Method and apparatus for rapid change of fluorescence bands in the detection of dyes in fluorescence microscopy
View Patent ↗A method is disclosed for operation of an image-generating optical system for detection of characteristic quantities of the wavelength-dependent behavior of an illuminated specimen, such as the emission behavior and/or absorption behavior and, in particular, the fluorescence and/or luminescence and/or phosphorescence and/or enzyme-active light emission and/or enzyme-active fluorescence, for the operation of a laser scanning microscope, comprising the steps of splitting the image point information of the specimen into spectral components in a spatially resolved manner on the detection side in dependence on wavelength and carrying out at least one summing for different spectral components.
1. A method for operation of an image-generating optical system for detection of characteristic quantities of the wavelength-dependent behavior of an illuminated specimen, such as the emission behavior and/or absorption behavior and, in particular, the fluorescence and/or luminescence and/or phosphorescence and/or enzyme-active light emission and/or enzyme-active fluorescence, for the operation of a laser scanning microscope, comprising the steps of:
splitting the image point information of the specimen into spectral components in a spatially resolved manner on the detection side in dependence on wavelength; and
carrying out at least one summing for different spectral components, wherein a change in at least one irradiation wavelength and/or irradiation intensity is carried out within a scanning process between different specimen regions and a summing of at least some of the respective spectral components is carried out for different specimen regions and/or irradiation wavelengths/intensities;
displaying the summed regions as images.
2. The method according to claim 1 , wherein a plurality of partial sums is formed and then added.
3. The method according to claim 1 , with an overlapping of partial sums of spectral components containing the overlapped signals of different fluorescence components.
4. The method according to claim 1 , further including determining spectral centroid formation for a plurality of spectral components.
5. The method according to claim 1 , with mathematical combination such as division and subtraction of partial sums or individual components and graphic representation of the combination.
6. The method according to claim 1 , for optical detection of characteristic quantities of the wavelength-dependent behavior of an illuminated specimen, such as the emission behavior and/or absorption behavior, in particular, the fluorescence and/or luminescence and/or phosphorescence and/or enzyme-active light emission and/or enzyme-active fluorescence, wherein the emission radiation is split spectrally by a dispersive element, detected in a spatially resolved manner and at least one sum signal of the emission radiation and/or of the absorbed radiation is determined electronically.
7. The method according to claim 1 , wherein the sum signal of the spectrally split emission radiation is determined for distinguishing different dyes and/or for determining the local dye composition of an image point when a plurality of dyes are used simultaneously and/or for determining the local shift of the emission spectrum depending on the local environment to which the dye or dyes is or are attached and/or for measuring emission ratio dyes for determining ion concentrations.
8. The method according to claim 1 , wherein the sum signal of the spectrally expanded, reflected, backscattered and/or transmitted excitation radiation of fluorochromes is carried out for distinguishing different dyes and/or for determining the local dye composition of an image point when a plurality of dyes are used simultaneously and/or for determining the local shift in the absorption spectrum depending on the local environment to which the dye or dyes is or are attached and/or for measuring the absorption ratio for determining ion concentrations.
9. The method according to claim 1 , wherein the composition of the sum signals can be varied during scanning as a function of the excitation parameters (multitracking).
10. The method according to claim 1 , wherein the composition of the sum signals can be varied during scanning as a function of the respective scanning position (ROI tracking).
11. The method according to claim 1 , wherein the emission radiation of the specimen is split by a dispersive element and is detected in a spatially resolved manner in at least one direction.
12. The method according to claim 1 , wherein a splitting of the fluorescence radiation is carried out.
13. The method according to claim 1 , wherein the radiation that is reflected, backscattered and/or transmitted by the specimen is split by a dispersive element and is detected in a spatially resolved manner in at least one direction for absorption measurement.
14. The method according to claim 1 , wherein the signals of detection channels are converted and digitally read out and summing is carried out digitally in a computer.
15. The method according to claim 1 , wherein the summing is carried out with analog data processing by a demultiplexer in combination with a summing amplifier.
16. The method according to claim 1 , wherein the signals of detector channels are influenced by a nonlinear distortion of the input signals.
17. The method according to claim 1 , further comprising integrating the signal from a detection channel over a predetermined time.
