Radiant energy – Photocells; circuits and apparatus – Optical or pre-photocell system
Reexamination Certificate
2001-04-09
2003-04-29
Pyo, Kevin (Department: 2878)
Radiant energy
Photocells; circuits and apparatus
Optical or pre-photocell system
Reexamination Certificate
active
06555811
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a scanning confocal optical microscope system.
BACKGROUND OF THE INVENTION
The confocal scanning optical microscope is now widely used. In its essentials it consists of a means for focussing a beam of light to a small spot on a specimen and means for collecting the emitted or reflected emission from that spot in order to build up an image by systematic scanning of the spot over a specimen. A defining feature of the confocal instrument is the presence of a beam-limiting aperture in front of the detector, which serves to limit detection of emitted light to that emerging from the immediate vicinity of the focus of illumination. White, in U.S. Pat. No. 5 032 720, taught the use of a variable iris as such an aperture, allowing a compromise to be sought between best optical depth discrimination, which is obtained with the aperture maximally closed, and high signal strength, which is obtained with it open. An analysis of this advantage has been published by Sandison et al, pp 39-51 in Handbook of Biological Confocal Microscopy, IInd Edn., Plenum Press, New York and London, 1995. White also taught the division of the emitted light in a confocal microscope into more than one beam according to wavelength, by means of chromatic reflectors. This principle has proved to have many applications, chiefly in the imaging of a plurality of fluorescent stains simultaneously present in the same specimen.
In a widely-used commercial form of White's instrument, there are two or three such beams, each passing light to a separate variable iris, i.e. there are two or three confocal beam-limiting apertures. The value of having more than one aperture is that the diameter may be set differently in each. This is of value because the above-mentioned compromise may be sought according to the brightness of each individual stain. Also, the theoretical optimum width of the aperture scales with wavelength, so a single diameter can never be precisely optimum for all wavelengths (van der Voort, H. T. M. & Brakenhoff, G. J. (1990), J. Microscopy 158, pp 43-54).
In White's microscope system, the separation of beams is achieved by the use of chromatic reflectors and further colour separation is achieved by means of barrier filters. Since it is accepted to be desirable to be able to distinguish colours, the use of a spectrometer in the emission path of a confocal microscope is an obvious development. Brakenhoff, in a diagram published on page 189 of Confocal Microscopy, edited by T. Wilson, Academic Press 1990, showed how a spectrometer could be used.
FIG. 1
of the accompanying drawings is redrawn from Brakenhoff's figure and serves to clarify the placement of components in a confocal microscope.
In
FIG. 1
, light from a laser
11
is passed through a lens
12
and a first aperture, consisting of a pinhole
13
, from which the light emerges as an expanding beam which is rendered parallel by lens
14
. From lens
14
the light is reflected by chromatic reflector
15
so that it passes into a microscope objective lens
16
and is brought to a focus, normally as a diffraction-limited spot on a specimen at
17
. Some of the light emitted from the specimen passes into lens
16
and the chromatic performance of the reflector
15
is chosen such that the emitted light is transmitted by this reflector
15
rather than reflected. The transmitted beam passes through a barrier filter
18
, which absorbs unwanted laser light, and is focussed by the lens
19
on a confocal aperture at
20
. It is essential for the proper functioning of a confocal microscope that this aperture lies in an optically conjugate position to the focus on the specimen; in other words the specimen is focussed on this aperture. In conventional optical terminology, the confocal aperture is an image plane stop.
FIG. 1
shows how the light which has passed through the confocal aperture at
20
is passed, in Brakenhoff's scheme, through a spectral dispersing means, such as a monochromator
21
, and the outgoing light is finally passed to a unitary detector such as a photomultiplier tube
22
. The photocurrent in the photomultiplier tube
22
is used as a measure of the intensity of the light in the range of wavelengths selected by the monochromator
21
and allows the construction of an image in computer memory if the spot of light is scanned systematically over the specimen.
In order to be able to record images from the spectrometer (photomultiplier tube
22
) at more than one wavelength simultaneously, it is a possible development of the system proposed by Brakenhoff that the spectrometer should be of the multichannel type. This was proposed explicitly by Engelhardt in PCT Application WO 95/07447. This combination of the known art of spectrometry with known apparatus for confocal microscopy works well and has the advantage of being more flexible than the fixed-reflector design taught by White. It is, however, inferior to White's design in that a plurality of confocal apertures, each for a different wavelength range, cannot be used. Since all the detected light is passed through a single confocal aperture the previously mentioned advantages of multiple apertures are lost.
The present invention aims to overcome this difficulty, allowing the use of multiple confocal apertures in conjunction with multichannel spectral detection. It effectively consists of a form of imaging spectrometer, which is simple in construction and easily integrated into a scanning confocal optical microscope system.
The Invention
According to one aspect of the invention, there is provided a scanning confocal microscope system which is confocal in operation, the system including a dispersive optical means which produces from the same restricted region of a specimen a plurality of separated optical images of differing wavelength ranges, and a beam-limiting aperture for each said image, all said apertures being located at foci which are conjugate with each other. All the apertures are located in image planes which are conjugate with the plane of focus in the specimen, and the central points of the apertures are conjugate with the point of illumination in the specimen. The above-mentioned beam-limiting image plane apertures function as confocal apertures.
Preferably, the restricted region or area of the specimen is imaged at a primary image plane and the dispersive optical means, in conjunction with focussing means, produces said images of differing wavelength ranges in secondary image planes conjugate with the primary image plane.
The system preferably includes a plurality of detectors receiving light through the respective beam-limiting apertures.
According to another aspect of the invention, there is provided a scanning confocal optical microscope system comprising a scanning confocal optical microscope which produces a beam of light forming an image of a restricted region of a specimen in a primary image plane, a dispersive optical means which receives the beam from the said primary image plane and produces, in secondary image planes each conjugate with the primary image plane, a plurality of separated secondary optical images of the same region of the specimen, said secondary images respectively comprising light of differing ranges of wavelengths, a beam-limiting aperture in each secondary image plane and a plurality of detectors receiving light through the respective beam-limiting apertures.
Preferably, the beam-limiting area of at least one of the beam-limiting apertures is adjustable or variable. This may be achieved by exchange of a beam-limiting aperture of one size by an aperture of a different size, or by making the beam-imiting area adjustable in size, e.g. in width or diameter. For example, at least one beam-limiting aperture may comprise a variable iris diaphragm, a system of movable jaws, or an adjustable slit.
The dispersive optical means may comprise one or more optical prisms or a diffraction grating or gratings, but an equivalent device or devices may alternatively be employed.
Conveniently, a fo
Medical Research Counsel
Pyo Kevin
Sohn Seung C.
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