Method and apparatus for screening chemical compounds
View Patent ↗Methods and apparatus for screening large numbers of chemical compounds and performing a wide variety of fluorescent assays, including live cell assays. The methods utilize a laser linescan confocal microscope with high speed, high resolution and multi-wavelength capabilities and real time data-processing. Imaging may be done at video-rates and with use of ultraviolet illumination.
1. A method of analyzing a biological sample, comprising the steps of:
(a) providing a biological sample comprised of at least one cell with a first and a second labeled species;
(b) acquiring images of the first and second labeled species in the biological sample;
(c) detecting at least one image object in the image of the first labeled species;
(d) generating an annular mask for the object detected in step (c) by dilating the object and comparing the dilated representation of the object with the non dilated object;
(e) generating an eroded mask for the object detected in step (c) by eroding the object;
(f) calculating registered intensity in the image of the second labeled species for the corresponding image areas defined by the annular mask and by the eroded mask respectively, and
(g) correlating the calculated intensity values for the image of the second labeled species in the respective image areas comprising graphic superimposition of the annular mask and the eroded mask, and calculating the ratio of eroded to annular averaged pixel intensities under the eroded and annular masks.
2. The method of claim 1 , wherein the detection step comprises thresholding.
3. The method of claim 1 , wherein steps (b)-(i) are performed on multiple target cells present in the biological sample.
4. The method of claim 3 , wherein the multiple target cells comprise a cluster of cells that are selected and identified with a boundary.
5. The method of claim 1 , wherein acquiring step includes acquiring a first digital image of a first labeled species and a second digital image of a second labeled species.
6. The method of claim 5 , wherein the one or both of the labeled species are cellular components tagged with a fluorescent label or a chromogenic label.
7. The method of claim 5 , wherein the intensities of one or both of the labeled species is calculated for all target cells in the image.
8. The method of claim 6 , further comprising the step of determining the cellular location of one or more of the cellular components tagged with a fluorescent label.
9. The method of claim 1 , further comprising the step of transforming at least one of the images into a binary image using a selection criteria, and wherein the image objects are defined as groups of contiguous value-1 pixels.
10. The method of claim 9 , wherein the dilation involves n dilation steps in which pixels of value 0 that touch 1-valued pixels are set to value 1, and wherein the erosion involves m erosion steps in which 1-value pixels touching 0-value pixels are set to 0.