Light modulated microarray reader and methods relating thereto

Details Light modulated microarray reader and methods relating thereto Light modulated microarray reader and methods relating thereto

2002-06-06

2004-09-21

Hannaher, Constantine (Department: 2878)

Radiant energy

Luminophor irradiation

C250S459100, C250S461100

Reexamination Certificate

active

06794658

ABSTRACT:

BACKGROUND
“Microarrays” are devices used in biotechnology and other science research, and can be made by putting a large number of tiny droplets of DNA (or other target such as cDNA or proteins) on a glass slide. Short pieces of DNA, called probes, are then applied to the DNAs on the slide. Typically, the probes are fluorescent, so they light up when short wavelength light is shone on them (the probes can also be labeled with other substances to reflect or otherwise emanate light when they are scanned). Microarrays can be used, for example, to study how large numbers of genes interact with each other (genes are made of DNA), or how a cell is able to simultaneously control vast numbers of genes.
The probes stick to the microarray wherever the probes find target stretches of DNA called complementary DNA strands. The microarrays are then put into a scanning microarray reader that measures the brightness of each fluorescent dot: the brighter the dot, the more probe (and thus the more target DNA or other biological material) is present. This can indicate, for example, how active the target is, or where it is on the slide.
Microarrays can be used, for example, to study genomic content, how large numbers of genes interact with each other (genes are made of DNA), or how a cell is able to simultaneously control vast numbers of genes (expression patterns). Different types of microarrays include, but are not limited to, cDNA arrays, oligonucleotide arrays and protein arrays.
Some general concepts about microarrays, and the microarray readers that measure the dots, are discussed in more scientific terms in the following paragraphs.
Fluorescence based microarray readers suffer from limited dynamic range with respect to the possible intensities of target spots in, for example, cDNA expression microarrays. Systems which use scanning spots and photomultiplier tubes (PMTs) for detection are reported to possibly have a ~10
6
dynamic range but may have a 10
4
to 10
5
dynamic range in practice. Most charge couple device (CCD) imaging microarray readers have about a ~10
3
dynamic range (12 bit-digitization, 4096 levels, 40,000-1,000,000 electron well depths, 200:1 up to 1000:1 possible signal-to-noise ratio).
The intensity of a fluorescent target spot on a microarray is a function of factors such as how long the target spot is sampled (data sampling time, which can be dwell time for scanning spot systems and integration time for CCD imaging systems), the intensity of the illumination (illumination intensity), the sensitivity of the detector (quantum efficiency, signal transducer, or measurement sensitivity), and the accuracy of digitization (pulse counting or voltage digitization). Coordination and control of these factors is difficult, so measuring spot intensity over a wide range is difficult.
Accordingly, there has gone unmet a need for improved methods of precisely measuring the brightness of the target spots on a microarray over a wide range of target spot intensity. The present invention provides this and other advantages.
SUMMARY
Target spots on a microarray that are too dim or too bright for the microarray reader to accurately measure are a problem, for example because they fall outside of certain threshold levels so the microarray reader cannot accurately measure them, or because target spots that are too bright can also hinder the measurement of neighboring target spots due to glare or other interference. Typically, the intensity of light emanating from target spots is proportional to the amount of light shown or incident on the target spots; the more light that is incident on the target spot (excitation or illumination light), the brighter the light coming from the target spot. The present invention takes advantage of this and adjusts the amount of light directed at specific non-acceptable target spots (for example, those spots which fall outside the dynamic range of the system in use), such that dim spots receive more excitation light and overly bright spots receive less.
Similarly, in conjunction with or instead of such actions, the present invention adjusts the amount of light received from specific non-acceptable target spots, such that the detector receives more light from dim spots and less light from overly bright spots. This increases or decreases, respectively, their measured brightness, which in turn effectively increases the range over which a microarray reader can accurately measure the spots, and can also improve the signal-to-noise ratio and other aspects of the measurements. In some embodiments, the present invention can increase the range of the microarray reader by up to about 1000 times or more, and improve the signal-to-noise ratio for target spots up to about 16 times or more.
In one aspect, the present invention provides automated methods of reading a microarray comprising, a) providing an initial representation of a microarray comprising a plurality of target spots illuminated by illumination light having a designated intensity; b) determining from the initial representation whether at least one of the target spots has an emanating light intensity that can be not between selected upper and lower threshold values, and designating such target spot a non-acceptable target spot; and, c) modulating the designated intensity of the illumination light via an automated upstream selective light modulator located in an illumination light path substantially at a conjugate image plane of the sample to provide a modulated illumination light and an adjusted target spot that emanates an adjusted light intensity between the selected upper and lower threshold values.
In some embodiments, the methods further comprise measuring the amount of modulation of the designated intensity of the illumination light and measuring the adjusted light intensity, then correlating the amount of modulation with the adjusted light intensity to provide a measure of the actual signal strength of the target spot. In this and other embodiments of the invention (unless expressly stated otherwise or clear from the context), all embodiments, aspects, features, etc., of the present invention can be mixed and matched, combined and permuted in any desired manner. The methods can further comprise determining an amount of a probe located at the adjusted target spot from the measure of the actual signal strength of the target spot. The methods are suitable for detecting any light emanating spot, such as reflective, fluorescent or other light.
The methods can be implemented according to various formulae. Such formulae include:
SS
(
x,y
)=
K*CCDS
(
x,y
)/
II
(
x,y
)   (1)
where,
SS(x,y) can be the actual signal strength of the target spot,
K can be a constant for the system,
(CCDS(x,y)) can be the adjusted light intensity, and
(II(x,y)) can be the modulated illumination light.
SS
(
x,y
)=
K*PB
(
II
(
x,y
),fluoro)*
CCDS
(
x,y
)/
II
(
x,y
)   (2)
where,
SS(x,y) can be the actual signal strength of the target spot,
K can be a constant for the system,
PB(II(x,y),fluoro) can be a photobleaching function based on illumination energy/intensity and a fluorophore being excited,
(CCDS(x,y)) can be the adjusted light intensity, and
(II(x,y)) can be the modulated illumination light.
SS
(
x,y
)=
K*PB
(
II
(
x,y
),fluoro,
x,y
)*
CCDS
(
x,y
)/
II
(
x,y
)   (3)
where,
SS(x,y) can be the actual signal strength of the target spot,
K can be a constant for the system,
PB(II(x,y),fluoro,x,y) can be a photobleaching function based on illumination energy/intensity, a fluorophore being excited, and a spatial variation term,
(CCDS(x,y)) can be the adjusted light intensity, and
(II(x,y)) can be the modulated illumination light.
The modulated illumination light can be modulated by changing its illumination intensity, by changing its duration of illuminating the target spot, or otherwise as desired. The initial representation can comprise a precompiled map of expected data for the target spots of the microarray, or an initial image of the plurality of target spots illuminated by the i

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