Method and apparatus for performing cell analysis based on simultaneous multiple marker emissions from neoplasia (CASMMEN)

• US 6,005,256 A
• Filed: 02/12/1998
• Issued: 12/21/1999
• Est. Priority Date: 02/12/1998
• Status: Expired due to Term

• Current Assignee: Raytheon Company (Rtx Corporation)
• Original Assignee: Raytheon Company (Rtx Corporation)
• Inventors: Akkapeddi, Prasad R., McGlynn, Charles L.
• Primary Examiner(s): LE, QUE TAN
• Application Number:
  US09/022,641
• Time in Patent Office: 677 Days
• Field of Search: 250/559.4, 250/221, 250/222.2, 250/461.1, 250/461.2, 356/346, 356/244, 356/246, 356/40, 356/39
• US Class Current: 250/559.4
• CPC Class Codes: G01N 2021/6441 with two or more labels
• 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

Abstract

Disclosed are a method and apparatus for determining whether one or more organic substances, namely, proteins (42) and associated cancer cells (40'"'"'), are present in a sample (4a). The method comprises a step of deploying a plurality of fluorescent materials (40) in the tissue sample, individual ones of the plurality of fluorescent materials (40) being selected for coupling to a specific type of substance (42) of interest. The fluorescent materials (40) generally emit characteristic wavelengths in response to being illuminated with excitation light (14a'"'"'-14c'"'"') having predetermined excitation wavelengths. A next step includes illuminating the sample (4a) with the excitation light (14a'"'"'-14c'"'"'). The excitation light (14a'"'"'-14c'"'"') causes any of the fluorescent materials (40) that are disposed within the illuminated portion of the sample (4a), and which are responsive to the excitation wavelengths, to emit their characteristic wavelengths. Further steps include simultaneously detecting the presence of the emitted characteristic wavelengths, and, in response to detecting the presence of the emitted characteristic wavelengths, indicating that at least one of the substances (42) is present within the sample (4a). The invention enables the sample (4a) to be analyzed for the presence of multiple types of proteins (42) and associated cancer cells (40'"'"') simultaneously, using either a narrow band or broad band of excitation wavelengths. The invention also enables the proteins (42) and cancer cells (40'"'"') to be identified using a spectral and temporal analysis. These identifications are made regardless of whether or not the fluorescent materials (40) emit similar or different wavelengths.

Claims

1. An imaging system for detecting the presence of organic substances in a sample, at least certain types of the organic substances being bound with specific types of fluorescent materials, the imaging system comprising:

a tunable excitation source for illuminating at least a portion of the sample with at least one excitation wavelength for causing one or more of the fluorescent materials to emit their respective characteristic wavelengths;

a photodetector array disposed for detecting the emitted characteristic wavelengths, said photodetector array being responsive to detecting said emitted characteristic wavelengths for outputting signals corresponding to detected ones of the emitted characteristic wavelengths;

an output-user interface; and

processing means coupled to an output of said photodetector array and to said output-user interface, said processing means being responsive to receiving at least one of said signals output by said photodetector array for controlling said output-user interface to generate a user-perceptible indicator indicating that at least one of said organic substances is present within the sample.

2. An imaging system as set forth in claim 1, wherein the organic substances include various types of proteins associated with corresponding types of cancer cells.

3. An imaging system as set forth in claim 1, wherein said imaging system further comprises:

a data table, said data table being bidirectionally coupled to said processing means, said data table storing a plurality of reference wavelength values and information identifying the organic substances, each of the reference wavelength values corresponding to at least a respective one of the organic substances;

storage means, said storage means being bidirectionally coupled to said processing means and including a plurality of groups of storage bins, each of said groups of storage bins corresponding to at least one of the reference wavelength values stored in said data table;

wherein said processing means is also responsive to receiving said signals output by said photodetector array for storing individual ones of these signals in corresponding ones of the storage bins, and wherein said processing means also correlates individual ones of said groups that include said corresponding ones of said storage bins to corresponding ones of the reference wavelength values and to corresponding ones of the organic substances identified by the information stored in the data table, said processing means also for controlling the output user-interface for presenting to a user a message identifying the corresponding ones of the organic substances as being present in the sample.

