Two-dimensional spectral imaging system
Systems, including methods, apparatus, and algorithms, for spectrally imaging a two-dimensional array of samples are disclosed. In one such system, a method of sample analysis includes directing light from a two-dimensional array of examination sites onto a detection area such that dispersed light of known spectral composition from each site falls on the detection area. The method further includes assigning a corresponding two-dimensional array of regions of the detection area for detecting spectra from the examination sites based, at least in part, on the known spectral composition. The method also includes placing samples into the two dimensional array of examination sites and obtaining spectral data for each sample based on the detection area.
1. A method of sample analysis, comprising:
directing light from a two-dimensional array of examination sites onto a detection area such that dispersed light of known spectral composition from each site falls on the detection area concurrently;
assigning a corresponding two-dimensional array of regions of the detection area for detecting spectra from the examination sites based, at least in part, on detection of the dispersed light of known spectral composition from one or more of the examination sites;
placing samples into the two dimensional array of examination sites; and
obtaining spectral data for each sample based on the two-dimensional array of regions of the detection area
wherein the two-dimensional array of regions defines a pair of orthogonal axes, wherein the step of directing light includes a step of dispersing the light to define a spectral axis for the detection area, and wherein the spectral axis is oblique to each of the orthogonal axes.
2. The method of claim 1 , wherein the step of directing includes a step of collectively resolving dispersed visible light relative to nondispersed visible light from the array of examination sites.
3. The method of claim 1 , further comprising a step of disposing one or more calibration standards in the examination sites, wherein the step of assigning is based on detection of light emitted by each calibration standard.
4. The method of claim 3 , wherein the step of disposing is performed before the step of placing.
5. The method of claim 3 , wherein the step of disposing includes a step of disposing a calibration standard in every examination site.
6. The method of claim 5 , wherein the step of disposing includes a step of disposing substantially the same calibration standard in every examination site.
7. The method of claim 3 , wherein the step of disposing includes a step of disposing different luminophores serially in an examination site.
8. The method of claim 3 , wherein the step of disposing includes a step of disposing a mixture of two or more luminophores in an examination site.
9. The method of claim 1 , further comprising a step of filtering light, wherein the step of filtering at least substantially generates the known spectral composition of the dispersed light.
10. The method of claim 1 , wherein the samples provide the dispersed light of known spectral composition.
11. The method of claim 1 , wherein the step of assigning includes (1) a step of collecting image data from the detection area, and (2) a step of processing the image data with an algorithm to identify features within the image data.
12. The method of claim 11 , wherein the step of processing the image data includes a step of collapsing the image data separately parallel to each of a pair of orthogonal axes to identify feature indicators within the collapsed image data.
13. The method of claim 11 , wherein the step of processing the image data includes a step of identifying a two-dimensional array of intensity maxima, minima, or both, that corresponds to the features within the image data.
14. The method of claim 11 , wherein the step of processing the image data includes a step of converting the image data to binary.
15. The method of claim 1 , wherein the step of obtaining spectral data for each sample includes (1) a step of detecting spectral image data collectively for the samples, and (2) a step of selecting a portion of the spectral image data corresponding to each sample.
16. The method of claim 1 , wherein the step of obtaining spectral data for each sample includes a step of selectively collecting the spectral data for each sample according to the regions of the detection area.
17. The method of claim 1 , wherein the step of obtaining spectral data is performed in exact correspondence with the regions of the detection area.
18. The method of claim 1 , wherein the step of obtaining spectral data is performed in exact correspondence with the regions of the detection area after applying a translational offset, a rotational offset, or both, to the array of regions.
19. The method of claim 18 , wherein the samples are held by a sample holder including at least one fiducial that is distinct from the wells, wherein the step of obtaining includes (1) a step of detecting a signal corresponding to the at least one fiducial, and (2) a step of moving the array of regions in relation to the detection area according to a position of the signal detected.
20. The method of claim 18 , further comprising a step of moving the array of regions based on signals detected from one or more of the samples.
21. The method of claim 20 , wherein the step of moving is based, at least in part, on detection of nondispersed light from one or more of the samples.
22. The method of claim 1 , wherein the step of obtaining spectral data is performed a plurality of times for the same samples to identify changes in the spectral data over time.
23. The method of claim 1 , wherein the samples include a set of luminophores, further comprising (1) a step of calibrating each region to determine a spectral characteristic of emitted light within the region from each luminophore disposed in an examination site corresponding to the region, and (2) a step of processing the spectral data based on the spectral characteristic to determine relative contributions of the luminophores to the spectral data of a sample in the examination site.
24. The method of claim 23 , wherein the luminophores have substantially overlapping spectral distributions within each region.
25. The method of claim 1 , wherein the spectral data for each sample are obtained from a portion of the detection area corresponding to one of the regions, wherein the portion detects a set of pixels, further comprising a step of binning subsets of the set of pixels such that the spectral data for each sample is simplified.
26. The method of claim 25 , wherein the subsets have a uniform size.
27. The method of claim 25 , wherein the samples include luminophores, and wherein the subsets are selected according to spectral distributions of emitted light from the luminophores within the region.
28. The method of claim 1 , wherein the step of assigning includes (1) a step of detecting image data collectively for dispersed light of known spectral distribution from each of the examination sites, and (2) a step of processing the image data to remove optical distortion.
29. The method of claim 28 , wherein the step of processing includes a step of processing the image data according to a polynomial function to remove optical distortion.