Fluorescence digital imaging microscopy system

• US 6,665,430 B2
• Filed: 08/13/2002
• Issued: 12/16/2003
• Est. Priority Date: 03/26/1996
• Status: Expired due to Fees

• Current Assignee: Childrens Hospital Los Angeles
• Original Assignee: Childrens Hospital Los Angeles
• Inventors: Reynolds, C. Patrick, Frgala, Tomas
• Primary Examiner(s): Patel, Jayanti K.
• Application Number:
  US10/217,721
• Publication Number:
  US 20020191824A1
• Time in Patent Office: 490 Days
• Field of Search: 382/100, 382/128, 382/129-134, 424/400, 424/145.1, 435/23, 435/226, 435/6, 435/69.1, 435/29, 435/183, 435/173.3, 514/12, 530/350
• US Class Current: 382/128
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16

Abstract

A method of preparing cell samples for viable cell number quantification with a fluorescence digital imaging microscopy system employing digital thresholding technique. The cell sample is stained with a first, fluorescent dye and treated with a second dye that is able to quench the fluorescence of the first dye. The fluorescent dye accumulates in viable cells only and is used to stain the viable cells. The second dye is excluded from viable cells but enters non-viable cells, thereby quenching the background fluorescence in non-viable cells and the medium. Two examples of dye combinations are described: fluorescein diacetate used as the fluorescent dye with eosin Y as the quenching dye; and calcein-AM used as the fluorescent dye with trypan blue as the quenching dye. By reducing the background fluorescence, the dynamic range and accuracy of viable cell number measurements are enhanced. In low viability cultures treated with fluorescein diacetate, background fluorescence completely masked viable cells, but digital thresholding and eosin treatment dramatically reduced background fluorescence, producing a linear response over 4 logs of viable cell density.

Claims

1. A method for reducing background fluorescence in a culture cell sample during quantification of viable cell numbers using fluorescent dyes, comprising:

treating the sample with eosin Y, wherein the eosin Y enters non-viable cells but is substantially excluded by viable cells, wherein the background fluorescence in the medium and non-viable cells in the sample is quenched; and

measuring an intensity of fluorescence of the treated sample.

2. The method of claim 1, wherein the sample is in a container, wherein the measuring step comprises recording a digital image of a defined area of the container using an imaging device, the digital image representing fluorescence light intensities of the defined area.

3. The method of claim 2, wherein the measuring step further comprises:

holding the container on a stage; and

moving the stage and the imaging device relative to each other in at least two horizontal directions to image a plurality of areas.

4. The method of claim 3, further comprising quantifying relative cell numbers in the sample.

5. The method of claim 4, wherein the quantifying step comprises manipulating digital images to reduce background fluorescence intensities in the images and acquiring information relating to relative cell numbers.

6. A method for reducing background fluorescence in a culture cell sample during quantification of viable cell numbers using fluorescent dyes, comprising:

treating the sample with eosin Y, wherein the sample is in a container, wherein the eosin Y enters non-viable cells but is substantially excluded by viable cells, wherein the background fluorescence in the medium and non-viable cells in the sample is quenched;

measuring an intensity of fluorescence of the treated sample; and

quantifying relative cell numbers in the sample.

7. The method of claim 6, wherein the measuring step comprises:

holding the container on a stage;

recording a digital image of a defined area of the container, the digital image representing fluorescence light intensities of the defined area;

moving the stage and an imaging device relative to each other in at least two horizontal directions to image a plurality of areas; and

controlling the movement of the stage and imaging device and controlling the recording of the digital images, wherein the relative movements between the stage and imaging device and the recording of the images are performed in a synchronized manner and according to a predetermined scanning pattern.

8. The method of claim 7, wherein the measuring step further comprises inputting parameters according to a format of a container having multiple wells.

9. The method of claim 8, wherein the quantifying step comprises manipulating the digital images to reduce background fluorescence intensities in the images and acquiring information relating to relative cell numbers.