Method and apparatus for analyzing individual cells or particulates using fluorescent quenching and/or bleaching
A method for analyzing a blood sample is provided that includes the steps of: providing a blood sample having one or more of each first and second constituents; admixing a colorant with the sample, which colorant is operative to cause the first constituents and second constituents to fluoresce and absorb light; illuminating at least a portion of the sample; e) imaging a portion of the sample; determining a fluorescence value for each the first constituents and second constituents; determining an optical density value for each of the first constituents and second constituents; and identifying the first constituents and the second constituents using the determined fluorescence and optical density values.
1. A method for analyzing a blood sample quiescently residing within an analysis chamber, which sample has at least one first constituent and at least one second constituent, and which second constituent is a different type than that of the first constituent, and which sample is mixed with a colorant, the method comprising:
illuminating at least a portion of the sample containing the first constituent and the second constituent with light at one or more wavelengths that cause the colorant to fluoresce, and with light at one or more wavelengths that are absorbed by the colorant;
imaging the at least a portion of the sample, including producing first image signals indicative of fluorescent emissions from the colorant disposed within the first constituent and second image signals from the colorant disposed within the second constituent, and including producing third image signals indicative of the optical density of at least one region within the first constituent and producing fourth image signals indicative of the optical density of at least one region within the second constituent;
determining at least one value representative of fluorescent emissions using the first image signals and at least one value representative of fluorescent emissions using the second image signals;
determining at least one value representative of optical density associated with the first constituent using the third image signals and at least one value representative of optical density associated with the second constituent using the fourth image signals; and
identifying the first constituent and the second constituent using the determined representative fluorescent emission values and the determined representative optical density values.
2. The method of claim 1 , wherein the first constituent and the second constituent are selected from the group consisting of eosinophils, neutrophils, and basophils.
3. The method of claim 1 , wherein the first and second constituents are selected from the group consisting of white blood cells and particulates.
4. The method of claim 3 , wherein the first and second constituents are each types of white blood cells.
5. The method of claim 4 , wherein the step of identifying the first constituents and the second constituents utilizes the determined representative fluorescent emission values and the determined representative optical density values of only a cytoplasmic region of the respective first constituent and second constituent.
6. The method of claim 3 , wherein the values representative of fluorescent emissions are determined on a per pixel basis.
7. The method of claim 3 , wherein the step of identifying the first constituent and the second constituent, includes determining a ratio of the at least one determined representative fluorescent emission value over the at least one representative determined optical density value for a particular constituent.
8. The method of claim 7 , wherein the ratio is an average of determined representative fluorescent emission values for the particular constituent over an average of determined representative optical density values for the particular constituent.
9. The method of claim 3 , wherein the first and second constituents are each types of particulates.
10. The method of claim 9 , wherein the first and second particulates are selected from a group consisting of platelets and hematoparasites.
11. A method for analyzing a blood sample quiescently residing within an analysis chamber, which sample has at least one first constituent and at least one second constituent, and which second constituent is a different type than that of the first constituent, and which sample is mixed with a colorant, the method comprising:
illuminating at least a portion of the sample containing the first constituent and the second constituent with light at one or more wavelengths that cause the colorant to fluoresce, and with light at one or more wavelengths that are absorbed by the colorant;
imaging the at least a portion of the sample, including producing first image signals indicative of fluorescent emissions from the colorant disposed within the first constituent and second image signals from the colorant disposed within the second constituent, and including producing third image signals indicative of the optical density of at least one region within the first constituent and producing fourth image signals indicative of the optical density of at least one region within the second constituent;
determining at least one value representative of fluorescent emissions using the first image signals and at least one value representative of fluorescent emissions using the second image signals;
determining at least one value representative of optical density associated with the first constituent using the third image signals and at least one value representative of optical density associated with the second constituent using the fourth image signals; and
distinguishing the first constituent from the second constituent using the determined representative fluorescent emission value and the determined representative optical density value.