Autofocus system for scanning microscopy having a volume image formation

• US 5,932,872 A
• Filed: 09/10/1997
• Issued: 08/03/1999
• Est. Priority Date: 07/01/1994
• Status: Expired due to Term

• Current Assignee: Q3DM, LLC
• Original Assignee: Jeffrey H. Price
• Inventors: Price, Jeffrey H.
• Primary Examiner(s): LE, QUE TAN
• Application Number:
  US08/913,647
• Time in Patent Office: 692 Days
• Field of Search: 250/201.3, 250/208.1, 250/234, 345/6, 382/154, 382/285, 358/450, 358/452
• US Class Current: 250/201.3
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G02B 21/002 Scanning microscopes scanning near field optical microscopes G01Q60/18
• CPC Class Codes: G02B 21/244 using image analysis techniques

Abstract

Reliable autofocus is required to obtain accurate measurements of fluorescent stained cellular components from a system capable of scanning multiple microscope fields. Autofocus could be performed directly with fluorescence images, but due to photobleaching and destructive fluorescence by-products, it is best to minimize fluorescence exposure for photosensitive specimens and live cells. This exposure problem could be completely avoided by using phase-contrast microscopy, implemented through the same optics as fluorescence microscopy. Functions for both phase-contrast and fluorescence autofocus were evaluated using the present invention and the suitability of phase-contrast autofocus for fluorescence microscopy was determined. The present autofocus system for scanning microscopy can be performed at least as fast as 0.25 s/field without loss of precision. The speed of autofocus can be further increased by a volume image which is obtained by observing an image object at each image plane of a plurality of image planes, where each image plane is vertically displaced with respect to each other image plane. An electronic image representation is obtained at each image plane. The image planes are scanned over the image object and the images arm aligned timewise in a buffer. The buffer holds a volume image comprising images at the image planes, aligned by the buffer. The image plane having the best focus is selected and a microscope objective is automatically positioned at the selected plane.

Claims

1. An apparatus for generating a volume image, comprising:

a plurality of imaging devices, in which at least one imaging device is positioned at a respective image plane of a plurality of image planes, where each image plane is displaced in a third dimension with respect to each other image plane;

an image forming apparatus coupled to the plurality of imaging devices for forming a plurality of electronic image representations representing images at respective image planes of the plurality of image planes; and

means coupled to the image forming apparatus for acquiring a volume image by storing successive pluralities of electronic image representations.

2. The apparatus of claim 1, further including processing means coupled to the image forming apparatus for determining an optical quality metric by combining electronic image representations.

3. The apparatus of claim 1, wherein the plurality of imaging devices includes a plurality of optical conductors, each optical conductor having a first end face positioned at a respective image plane and a second end face coupled to the image forming apparatus.

4. The apparatus of claim 1, wherein the plurality of imaging devices are disposed in a substantially elongate array.

5. The apparatus of claim 1, wherein the image forming apparatus includes a plurality of charge-coupled device (CCD) cameras.

6. The apparatus of claim 5, wherein the plurality of imaging devices includes a plurality of optical conductors, each optical conductor having a first end face positioned at a respective image plane of the plurality of image planes and a second end face coupled to a respective CCD camera.

7. The apparatus of claim 6, wherein each first end face is offset in the third dimension from each other first end face.

8. The apparatus of claim 1, wherein the plurality of imaging devices includes an elongate array of photo electric devices.

9. The apparatus of claim 8, wherein the elongate array of photo electric devices is tilted with respect to an image object plane, each image plane of the plurality of image planes being intersected by at least one photo electric device of the elongate array of photo electric devices.

10. The apparatus of claim 2, wherein the processing means includes:

a plurality of image buffers, each image buffer for storing a plurality of electronic representations of at least a portion of an image;

a high-pass filter coupled to a respective buffer for providing a filtered electronic representation; and

means coupled to the high-pass filter for combining a plurality of filtered electronic representations to produce the optical quality metric.

11. The apparatus of claim 10, wherein te means for combining includes:

means for obtaining augmented intensity values for the filtered electronic representations; and

means for indicating a value of the optical quality metric for each image based upon the augmented intensity values.

12. The apparatus of claim 1, further including:

an optical high pass filter positioned between the plurality of imaging devices and an image object plane; and

processing means coupled to the image forming apparatus for determining an optical quality metric by combining electronic range image representations.

13. The apparatus of claim 12, wherein the processing means includes:

a plurality of image buffers, each image buffer for storing a plurality of filtered electronic image representations of at least a portion of an image; and

means coupled to the plurality of image buffers for combining a plurality of filtered electronic image representations to produce the optical quality metric.

