Radiant energy – Photocells; circuits and apparatus – Photocell controls its own optical systems
Patent
1997-09-10
1999-08-03
Le, Que T.
Radiant energy
Photocells; circuits and apparatus
Photocell controls its own optical systems
2502081, 345 6, G01J 120
Patent
active
059328720
DESCRIPTION:
BRIEF SUMMARY
MICROFICHE APPENDIX
A Microfiche Appendix containing computer source code is attached. The Microfiche Appendix comprises one (1) sheet of microfiche having 22 frames, including one title frame.
The Microfiche Appendix contains material which is subject to copyright protection. The copyright owner has no objection to the reproduction of such material, as it appears in the files of the Patent and Trademark Office, but otherwise reserves all copyright rights whatsoever.
1. TECHNICAL FIELD
The present invention relates to autofocusing and, more particularly, to a system for microscope autofocusing.
2. BACKGROUND ART
Autofocus is a requirement for any fully automated microscope-based image processing system that must scan areas larger than a single field. Experience has shown that it is not possible to maintain focus simply by determining the best foci at two points on a microscope slide and scanning along the line between them in three dimensional space. This may be due to many causes, including mechanical instability of the microscope and irregularity of glass slide surfaces. For example, thermal expansion could account for several microns of instability in microscopes with lamps acting as unevenly distributed heat sources. Using the coefficient of thermal expansion for aluminum, a 1.0.degree. C. increase causes 0.6 micrometer (.mu.m) of expansion for each 25 millimeters (mm) length between the objective and stage in a microscope. Mechanical instability may also arise from gear slippage and settling between moving components in the stage. Microscope slide surface irregularity is another source of error. Standard optical quality mirror flatness is about 1.5 .mu.m over 25 mm. Given that mirrors are ground glass and microscope slides are float glass, microscope slide surface irregularity could be much greater. According to the definition by others, such as Francon (Frangon M: Progress in Microscopy. Row, Peterson, Evanston, Ill., 1961), the theoretical microscope depth of field for an objective with numerical aperture (NA) 0.75 is 0.74 .mu.m at a wavelength of 500 nm. Best focus can vary through a range of about 25 .mu.m in a horizontal scan of 50 mm across a microscope slide. Whatever the source of instability, autofocus can compensate given that the positional variations have relatively long time constants.
Most autofocus methods fall into two categories: position sensing and image content analysis. Position sensing methods, such as interferometry, require independent calibration of the best focus location and, more importantly, a single well-defined surface from which to reflect light or sound. In light microscopy there are often two reflective surfaces, the coverslip and slide. In addition, tissue specimens can have significant depth and best focus is not necessarily achieved at the surface of the glass. These problems make absolute position sensing methods impractical for use in light microscopy. Image content analysis functions, such as used by the present invention for autofocusing the microscope, on the other hand, depend only on characteristics measured directly from the image. Best focus is found by comparison of these characteristics in a series of images acquired at different vertical positions. This method of autofocus requires no independent reference and is not affected significantly by the second reflective surface. Its most important limitation is speed, which is dependent on the video rate, the vertical repositioning time, function calculation time and search range.
Image content autofocus functions have previously been compared for brightfield microscopy, but apparently not for fluorescence or phase-contrast microscopy. For example, Groen, Young and Ligthart (Groen FCA, Young IT, Ligthart G: A comparison of different focus functions for use in autofocus algorithms. Cytometry 6:81-91, 1985) compared 11 autofocus functions under brightfield using an electron microscope grid and a metaphase spread, and Vollath (Vollath D: Automatic Focusing by Correlative Methods. J Microsc 147:279-288, 1987) te
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Le Que T.
Price Jeffrey H.
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