Computer interfaced scanning fluorescence lifetime microscope applied to directed evolution

• US 7,193,706 B2
• Filed: 08/02/2001
• Issued: 03/20/2007
• Est. Priority Date: 08/02/2000
• Status: Expired due to Fees

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Bowen, Benjamin P., Scruggs, Allan, Woodbury, Neal W. T.
• Primary Examiner(s): Le; Long V.
• Assistant Examiner(s): Yang; N
• Application Number:
  US10/343,412
• Publication Number:
  US 20030148393A1
• Time in Patent Office: 2,056 Days
• Field of Search: 356/317, 356/210, 435/85.5, 435/173.1, 435/7.2, 435/173.4, 435/40.2, 514/183, 514/31, 514/410, 514/561, 514/152, 514/192, 424/85.5, 424/577, 424/600, 604/20, 604/21, 607/88, 607/89, 522/25, 522/26, 522/2
• US Class Current: 356/317
• CPC Class Codes: G01N 2021/6482 Sample cells, cuvettes
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• 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 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/567 utilising isolate of tissue or organ as binding agent
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence

Abstract

This invention provides a method for screening large numbers of individual cells or colonies of cells using scanning microscopy coupled with fluorescence lifetime measurement and analysis, using time-correlated single photon counting. This invention further provides an automated method for selecting cells that exhibit desired characteristics. The method uses the scanning microscope system to focus a laser beam onto a surface upon which cells are immobilized on the timescale of the procedure. The cells that are illuminated in this way are killed or their growth is inhibited. The focused laser beam is scanned across the surface and turned on and off during the scanning process such that only non-irradiated cells survive, resulting in a patterned cell growth This invention further provides a computer-controlled projection device, such as a micro-mirror array or a liquid crystal display system, which is sued to project an image onto the cells.

Claims

1. An automated method for screening and selecting cells, the method comprising the steps of:

providing a substrate with multiple locations;

immobilizing a plurality of cells exhibiting an imagable property when excited, either onto or within said substrate;

detecting the imagable property by scanning a first light beam across the substrate to excite the imagable property of the cells;

recording the imagable property;

determining an excited state lifetime of the imagable property for each of the cells;

identifying and recording locations containing cells expressing a desired characteristic and locations not containing cells expressing the desired characteristic, based on the excited state lifetimes of the cells;

generating a kill image from the excited state lifetimes and the locations of the cells;

applying a sensitizing agent to the substrate, wherein the sensitizing agent is selected to render the cells sensitive to light at a particular sensitizing wavelength; and

scanning a second light beam, according to the kill image, having a wavelength that is a multiple of the sensitizing wavelength across the substrate through a high speed shutter, wherein the shutter is open only when the light beam is positioned over locations not containing cells expressing the desired characteristic of the imagable property to thereby kill the cells in such locations.

2. The method of claim 1 wherein the sensitizing agent is a DNA intercalating dye.

3. The method of claim 2 wherein the DNA intercalating dye is ethidium bromide.

4. The method of claim 1 wherein the sensitizing agent is a porphyrin.

5. The method of claim 1 wherein the sensitizing agent generates reactive oxygen species upon absorption of light.

14. The method of claim 1 wherein the first and second light beam are produced by the same light source.

6. An automated method for screening and selecting cells, the method comprising the steps of:

providing a substrate with multiple locations;

immobilizing a plurality of cells exhibiting an imagable property when excited, either onto or within said substrate;

detecting the imagable property by scanning a light beam across the substrate to excite the imagable property of the cells;

recording the imagable property;

determining an excited state lifetime of the imagable property for each of the cells;

identifying and recording locations containing cells expressing a desired characteristic and locations not containing cells expressing the desired characteristic, based on the excited state lifetimes of the cells;

generating a kill image from the excited state lifetimes and the locations of the cells;

applying a sensitizing agent to the substrate, wherein the sensitizing agent is selected to render the cells sensitive to light at a particular sensitizing wavelength; and

projecting light, according to the kill image, having a wavelength that is a multiple of the sensitizing wavelength only onto those locations not containing cells expressing the desired characteristic of the imagable property to thereby selectively kill those cells.

7. The method of claim 6 wherein the sensitizing agent is a DNA intercalating dye.

8. The method of claim 7, wherein the DNA intercalating dye is ethidium bromide.

9. The method of claim 6 wherein the sensitizing agent is a porphyrin.

10. The method of claim 6 wherein the sensitizing agent generates a reactive oxygen species upon absorption of light.

11. The method of claim 6, wherein the imagable property of the cells is sensed and recorded by a charge couple device based (CCD) camera.

12. The method of claim 6, wherein a computer-controlled projection device is used to project the light onto the cells not exhibiting the desired characteristic of the imagable property.

13. The method of claim 6, wherein the computer-controlled projection device is a digital light processor.