Three-dimensional imaging using swept confocally aligned planar excitation with asymmetrical magnification
Implementing swept, confocally aligned planar excitation (SCAPE) imaging with asymmetric magnification in the detection arm provides a number of significant advantages. In some preferred embodiments, the asymmetric magnification is achieved using cylindrical lenses in the detection arm that are oriented to increase the magnification of the intermediate image in the width direction but not in the depth direction. SCAPE imaging may also be improved by using an SLM to modify a characteristic of the sheet of excitation light that is projected into the sample. Additional embodiments include a customized version of SCAPE that is optimized for imaging the retina at the back of an eyeball in living subjects.
1. An imaging apparatus comprising:
a first set of optical components having a proximal end and a distal end, wherein the first set of optical components includes an objective disposed at the distal end of the first set of optical components;
a second set of optical components having a proximal end and a distal end, wherein the second set of optical components includes an objective disposed at the distal end of the second set of optical components, wherein the second set of optical components has a first magnification in a first radial direction and a second magnification in a second radial direction that is perpendicular to the first radial direction, and wherein the first magnification is at least 1.5 times the second magnification;
a scanning element that is disposed proximally with respect to the proximal end of the first set of optical components and proximally with respect to the proximal end of the second set of optical components,
wherein the scanning element is arranged to route a sheet of excitation light so that the sheet of excitation light will pass through the first set of optical components in a proximal to distal direction and project into a sample that is positioned distally beyond the distal end of the first set of optical components, wherein the sheet of excitation light is projected into the sample at an oblique angle, and wherein the sheet of excitation light is projected into the sample at a position that varies depending on an orientation of the scanning element,
wherein the first set of optical components routes detection light from the sample in a distal to proximal direction back to the scanning element, and
wherein the scanning element is also arranged to route the detection light so that the detection light will pass through the second set of optical components in a proximal to distal direction and form an intermediate image plane at a position that is distally beyond the distal end of the second set of optical components; and
a light detector array arranged to capture images of the intermediate image plane.
2. The apparatus of claim 1 , wherein the intermediate image plane is stationary.
3. The apparatus of claim 1 , wherein the detection light arriving from the sample has a depth dimension and a width dimension that is perpendicular to the depth dimension,
wherein the magnification in the first radial direction in the second set of optical components corresponds to magnification of the width dimension of the detection light,
wherein the first set of optical components has a uniform magnification in all radial directions, and
wherein the uniform magnification of the first set of optical components is the same as the second magnification of the second set of optical components.
4. The apparatus of claim 3 , wherein the first magnification is at least 2 times the second magnification.
5. The apparatus of claim 4 , wherein the first set of optical components comprises a first set of spherical optical components, and wherein the second set of optical components comprises (a) a second set of spherical optical components with a magnification that matches the first set of spherical optical components and (b) a set of cylindrical optical components.
6. The apparatus of claim 1 , wherein the detection light arriving from the sample has a depth dimension and a width dimension that is perpendicular to the depth dimension, and wherein the magnification in the first radial direction in the second set of optical components corresponds to magnification of the width dimension of the detection light.
7. The apparatus of claim 6 , wherein the light detector array comprises a 2D image sensor with pixels arranged in a plurality of readout rows, and the light detector array is oriented so that each of the plurality of readout rows corresponds to a respective different position in the depth direction of the detection light.
8. The apparatus of claim 6 , wherein the light detector array comprises a 2D image sensor with pixels arranged in a plurality of readout rows, and the light detector array is oriented so that each of the plurality of readout rows corresponds to a respective different position in the depth direction of the detection light, and
wherein the captured images of the intermediate image plane are arranged in frames, and each frame includes data from not more than half of the rows.
9. The apparatus of claim 8 , wherein each frame includes data from not more than one quarter of the rows.
10. A method of imaging a sample comprising:
projecting a sheet of excitation light into a sample, wherein the sheet of excitation light is projected into the sample at an oblique angle, and wherein the sheet of excitation light is projected into the sample at a position that varies with time;
routing detection light arriving from the sample into a proximal end of an optical system that has a first magnification in a first radial direction and a second magnification in a second radial direction that is perpendicular to the first radial direction, wherein the first magnification is at least 1.5 times the second magnification;
forming a stationary intermediate image plane at a distal end of the optical system; and
capturing images of the intermediate image plane at a plurality of times.
11. The method of claim 10 , wherein the detection light arriving from the sample has a depth dimension and a width dimension that is perpendicular to the depth dimension, and wherein the magnification in the first radial direction in the optical system corresponds to magnification of the width dimension of the detection light.
12. The method of claim 10 , wherein the first magnification is at least 2 times the second magnification.
13. The method of claim 10 , wherein the sheet of excitation light is projected into the sample at a position that varies with time depending on an orientation of a scanning element,
wherein the routing step is implemented by the scanning element, and
wherein each of the images of the intermediate image plane corresponds to a different orientation of the scanning element.
14. An imaging apparatus comprising:
a first set of optical components having an objective, wherein the first set of optical components is arranged to (a) route excitation light into the objective so as to generate a sweeping sheet of excitation light through the objective and (b) simultaneously route image light returning through the objective along a detection path;
a second set of optical components disposed in the detection path arranged to receive light from the first set of optical components and produce an asymmetrically magnified oblique real image by magnifying in a first radial direction at a power of at least 1.5 times that in a second radial direction perpendicular to the first radial direction; and
a light detector array positioned to sample the oblique real image.
15. The apparatus of claim 14 , wherein the oblique real image has a first dimension whose pixels resolve light from multiple depths along an optical axis in front of the objective and a second dimension perpendicular the first dimension whose pixels resolve light from multiple positions along an axis transverse to the optical axis.
16. The apparatus of claim 14 , wherein the second set of optical components produce the asymmetrically magnified image by magnifying in the first radial direction at a power of at least 2 times that in the second radial direction.
17. The apparatus of claim 14 , wherein the detection path includes a scanning element that routes the image light from the first set of optical components into the second set of optical components, wherein the scanning element also routes the sheet of excitation light into the first set of optical components.
18. The apparatus of claim 17 , wherein the first set of optical components provides symmetric magnification between the objective and the scanning element.
19. The apparatus of claim 14 , wherein the light detector array comprises a 2D image sensor.
20. The apparatus of claim 19 , further comprising a sampling controller that reads out the pixels of the light detector array row by row, wherein the rows correspond to the second dimension.
21. The apparatus of claim 20 , wherein the sampling controller reads out only a fraction of the total number of rows of the light detector array for each of position of the scanning element.