Radiant energy – Photocells; circuits and apparatus – Photocell controls its own optical systems
Reexamination Certificate
1999-06-23
2003-04-15
Stafila, Michael P. (Department: 2877)
Radiant energy
Photocells; circuits and apparatus
Photocell controls its own optical systems
Reexamination Certificate
active
06548796
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to confocal imaging systems, and more particularly to a confocal macroscope.
BACKGROUND
Confocal microscopes with coherent optical illumination are capable of producing very thin optical sectioning yielding sharp 3-D image volume data sets and an image of a specimen with much better contrast between fine details than is possible with non-confocal imaging systems such as wide-field instruments known to those skilled in the art. Confocal microscopes are employed to produce images of many types of specimens such as biological materials and semiconductor devices.
A schematic diagram of the essential components of a conventional confocal microscope
100
is shown in
FIG. 1. A
light source
110
, such as a laser in the instance of monochromatic illumination, generates light that is deflected off of a beamsplitter
114
, which directs the light toward an objective lens
116
. The objective lens
116
focuses the light at a focal point
118
in a specimen
120
. The focal point
118
is a small illuminated area in a focal plane, also called an object plane
122
, in the specimen
120
. In the instance of fluorescent imaging, if the specimen
120
is stained with fluorescent dye that is illuminated with a wavelength near its excitation maximum, then it will emit fluorescent light of a Stokes-shifted wavelength. Fluorescent molecules at the focal point
118
emit Stokes-shifted light rays toward the objective lens
116
which focuses the emitted rays into a confocal pinhole in a conjugate image plane. The confocal pinhole is also called an image pinhole
130
and is located in a plate
132
placed in the conjugate image plane for the focal point
118
. In the instance of fluorescent imaging the beamsplitter
114
transmits the fluorescent light to the image pinhole
130
, and the fluorescent light passing through the image pinhole
130
is detected by a photodetector
140
such as a photomultiplier tube (PMT). The photodetector
140
generates a signal indicating an intensity of the fluorescent light passing through the image pinhole
130
, and the signal is processed by an appropriate data processing system (not shown). An image of the specimen
120
in the object plane
122
is generated by moving the focal point
118
relative to the specimen
120
such that the focal point
118
traverses the object plane
122
in the specimen
120
in a pattern such as a raster pattern. The data processing system assembles the signal from the photodetector
140
to generate the image. Images of different sectional depths of the specimen
120
may be generated by moving the object plane
122
relative to the specimen
120
.
If the specimen
120
is reflective then the illumination light is reflected back toward the objective lens
116
and the beamsplitter
114
to be focused on the image pinhole
130
and detected by the photodetector
140
. An example of a reflective specimen
120
is an integrated circuit wafer specimen.
Beam-scanning or stage-scanning confocal microscopes differ from wide-field instruments in two major aspects: an illumination spot and an image pinhole. First, in the confocal microscopes rays of light impinging on a specimen from an objective lens are converged along a cone to a single focal point or apex in an object plane in the specimen. This is in contrast to a wide-field instrument where, in each instant, the entire area circumscribed by the field-of-view of the objective lens is illuminated simultaneously. This area includes information from points extending through the entire depth of the specimen, including points above and below the object plane of the objective lens. One advantage of the beam-scanning or stage-scanning confocal microscopes is that all of the light is focused on the focal point in the object plane to produce a much more intense excitation of each scanned point of the specimen, with greater spatial specificity of the area being excited.
A second advantage of the beam-scanning or stage-scanning confocal microscopes is the pinhole in the emission/detection path. Some of the rays of light emanating from the object plane as a result of the illumination light will retrace the path of the impinging path through the objective lens to be collected at a point in the conjugate image plane. The confocal pinhole or image pinhole at the conjugate image plane acts as a spatial-filter to remove out-of-focus rays of light which emanated from points above, below, or to the side of the focal point or apex in the specimen. A single focal point in the specimen is examined at a time. If the focal point of the objective lens is scanned over the specimen at different object planes, then a three-dimensional data set of the specimen may be obtained. The greater intensity of confocal illumination and a segregation of adjacent object planes through which the focal point is scanned allow for the generation of low-distortion images of slices of a thick specimen such as a biological tissue section.
The intensity of illumination in a confocal microscope is enhanced if the excitation light source is a laser such as the laser
110
shown in FIG.
1
. Arc-lamps normally used in wide-field instruments have much less optical power at a given excitation wavelength. Arc-lamps are also not as capable as a laser of providing a narrow excitation wavelength while excluding other wavelengths or colors, as arc-lamps emit wavelengths throughout a very broad spectrum. Lasers produce just a few colors or discrete wavelength lines with negligible energy in other spectral regions.
The confocal microscope has undergone many exciting and ingenious changes since its conception by M. Minsky, described in U.S. Pat. No. 3,013,467, with its defining characteristic being a detector pinhole. Minsky used arc-lamp illumination and a pair of orthogonally oriented, electromechanically oscillated tuning forks to translate a specimen. Advances in confocal designs are disclosed in Sheppard et al. “A Scanning Optical Microscope For The Inspection Of Electrical Devices” Microcircuit Eng., Cambridge, 1980, p.447-454 and in Marsman et al. “Mechanical Scan System for Microscopic Applications”; Rev. Sci. Instrument, 1047-1052, 54(8). These confocal microscopes use resonant galvanometers to oscillate the specimen, incorporate laser illumination and PMT detection, scan in real-time, and are used for observing the functional processes of living cells. They are limited to scanning areas of only about 1 mm on a side. The confocal microscopes described so far are categorized as “stage-scanners”, because they move the specimen on a support stage with respect to a fixed optical beam.
Laser beam scanning confocal microscopes, also called beam scanners, are described in Åslund et al., “PHOIBOS, A Microscope Scanner Designed For Micro-Fluorometric Applications, Using Laser Induced Fluorescence, Proceedings of the Third Scandinavian Conference on Image Analysis, Copenhagen, Denmark (1983). Beam scanners angularly deflect an illumination and detection beam with respect to a central axis of an objective lens, using tilting mirrors or acousto-optic devices. Beam scanners have an advantage in that a specimen is not jostled during scanning, and the specimen position may be adjusted without disturbing the scanner. However, spherical and chromatic aberrations in the objective lens are accentuated as the beam is deflected towards the periphery of the field. The field of view is both delimited and restricted by the diameter of the exit pupil of the objective lens which is typically less than half a millimeter. This produces an image that is bowl-shaped which may extend out of the specimen, and is not a flat-field scan. The introduction of beam scanners was contemporaneously accompanied by the implementation of digital storage.
Beam scanners and early stage scanner designs have drawbacks due to an angular scan, nonlinear velocity, and a curved scan path. These result in images that are irregular in shape, flawed quantitatively, and limited in field-of-view. This is partly at
Elde Robert
Silvermintz Lawrence S.
Regents of the University of Minnesota
Schwegman Lundberg Woessner & Kluth P.A.
Stafila Michael P.
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