18. The method according to claim 1 , wherein the characteristic or response curve of an amplifier is influenced.
19. The method according to claim 1 , wherein the sum signal is used for generating an image.
20. The method according to claim 1 , wherein a color-coded fluorescence image is generated.
21. The method according to claim 1 , wherein a superposition is carried out with additional images.
22. The method according to claim 1 , wherein the sum signals are combined with a lookup table.
23. The method according to claim 1 , wherein representation of different dyes and/or the spread of the generated image is carried out by the lookup table.
24. The method according to claim 1 , wherein a comparison of the measured signal with a reference signal is carried out via comparators in detection channels and in case the reference signal is not reached and/or is exceeded a change in the operating mode of the detection channel is carried out.
25. The method according to claim 1 , wherein the respective detection channel is switched off and/or not taken into account.
26. The method according to claim 25 , wherein the spectral region of interest is narrowed in this way.
27. The method according to claim 1 , wherein the signals of the detection channels are generated by at least one integrator circuit.
28. The method according to claim 1 , wherein the signals of the detection channels are generated by photon counting and subsequent digital-to-analog conversion.
29. The method according to claim 28 , wherein photon counting is carried out in time correlation.
30. The method according to claim 1 , for detection of single-photon and/or multiphoton fluorescence and/or fluorescence excited by entangled photons.
31. The method according to claim 1 , with parallel illumination and detection, such as in ingredient screening, wherein the specimen is a microtiter plate.
32. The method according to claim 1 , incorporated in a microscope.
33. The method according to claim 1 , for detection in a nearfield scanning microscope.
34. The method according to claim 1 , for detection of a single-photon and/or multiphoton dye fluorescence in a fluorescence-correlated spectroscope.
35. The method according to claim 1 , employing confocal detection.
36. The method according to claim 1 , employing a scanning arrangement.
37. The method according to claim 1 , employing illumination means with an X-Y scanner.
38. The method according to claim 1 , employing an X-Y scan table.
39. The method according to claim 1 , employing nonconfocal detection.
40. The method according to claim 1 , employing a scanning arrangement.
41. The method according to claim 1 , employing descanned detection.
42. The method according to claim 1 , employing brightfield imaging.
43. The method according to claim 1 , employing point imaging.
44. The method according to claim 1 , employing non-descanned detection.
45. The method according to claim 1 , employing non-scanning, confocal or nonconfocal detection and point imaging or brightfield imaging.
46. An arrangement for optical detection of characteristic quantities of the wavelength-dependent behavior of an illuminated specimen, such as the emission behavior and/or absorption behavior and, in particular, the fluorescence and/or luminescence and/or phosphorescence and/or enzyme-active light emission and/or enzyme-active fluorescence, comprising:
a dispersive element for splitting the emission radiation spectrally;
a detector for detecting a radiation signal; and
means for electronically determining at least one sum signal of the emission radiation and/or of the absorbed radiations;
wherein a change in at least one irradiation wavelength and/or irradiation intensity is carried out within a scanning process between different specimen regions and a summing of at least some of the respective spectral components is carried out for different specimen regions and/or irradiation wavelengths/intensities.
47. The arrangement according to claim 46 , wherein the sum signal of the spectrally split emission radiation is determined for distinguishing different dyes and/or for determining the local dye composition of an image point when a plurality of dyes are used simultaneously and/or for determining the local shift of the emission spectrum depending on the local environment to which the dye or dyes is or are attached and/or for measuring emission ratio dyes for determining ion concentrations.
48. The arrangement according to claim 46 , wherein the sum signal of the spectrally expanded, reflected, backscattered and/or transmitted excitation radiation of fluorochromes is carried out for distinguishing different dyes and/or for determining the local dye composition of an image point when a plurality of dyes are used simultaneously and/or for determining the local shift in the absorption spectrum depending on the local environment to which the dye or dyes is or are attached and/or for measuring the absorption ratio for determining ion concentrations.
49. The arrangement according to claim 46 , wherein the composition of the sum signals can be varied during scanning (multitracking).
50. The arrangement according to claim 46 , wherein the composition of the sum signals can be varied during scanning (ROI tracking).
51. The arrangement according to claim 46 , wherein the emission radiation of the specimen is split by a dispersive element and is detected in a spatially resolved manner in at least one direction.
