Fluorescence imaging autofocus systems and methods
Quantitative fluorescence imaging systems and methods using angular illumination to obtain automatic focus information. Laser scanning (e.g., point or line scanning) with angular illumination in combination with an area imaging sensor, such as with a bi-telcentric scanner, is used to determine sample height (relative to a detection axis orthogonal to a platform holding the sample) and also correct for sample height in subsequent scans.
1. A fluorescence imaging system, comprising:
a sample platform for holding a fluorescent material;
a light detector having an array of sensing locations for detecting light emitted from the fluorescent material;
an optical imaging system positioned between the sample platform and the light detector and configured to focus light emitted from field points on the sample platform onto the light detector, wherein contiguous field points on the sample platform are simultaneously imaged onto contiguous sensing locations on the light detector;
an illumination system including a light source that emits excitation light in an absorption band of the fluorescent material, wherein the illumination system provides a beam of illumination that impinges on the fluorescent material at an angle relative to a detection axis, wherein a plane of incidence of the beam includes the detection axis and a scan direction, wherein the scan direction is perpendicular to the detection axis;
a scanning mechanism that enables continuous scanning of the beam of illumination relative to the sample platform along the scan direction; and
an intelligence module coupled to the light detector and configured to determine variations along the scan direction of a height of the fluorescent material parallel to the detection axis based on variations along the scan direction of positions on the light detector of maximum illuminance detected by the light detector.
2. The imaging system of claim 1 , further including a memory attached to the intelligence module for storing a height map of the fluorescent material on the platform.
3. The imaging system of claim 1 , wherein the optical imaging system comprises a bi-telecentric optical imaging system.
4. The imaging system of claim 3 , wherein the bi-telecentric optical imaging system comprises an Offner relay mirror system arrangement comprising a first mirror element having a spherical mirror surface and a second mirror element having a spherical mirror surface, wherein an entry aperture stop and an exit aperture stop each comprise a portion of the first mirror element.
5. The imaging system of claim 4 , wherein the first mirror element presents a convex-shaped mirror surface, and wherein the second mirror element presents a concave-shaped mirror surface.
6. The imaging system of claim 3 , wherein the bi-telecentric optical imaging system comprises a bi-telecentric lens arrangement, wherein an entry aperture stop includes a first refractive lens element and wherein an exit aperture stop includes a second refractive lens element.
7. The imaging system of claim 1 , wherein the scanning mechanism adjusts one of the sample platform along the scan direction or the light detector and illumination beam along the scan direction.
8. The imaging system of claim 1 , wherein the light source comprises a diode laser and wherein the light detector comprises a CCD array detector.
9. The imaging system of claim 1 , wherein the beam of illumination has one of a spot shaped profile and a line-shaped profile.
10. The imaging system of claim 1 , wherein the scan direction is parallel to the sample platform.
11. A method of determining height variations of a fluorescent material on a sample platform, wherein the fluorescent material absorbs light in an absorption band of wavelengths and emits fluorescent light in an emission band of wavelengths, the method comprising:
a) illuminating the fluorescent material on the sample platform with an illumination beam having excitation light in the absorption band, the beam of illumination impinging on the fluorescent material at an angle relative to a detection axis, wherein a plane of incidence of the beam includes the detection axis and a scan direction, wherein the scan direction is perpendicular to the detection axis;
b) scanning the illumination beam along the scan direction;
c) detecting emissions from the first portion of fluorescent material using a detector system including a light detector having an array of sensing locations, and an optical imaging subsystem positioned between the sample platform and the light detector and configured to focus light emitted from field points on the sample platform onto the light detector, wherein contiguous field points on the sample platform are simultaneously imaged onto contiguous sensing locations on the light detector; and
d) determining variations along the scan direction of a height of the fluorescent material parallel to the detection axis based on variations along the scan direction of positions on the light detector of maximum illuminance detected by the light detector.
12. The method of claim 11 , wherein scanning includes moving one or both of a) the sample platform, and b) the illumination beam and the light detector along the scan direction.
13. The method of claim 11 , wherein the scan direction is parallel with a surface defined by the sample platform.
14. The method of claim 11 , further including storing to a memory a height map of the fluorescent material on the platform.
15. The method of claim 11 , further including rescanning the beam of illumination along the scan direction and adjusting a position of the platform as the scanning occurs based on the determined variations in height of the fluorescent material so as to compensate for the determined variations in height of the fluorescent material.
16. The method of claim 11 , wherein the optical imaging system comprises a bi-telecentric optical imaging system.
17. The method of claim 11 , wherein the light source comprises a diode laser and the light detector comprises a CCD array detector.
18. The method of claim 11 , wherein the illumination beam has one of a spot shaped profile and a line-shaped profile.
19. A fluorescence imaging system, comprising:
a sample platform for holding a fluorescent material;
a light detector having an array of sensing locations for detecting light emitted from the fluorescent material;
an optical imaging system positioned between the sample platform and the light detector and configured to focus light emitted from field points on the sample platform onto the light detector, wherein contiguous field points on the sample platform are simultaneously imaged onto contiguous sensing locations on the light detector;
an illumination system including a light source that emits excitation light in an absorption band of the fluorescent material, wherein the illumination system provides a beam of illumination that impinges on the fluorescent material at an angle relative to a detection axis, wherein a plane of incidence of the beam includes the detection axis and a scan direction, wherein the scan direction is perpendicular to the detection axis;
a scanning mechanism that enables continuous scanning of the beam of illumination relative to the sample platform along the scan direction; and
an intelligence module coupled to the light detector and configured to determine a difference in height of the fluorescent material at an imaging location on the sample platform relative to a previous imaging location based on a distance along the scan direction between positions on the light detector of a maximum illuminance detected by the light detector at the imaging location and the previous imaging location.