4. An imaging system as set forth in claim 1, and further comprising:

a data table, said data table being bidirectionally coupled to said processing means, said data table storing a plurality of reference wavelength values and information identifying the organic substances, each of the reference wavelength values being associated with at least one of the organic substances, said data table also storing reference decay time constant values, each of the reference decay time constant values corresponding to a respective one of the reference wavelength values and to a respective one of the organic substances; and

storage means, said storage means being bidirectionally coupled to said processing means and including a plurality of groups of storage bins, each of the groups of storage bins corresponding to at least one of the reference wavelength values stored in said data table;

wherein said processing means is also responsive to receiving said signals output by said photodetector array for storing individual ones of these signals in corresponding ones of the storage bins, said processing means also for calculating respective emission decay time constant values based on respective ones of the signals stored in the storage bins, said processing means also for determining whether individual ones of the groups of storage bins having signals stored therein correspond to respective ones of said reference wavelength values which are associated with more than one of the organic substances, wherein said processing means is responsive to determining that individual ones of these groups of storage bins correspond to respective ones of these reference wavelength values for correlating the calculated emission decay time constant values to corresponding ones of the reference decay time constant values and to corresponding ones of the organic substances, and wherein said processing means is also for controlling the output user-interface for presenting to a user a message identifying the corresponding ones of the organic substances as being present in the sample.

5. An imaging system as set forth in claim 1, wherein said tunable excitation source includes a source illuminator for generating a pulsed beam that includes a plurality of wavelengths, the plurality of wavelengths including the at least one excitation wavelength, and wherein said imaging system further comprises:

first means disposed for receiving said pulsed beam from said source illuminator, said first means for selecting the at least one excitation wavelength for being output from said first means as a resultant beam;

second means disposed for receiving the resultant beam output by said first means, said second means for converting the resultant beam to multiple image beams; and

a first focussing optic for focussing the multiple image beams to said portion of said sample to illuminate said portion of said sample.

6. An imaging system as set forth in claim 5, wherein said first focussing optic includes an objective.

7. An imaging system as set forth in claim 5, wherein said first means includes:

a slit optic;

a second focussing optic;

a grating; and

a collimating lens disposed between said grating and said source illuminator for receiving said pulsed beam from said source illuminator and for collimating said pulsed beam to provide a resultant, collimated pulsed beam to said grating;

wherein said grating diffracts said resultant, collimated pulsed beam to provide a beam portion to said second focussing optic, wherein said second focussing optic collimates said beam portion and provides a further beam portion to said slit optic, said further beam portion including a band of wavelengths that includes the at least one excitation wavelength, and wherein said slit optic is responsive to receiving said further beam portion for selecting the at least one excitation wavelength for being output from said slit optic as said resultant beam.

8. An imaging system as set forth in claim 5, wherein said first means includes:

a slit optic;

a second focussing optic;

a mirror; and

a collimating lens disposed between said mirror and said source illuminator for receiving said pulsed beam from said source illuminator and for collimating said pulsed beam to provide a resultant, collimated pulse beam to said mirror;

wherein said mirror reflects said resultant, collimated pulsed beam to said second focussing optic as a beam portion, wherein said second focussing optic collimates said beam portion and provides a further beam portion to said slit optic, said further beam portion including a band of wavelengths that includes the at least one excitation wavelength, and wherein said slit optic is responsive to receiving said further beam portion for selecting the at least one excitation wavelength for being output from said slit optic as said resultant beam.