52. The arrangement according to claim 46 , wherein a splitting of the fluorescence radiation is carried out.
53. The arrangement according to claim 46 , wherein the radiation that is reflected or transmitted by the specimen is split by a dispersive element and is detected in a spatially resolved manner in at least one direction for absorption measurement.
54. The arrangement according to claim 46 , wherein the signals of detection channels are converted and digitally read out and summing is carried out digitally in a computer.
55. The arrangement according to claim 46 , wherein the summing is carried out with analog data processing by a demultiplexer in combination with a summing amplifier.
56. The arrangement according to claim 46 , wherein the signals of detector channels are influenced by a nonlinear distortion of the input signals.
57. The arrangement according to claim 46 , further comprising integrating the signal from a detection channel over a predetermined time.
58. The arrangement according to claim 46 , wherein the characteristic or response curve of an amplifier is influenced.
59. The arrangement according to claim 46 , wherein the sum signal is used for generating an image.
60. The arrangement according to claim 46 , wherein a color-coded fluorescence image is generated.
61. The arrangement according to claim 46 , wherein a superposition is carried out with additional images.
62. The arrangement according to claim 46 , wherein the sum signals are combined with a lookup table.
63. The arrangement according to claim 46 , wherein representation of different dyes and/or the spread of the generated image is carried out by the lookup table.
64. The arrangement according to claim 46 , wherein a comparison of the measured signal with a reference signal is carried out via comparators in detection channels and in case the reference signal is not reached and/or is exceeded a change in the operating mode of the detection channel is carried out.
65. The arrangement according to claim 46 , wherein the respective detection channel is switched off and/or not taken into account.
66. The arrangement according to claim 65 , wherein the spectral region of interest is narrowed in this way.
67. The arrangement according to claim 46 , wherein the signals of the detection channels are generated by at least one integrator circuit.
68. The arrangement according to claim 46 , wherein the signals of the detection channels are generated by photon counting and subsequent digital-to-analog conversion.
69. The arrangement according to claim 68 , wherein photon counting is carried out in time-correlated manner.
70. The arrangement according to claim 46 , for detection of single-photon and/or multiphoton fluorescence and/or fluorescence excited by entangled photons.
71. The arrangement according to claim 46 , with parallel illumination and detection, such as in ingredient screening, wherein the specimen is a microtiter plate.
72. The arrangement according to claim 46 , incorporated in a microscope.
73. The arrangement according to claim 46 , for detection in a nearfield scanning microscope.
74. The arrangement according to claim 46 , for detection of a single-photon and/or multiphoton dye fluorescence in a fluorescence-correlated spectroscope.
75. The arrangement according to claim 46 , employing confocal detection.
76. The arrangement according to claim 46 , employing a scanning arrangement.
77. The arrangement according to claim 46 , employing an X-Y scanner in the illumination means.
78. The arrangement according to claim 46 , employing an X-Y scan table.
79. The arrangement according to claim 46 , employing nonconfocal detection.
80. The arrangement according to claim 46 , employing a scanning arrangement.
81. The arrangement according to claim 46 , employing descanned detection.
82. The arrangement according to claim 46 , employing brightfield imaging.
83. The arrangement according to claim 46 , employing point imaging.
84. The arrangement according to claim 46 , employing non-descanned detection.
85. The arrangement according to claim 46 , employing non-scanning, confocal or nonconfocal detection and point imaging or brightfield imaging.
86. A method for operation of an image-generating optical system for detection of characteristic quantities of the wavelength-dependent behavior of an illuminated specimen, such as the emission behavior and/or absorption behavior and, in particular, the fluorescence and/or luminescence and/or phosphorescence and/or enzyme-active light emission and/or enzyme-active fluorescence, for the operation of a laser scanning microscope, comprising the steps of:
splitting the image point information of the specimen into spectral components in a spatially resolved manner on the detection side in dependence on wavelength; and
carrying out at least one summing for different spectral components, wherein a change in at least one irradiation wavelength and/or irradiation intensity is carried out within a scanning process between different specimen regions and a summing of at least some of the respective spectral components is carried out for different specimen regions and/or irradiation wavelengths/intensities;
wherein the composition of the sum signals can be varied during scanning as a function of the excitation parameters (multitracking) or as a function of the respective scanning position (ROI tracking);
displaying the summed regions as images.