Method for measuring luminescence at a luminescence detection workstation
In a luminescence detecting apparatus and method for analyzing luminescent samples, luminescent samples are placed in a plurality of sample wells in a tray, and the tray is placed in a visible-light impervious chamber containing a charge coupled device camera. In the chamber, light from the luminescent samples pass through a collimator, a Fresnel field lens, an infrared filter, and a camera lens, whereupon a focused image is created by the optics on the camera. The use of an infrared filter suppresses stray IR radiation resulting from plate phosphorescence (which can result in abnormally high backgrounds and/or alteration of the image received by the camera).
1. A method for detecting light emitted from biological samples in wells, the method comprising:
filtering light emitted by the samples prior to reaching a detector;
focusing light emitted by the samples onto the detector to form well images;
for each of a plurality of well images, determining the edge of the well image on the detector, determining dark current within the well image, and determining pixel saturation within the well image; and
calibrating for spectral uniformity between images for different wells.
2. The method of claim 1 , wherein the detector is a charge coupled device (CCD).
3. The method of claim 2 , further comprising:
identifying outlier pixels in a well image that have intensities significantly different than neighboring pixels;
averaging the intensity of the neighboring pixels; and
replacing the intensity data for the outlier pixels with the average intensity of the neighboring pixels.
4. The method of claim 2 , further comprising:
subtracting the dark current within the well image from the well image data.
5. The method of claim 4 , wherein calibrating for spectral uniformity between images for different wells comprises:
generating data on the CCD using uniform input illumination; and
calibrating the image data using the reciprocal of the uniform input illumination data.
6. The method of claim 2 , wherein;
a first image of the well is taken at a first integration time;
a second image of the well is taken at a second integration time greater than the first integration time; and
a third image of the well is taken at a third integration time less than the second integration time.
7. The method of claim 6 , further comprising:
determining the number of saturated pixels in the second image;
using data from the second image as the image data for the well if the number of saturated pixels is less than or equal to a predetermined value; and
averaging the data from the first and third images and using the averaged data as the image data for the well if the number of saturated pixels exceeds the predetermined value.
8. The method of claim 1 , wherein filtering comprises filtering infrared emissions.
9. The method of claim 1 , further comprising collimating light emitted by the samples.
10. The method of claim 1 , wherein focusing comprises focusing light emitted by the samples with a first lens.
11. The method of claim 10 , wherein the first lens is a Fresnel lens.
12. The method of claim 10 , wherein focusing further comprises focusing light emitted by the samples with a second lens after focusing with the first lens.
13. The method of claim 12 , wherein the second lens is a camera lens.
14. The method of claim 12 , wherein the first lens is a Fresnel lens and the second lens is a camera lens.
15. The method of claim 12 , further comprising collimating light emitted by the samples after focusing with the first lens and before focusing with the second lens.
16. The method of claim 15 , wherein the first lens is a Fresnel lens and the second lens is a camera lens.
17. The method of claim 10 , further comprising collimating light emitted by the samples after focusing with the first lens.