic acids using known sequences to study genetic variation, differential expression, etc. High throughput systems have taken advantage of automation to increase sample analytical rate. Optical analytical systems may further increase sample processing throughput by designing systems with multiplex capability. For example, the ability to distinguish multiple dyes at a single dye location allows a number of dyes, each associated with a unique probe or binding agent to be used in a single assay. The combination of markers may be used at a single target loci and distinguished by the analytical system by the unique dye emission wavelength profile. This further increases the information gained from each analytical scan and increases analytical throughput. However, this would only be possible in an analytical system in which the illumination and detection optics were adapted for use with a number of different emission profiles and could operate over a wide spectral range.
In a number of scanning fluorescent analytical instruments, the excitation light and the generated fluorescent emission share a pathway and must be optically separated. Some mechanism is needed to separate an excitation laser beam from a spectrally shifted collinear, counter propagating collected fluorescent light. Typical systems use a dichroic mirror to reflect wavelengths of the laser light and transmit wavelengths of the emission light. One example is seen in Baer et al. (U.S. Pat. No. 5,547,849). A laser produces coherent light that is directed onto a dichroic mirror. The mirror directs light of the wavelengths produced by the laser through an objective lens and onto a sample. The focused beam waist is directed onto a layer to be scanned. The emitted fluorescent light is collected by the objective lens and transmitted as a collimated retrobeam to the dichroic mirror. The mirror is selected to transmit the wavelengths of the collected fluorescent light to detection optics. An alternative system is seen in Kamentsky (U.S. Pat. No. 5,072,382). In this system a laser produces an illumination beam that is directed through a dichroic mirror and onto a focus lens. The focus lens focuses the illumination light onto a sample, exciting fluorescence. The lens collects excited fluorescent light, which is transmitted as a retrobeam to the dichroic mirror. Light of the fluorescent wavelengths is reflected by the mirror onto detection optics.
These arrangements allow the rapid scanning of a sample and detection of emitted light. However, these systems have several significant drawbacks. First, the coatings on dichroic mirrors may be angle sensitive when the coatings must filter or reflect multiple sets of wavelengths. A slight variation in angle can result a significant change in the wavelengths transmitted and reflected by the mirror coating. In such cases, the positioning of the mirror must be precise for proper functioning. Second, the coatings are designed to transmit and reflect specific spectral bands. This severely limits the illumination and emission wavelengths that may be used in the optical scanning system. Changing the excitation laser wavelength or the fluorescent dye (having an alternative emission profile) would require replacement of the dichroic mirror. For any one dichroic mirror, the system is locked into specific wavelength choices. Third, the coatings that are designed to transmit or reflect multiple excitation wavelengths must still be selected for specific wavelength bands. It is common that the excitation wavelength will encroach on an emission profile of a fluorescence dye. Since the dichroic mirror transmits only a selected range of wavelengths, some emission intensity outside of this range is lost. The separation of excitation and emission wavelengths may result in a substantial loss of the collected fluorescence due to the dichroic mirror not transmitting

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