14. The apparatus of claim 13 wherein the optical quality metric is image sharpness.

15. A method for volume imaging using a plurality of imaging devices in which at least one imaging device is positioned at an image plane of a plurality of image planes, where each image plane is displaced in a third dimension with respect to each other image plane, the method comprising the steps of:

orienting the plurality of imaging devices such that the imaging devices observe an image object in an image object plane;

using the plurality of imaging devices, obtaining a plurality of images of the image object, each image of the plurality of images obtained in a respective imaging device;

converting the plurality of images into a plurality of electronic image representations;

storing the plurality of electronic image representations; and

obtaining a volume image by storing successive pluralities of electronic representations.

16. The method of claim 15, wherein the plurality of imaging devices includes a plurality of optical conductors, each optical conductor having a first end face positioned at a respective image plane, the step of orienting including positioning the first end faces with respect to the image object plane such that each image plane is substantially parallel to and vertically displaced from the image object plane.

17. The method of claim 15, wherein the plurality of imaging devices includes a plurality of photo electric devices disposed in an elongate array having a longitudinal dimension, the step of orienting including disposing the elongate array of photo electric devices such that an extension of the longitudinal dimension intersects the image object plane at a non-orthogonal angle.

18. An autofocus apparatus for autofocusing a microscope having a focus mechanism, the apparatus comprising:

a plurality of the imaging devices, at least one imaging device of the plurality of imaging devices positioned at each respective image plane of a plurality of image planes, where each image plane is displaced in a third dimension with respect to each other image plane;

an image forming apparatus coupled to the plurality of imaging devices to form a plurality of electronic image representations, each electronic image representation representing an image at a respective two-dimensional image plane of the plurality of image planes;

a storage for forming a volume image by retaining successive pluralities of electronic image representations;

a processor coupled to the storage for generating a focus location signal by applying a measurement function to the electronic image representations; and

means for causing the focus mechanism to adjust the focus of the microscope in response to the focus location signal.

19. The autofocus of claim 18, wherein the plurality of imaging devices includes a plurality of optical conductors, each optical conductor having a first end face positioned at a respective image plane and a second end face coupled to the image forming apparatus.

20. The autofocus apparatus of claim 18, wherein the image forming apparatus includes a plurality of charge-coupled device (CCD) cameras.

21. The autofocus apparatus of claim 20, wherein the plurality of imaging devices includes a plurality of optical conductors, each optical conductor having a first end face positioned at a respective image plane of the plurality of image planes and a second end face coupled to a respective CCD camera.

22. The autofocus apparatus of claim 18, wherein the plurality of imaging devices includes an elongate array of photo electric devices.

23. The autofocus of claim 22, wherein the elongate array of photo electric devices is tilted with respect to an image object plane at which an image object located, each image plane of the plurality of image planes being intersected by at least one photo electric device of the elongate array of photo electric devices.

24. The autofocus apparatus of claim 18, wherein the processor includes:

a plurality of image buffers, each image buffer for storing a plurality of electronic representations of at least a portion of the image each electronic representation representing an image of the portion at a respective image plane of the plurality of image planes;

highpass filter means coupled to an image buffer for providing a plurality of filtered electronic image representations; and

means coupled to the high-pass filter means for combining a plurality of filtered electronic image representations to produce a focus metric.

25. The autofocus apparatus of claim 18, further including an optical filter positioned between the plurality of imaging devices and an image object plane.

26. The autofocus apparatus of claim 25, wherein the processing means includes:

a plurality of image buffers, each image buffer for storing a plurality of filtered electronic image representations of at least a portion of an image; and

means coupled to the plurality of image buffers for combining a plurality of filtered electronic image representations to produce the optical quality metric.

27. The apparatus of claim 26, wherein the optical quality metric is image sharpness.

28. The autofocus apparatus of claim 24, wherein the means for combining includes:

means for obtaining augmented intensity values for the filtered electronic image representations; and

means for indicating a value of the focus metric for each image based upon the augmented intensity values.

29. A method for autofocus for a microscope having a focus mechanism, the method comprising the steps of:

acquiring a plurality of electronic image representations of an image object on an image object plane, wherein each electronic image representation represents an image of the image object at a respective image plane of a plurality of image planes in which each image plane is displaced in a third dimension with respect to every other image plane;

forming a volume image by storing successive pluralities of electronic image representations;

applying stored electronic image representations to a highpass filter to obtain a plurality of filtered electronic image representations;

applying a measurement function to the plurality of filtered electronic image representations to obtain a plurality of image sharpness values;

combining the image sharpness values by relating the image sharpness values to image plane positions;

selecting an image plane position related in the combining step to a high image sharpness value; and

adjusting focus of the microscope based on the selected image plane position.