Method and apparatus for fluorescent confocal microscopy
View Patent ↗A new and improved confocal fluorescence microscope is presented. The new microscope has significant advantages relative to existing implementations of microscope confocal imagers. In common with previous confocal imagers the instant invention has the advantages relative to conventional wide-field and confocal fluorescence imagers, however it addresses the drawbacks of confocal technology in terms of cost and complexity, and provides significant savings in both due to the simplicity of the components and the elimination of the need of, in particular, spatial filters such as pinholes or slits.
1. In an apparatus for confocal fluorescent microscopy comprising:
at least one optical source which provides excitation radiation to an illumination area on a target and at least one detector which detects fluorescent emission from the target;
wherein the at least one optical source includes a line forming means to illuminate a line portion of the target; and
the at least one detector is capable of random access reading of said fluorescence emission wherein a region of interest of the at least one detector is configured to be optically conjugated with the illumination area of the line portion of the target and said region of interest is moving in synchronization with the illumination area on the target.
2. The apparatus of claim 1 , wherein said at least one optical source is selected from the group consisting of lasers, laser diodes, light emitting diodes, lamps, and combinations thereof.
3. The apparatus of claim 2 , wherein said optical source includes two or more lasers.
4. The apparatus of claim 3 , wherein the lasers emit at different wavelengths.
5. The apparatus of claim 1 , wherein said at least one detector includes one or more two dimensional pixel-based optical receivers capable of independent reset and readout of such pixels.
6. The apparatus of claim 5 , wherein the at least one detector includes a CMOS detector.
7. The apparatus of claim 1 , wherein the line forming means is selected from the group consisting of Powell lenses, cylindrical lenses, diffraction gratings, holographic elements, and combinations thereof.
8. The apparatus of claim 1 , wherein the line forming means produces a line of light on the target which is moved across the target by one or more beam deflecting devices.
9. The apparatus of claim 2 , wherein said radiation is from more than one or more sources and said radiation from said one or more sources is coupled to provide illumination of the target by an optical selection device selected from the group consisting of dispersive prisms with optical shutters, reflective gratings with optical shutters, dichroic mirrors with optical shutters, fiber optic switches, and rotating mirrors.
10. The apparatus of claim 8 , wherein the beam deflecting devices include one or more galvanometers.
11. The apparatus of claim 8 , wherein the at least one detector is a CMOS detector which further comprises a rolling shutter means.
12. The apparatus of claim 11 , wherein the rolling shutter means has a width of less than or equal to the width of the image of the line on the target.
13. The apparatus of claim 12 , wherein the rolling shutter means is optically conjugated to the line on the target.
14. The apparatus of claim 11 , wherein reading of said fluorescent emission is synchronized with the scanning of said area illuminated.
15. The apparatus of claim 1 , wherein the target includes one or more fluorescent markers.
16. The apparatus of claim 1 , wherein the target is on a microscope slide or a microtiter plate.
17. The apparatus of claim 1 , further comprising a mirror centered on the rear of an objective to couple the excitation light into the illumination and to scan said illumination area across the target.
18. The apparatus of claim 8 , further comprising one or more optical filters in front of the at least one detector which efficiently transmit light at the emission wavelength of the target and reflect or attenuate light at the excitation wavelength of the target.
19. The apparatus of claim 18 , wherein said one or more filters are linear variable filters.
20. A method of obtaining an image of a target comprising:
providing an optical source that provides an excitation light;
scanning said target with the excitation light of a wavelength corresponding to an excitation wavelength of said target;
collecting fluorescent emission as said excitation light moves across said target;
providing the optical source with a line forming means to illuminate a line portion of the target;
providing at least one detector capable of random access reading of the fluorescent emission;
providing a region of interest of the at least one detector that is configured to be optically conjugated with illumination area on the target and said region of interest is moving in synchronization with the illumination area of the line portion on the target; and
providing an image of the target on a display.
21. The method of claim 20 , wherein said optical source is selected from the group consisting of lasers, laser diodes, light emitting diodes, lamps, and combinations thereof.
22. The method of claim 20 , wherein said at least one detector includes one or more two dimensional pixel-based optical receivers capable of independent reset and readout of such pixels.
23. The method of claim 22 , wherein said at least one detector includes a CMOS detector.
24. The method of claim 23 , wherein the CMOS detector further comprises a rolling shutter means.
25. The method of claim 24 , wherein the rolling shutter has a width of less than or equal to the width of the image of a line on the target.
26. The method of claim 24 , wherein the rolling shutter is optically conjugated to a line on the target.
27. The method of claim 20 , wherein the at least one optical source includes a means to illuminate substantially all of an area to be imaged.
28. The method of claim 20 , wherein the line forming means is selected from the group consisting of Powell lenses, cylindrical lenses, diffraction gratings, holographic elements, and combinations thereof.
29. The method of claim 20 , further comprising one or more optical filters in front of the at least one detector which efficiently transmit light at the emission wavelength of the target and reflect or attenuate light at the excitation wavelength of the target.
30. The method of claim 29 , wherein said one or more filters are linear variable filters.
31. The method of claim 30 , wherein said one or more filters are moved to track the movement of said excitation light across said target.
32. The method of claim 20 , wherein the target is a on a microscope slide or a microtiter plate.
33. A method of imaging a subject including one or more of targets marked by one or more fluorescent markers, said method comprising:
a. emitting an electromagnetic radiation from one or more optical sources having emission spectra at least partially overlapping with absorption bands of the fluorescent markers;
b. applying a beam-shaping optical system to control a shape of an illumination zone on a subject having a shape of a line;
c. collecting fluorescent emission from the fluorescent markers located within the illumination zone on the subject using a light collection system that can be the same or different with the illumination system;
d. placing one or more two-dimensional pixel-based detectors that are capable of an independent reset and readout of pixels in a position optically conjugated to the imaging area;
e. adjusting the shape, the size and location of the region of interest within the optical detector to be equal or smaller than an image of optically conjugated illumination zone of the subject;
f. detecting the fluorescent emission that is delivered to the region of interest of the detector by reading the signals from the pixels located within the region of interest;
g. resetting the pixels of the detector that are located outside of the region of interest of the detector that are not optically conjugated with the illumination zone;
h. scanning the illumination zone within the imaging area of the subject using a beam scanning system;
i. moving the region of interest of the detector to maintain the optical conjugation with the illumination zone using random access to the pixels of the optical detector;
j. forming an image of the imaging area of the subject from the signals (sub-images) from the region of interest of a receiver obtained during the scanning of the illumination zone across the imaging area;
k. repeating the method from step (a) for plurality of optical sources for the sequential image acquisition for plurality of optical sources; and
l. providing the imaging area on a display.
34. The method of claim 33 , further comprising one or more fluorescent markers, said method comprising:
filtering the fluorescent emission using longpass, bandpass, or linear variable optical filters.
35. The method of claim 33 , wherein said at least one optical source is selected from the group consisting of lasers, laser diodes, light emitting diodes, lamps, and combinations thereof.
36. The method of claim 33 , wherein said one or more detectors is selected from the group consisting of CMOS detectors and CCD detectors.
37. The method of claim 33 , wherein the target is on a microscope slide or a microtiter plate.