9. An imaging system as set forth in claim 5, wherein said second means includes:

an optical mask, the optical mask including a plurality of rows of slits, wherein adjacent ones of the rows of slits are separated from one another by predetermined distances, and wherein the slits of individual ones of the plurality of rows are horizontally offset from the slits of other ones of the plurality of rows;

a cylindrical lens disposed for receiving the resultant beam output by said first means, said cylindrical lens for outputting a further beam in response to receiving said resultant beam;

a lenslet array disposed between said cylindrical lens and said optical mask, said lenslet array comprising a plurality of lenslets, said lenslet array for receiving said further beam from said cylindrical lens, wherein the further beam received by the lenslet array illuminates said plurality of lenslets, and wherein in response to said lenslet array being illuminated by said further beam, individual ones of said lenslets focus a respective portion of said further beam to a respective one of the plurality of rows of slits of said optical mask; and

wherein in response to receiving respective portions of said further beam from said lenslets, respective ones of said plurality of rows of slits transmit a respective plurality of the multiple image beams to said first focussing optic, and wherein in response to receiving respective pluralities of the multiple image beams, said first focussing optic focusses respective ones of these pluralities of multiple image beams to respective portions of said sample.

10. An imaging system as set forth in claim 1, wherein said photodetector array includes a radiation sensitive surface having a plurality of rows of radiation detection elements, and wherein said imaging system further comprises means for directing those ones of said emitted characteristic wavelengths that are within predetermined wavelength bands to particular ones of said rows of radiation detection elements which correspond to these predetermined wavelength bands.

11. An imaging system as set forth in claim 10, wherein said characteristic wavelengths emitted by said fluorescent materials are part of radiation beams emitted by said fluorescent materials, and wherein said directing means further comprises:

a collimating lens;

a diffraction device;

a second focussing optic;

a slit optic, the slit optic including a plurality of rows of slits, wherein adjacent ones of the rows of slits are separated from one another by predetermined distances, and wherein the slits of individual ones of the plurality of rows are horizontally offset from the slits of other ones of the plurality of rows;

splitting means disposed for receiving said radiation beams, said splitting means for splitting said radiation beams to provide resultant beam portions, said splitting means also for focussing said resultant beam portions to said slit optic;

wherein in response to said slit optic receiving said resultant beam portions, said rows of slits transmit corresponding beams to said collimating lens, said collimating lens being responsive to receiving said corresponding beams from said slit optic for collimating said corresponding beams to provide a collimated beam to said diffraction device, wherein said diffraction device is responsive to receiving said collimated beam for separating wavelengths included in said collimated beam into corresponding ones of said predetermined wavelength bands, said diffraction device also for providing a diffracted beam that includes separated ones of said wavelengths to said second focussing optic, said separated wavelengths including said emitted characteristic wavelengths, and wherein said second focussing optic is responsive to receiving said diffracted beam for directing said emitted characteristic wavelengths to said rows of radiation detection elements.

12. An imaging system as set forth in claim 11, wherein said diffraction device includes a prism.

13. An imaging system as set forth in claim 11, wherein said diffraction device includes a grating.

14. An imaging system as set forth in claim 1, wherein said processing means is responsive to receiving said signals output by said photodetector array for detecting amplitudes of said signals, said processing means also determining whether at least one of the detected amplitudes exceeds a predetermined threshold level, wherein said processing means is responsive to determining that at least one of said amplitudes exceeds said predetermined threshold level for controlling said output user-interface for generating said user-perceptible indicator.

15. An imaging system for detecting the presence of organic substances in a sample, at least certain types of the organic substances being bound with specific types of fluorescent materials, the imaging system comprising:

an output user-interface;

a tunable excitation source for illuminating at least a portion of said sample with at least one excitation wavelength for causing one or more of the fluorescent materials to emit their respective characteristic wavelengths;

means for spatially dispersing the emitted characteristic wavelengths as a function of wavelength;

a photodetector array disposed for simultaneously detecting dispersed ones of the emitted characteristic wavelengths, and for outputting signals corresponding to detected ones of these dispersed emitted characteristic wavelengths; and

processing means coupled to an output of said photodetector array and to said output user-interface, said processing means being responsive to receiving at least one of said signals output by said photodetector array for controlling said output user-interface for generating an indicator indicating that at least one of said organic substances is present within the sample.