Method and device for conducting the spectral differentiating, imaging measurement of fluorescent light

• US 20050243313A1
• Filed: 05/15/2003
• Published: 11/03/2005
• Est. Priority Date: 05/21/2002
• Status: Active Grant

• Current Assignee: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e)
• Original Assignee: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e)
• Inventors: Neher, Erwin, Neher, Richard
• Granted Patent: US 7,304,733 B2
• Time in Patent Office: Days
• US Class Current: 356/317
• CPC Class Codes: G01J 3/4406 Fluorescence spectrometry
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/6471 Special filters, filter wheel
• 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

Abstract

The invention relates to a method for conducting the spectral differentiating, imaging measurement of fluorescent light, according to which a sample containing fluorophores of different species is irradiated with excitation light of at least one excitation channel that is defined by its spectral properties and/or the excitation time. The fluorescent light emitted by the sample is received by at least one detection channel, which is defined by its spectral detection characteristic and/or the detection time, and is converted into a digital signal. The digital signal is stored for further processing in a memory unit of a digital data processing system. The inventive method is characterized in that the properties of a number of measuring channels, which are defined as specific combinations each time consisting of an excitation channel and a detection channel, are automatically set before conducting the measurement according to the result of a mathematical optimization process, which is performed by a calculating unit of the digital processing system and which takes into account the fluorescence characteristics of at least some of the fluorophores presumed by the user to be in the sample, or said properties are given according to instructions for manually setting by the user. The invention also relates to a device for carrying out the inventive method.

Claims

1. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

23-44. -44. (canceled)

45. A method for image producing measurement of fluorescent light wherein

a sample, containing a fluorophore of at least one species, is irradiated with exciting light at at least one excitation channel defined by its spectral properties, and

the fluorescent light emitted by the sample is received by at least one detection channel defined by its spectral detection characteristic and is converted into a digital signal,

wherein the digital signal is stored in a memory unit of a digital data processing unit for further processing, wherein, prior to carrying out the measurement, the properties of the at least one excitation and the at least one detection channel are predetermined according to the result of a mathematical optimization process carried out by a computing unit of the digital data processing unit, which takes into consideration the fluorescence characteristics of at least one of the fluorophores presumed by the user to be in the sample,

wherein for carrying out a spectrally differentiating measurement of a sample, which contains fluorophores of different species, in the framework of the optimization process

a) a linear system of equations is set up,

which represents the coefficients of spectral characteristics of measurement channels, which are respectively defined as the combination of one excitation channel and one detection channel,

and which describes the relationship between a user-presumed chemical composition of the sample and the signal calculated based on the measurement channel characteristics to be optimized,

b) the coefficients are varied, until a clear solution to the equation system becomes possible,

c) the coefficients calculated as a result of the optimization process form the basis of the input of the characteristics of the excitation and detection channels.

5. A method according to claim 45, wherein the uniqueness of the solution of the linear system of equations is optimized with consideration of one or more additional conditions that can be introduced by the user.

6. A method according claim 45, wherein the mathematical optimization method includes the optimization of a condition number of a matrix expression containing a matrix formed from the coefficients of the linear system of equations.

7. A method according to claim 6, wherein the matrix elements of the matrix expression are weighted for minimization of the quantity χ

² corresponding to the measurement errors expected by the user.

8. A method according to claim 6, wherein the matrix expression containing the coefficient matrix is essentially the left-side matrix product of the coefficient matrix with its transposed form.

9. A method according to claim 6, wherein the condition number contains essentially the determinant of the matrix expression.

10. A method according to claim 6, wherein the condition number contains essentially the trace of the matrix expression.

11. A method according to claim 6, wherein the condition number corresponds essentially to the quantity

$\det /{\left(\frac{N}{n-1}\right)}^{\left(n-1\right)/2},$

where det is the determinant, N is the trace and n is the dimension of the matrix expression.

12. A method according to claim 6, wherein the condition number contains essentially the ratio of the smallest to the largest intrinsic value of the matrix expression.

13. A method according to claim 6, wherein the condition number to be optimized can be chosen by the user.

14. A method according to claim 13, wherein the condition number to be optimized is chosen by the user by entering a global characterization of the signal to be expected into the digital data processing installation and then the condition number is determined automatically.

15. A method according to claim 45, wherein the mathematical optimization method contains a second optimization step in which the noise of the expected signal is optimized by variation of coefficients which describe the properties of the measurement channels.

16. A method according to claim 15, wherein the noise of the signal to be expected is optimized with consideration of one or more additional conditions that can be introduced by the user.

17. A method according to claim 5, wherein a maximum limit or the optimum for the bleaching of one or several fluorophores is used as additional condition.

18. A method according to claim 5, wherein the minimization of the noise of a signal of a given intensity is used as additional condition.

19. A method according to claim 5, wherein the maximum spectral resolution of different fluorophores in a region of a previously recorded test image is used as additional condition.

20. A method according to claim 5, wherein the minimization of a relative error is used as additional condition.

21. A method according to claim 5, wherein the optimization method includes an iterative, dialog-controlled process for the definition of the additional conditions.

22. A method according to claim 5, wherein the automatic adjustment of the measurement channel properties is carried out by starting and/or motor motion of AOTFs, LCTFs, cut-off filters, band pass filters or neutral gray filters and/or beam splitters.