 |
Description  |
|
|
BACKGROUND OF THE INVENTION
1. Field of the invention
This invention relates to an optical confocal scanning microscope.
2. Description of the Related Art
U.K. Patent Publication No. 2 184 321A discloses a confocal scanning
optical microscope which is especially for the study of fluorescent or
reflecting specimens. This instrument depends upon the focussing of light
upon a single spot scanned over the specimen, which illuminated spot,
after de-scanning, is imaged on a confocal aperture in front of a
detector.
In the case where an image is to be formed of fluorescence from a specimen,
the wavelength of the light directed on to the specimen is selected in
such a way as to excite fluorescence. The emitted light is separated from
the exciting light by a suitable beam splitter and is passed through
wavelength-selective filters in such a way that the detector responds only
to the light emitted by fluorescence. Instruments based on this design are
commercially available. They contain a provision for subdividing the
emitted light into beams of different wavelength ranges by a suitable beam
splitter and filters. After this division, two dyes can be employed which
emit different colours of fluorescence which can be distinguished at two
detectors. Alternatively, a reflectance image can be obtained at the same
time as a fluorescence image by the use of suitable beam-splitters, in
accordance with accepted optical practice.
The prior art instruments work satisfactorily but all confocal scanning
microscopes which rely on the use of a single scanning spot suffer from
the defect that all the spectral selectivity of the system lies in the
separation of the emitted or reflected beam into fractions of different
wavelength. If there is considerable overlap between the fluorescent
emission spectra of two dyes, they cannot be distinguished. For example,
Bacallao et al comment in the Handbook of Confocal Microscopy, Plenum
Press, 1990, that the commonly-used dyes fluorescein and rhodamine cannot
be separated effectively in a system of this type. In order to achieve
acceptable separation, it is necessary to vary the wavelength of
excitation. This can be done by changing from one type of laser light to
another, of spectrally different properties. First an image is obtained by
operating the system with one type of excitation, and then a second image
is obtained with a different type of exciting beam. This operation is slow
and cumbersome.
Awamura, Ode and Yonezawa have described a microscope in which red, green
and blue laser beams are scanned independently over the specimen, and the
reflected beams are separated by dichroic filters and each executes a
scanning motion over one of three separate linear CCD detector arrays. The
description was published in the Proceedings of SPIE, The International
Society for Optical Engineering (1987) Volume 765 pp 53-60. In principle,
the system of Awamura et al might be used as a fluorescence microscope. It
would then allow more than one type of dye to be excited in rapid
succession during each line scan. However, in the case of two dyes with
identical emission spectra, or a single ratiometric dye where the emission
spectrum was to be monitored in a single waveband, the system of Awamura
et al offers no advantage over that of White (U.K. Patent Application No.
2 184 321A), since neither system is capable of separating the two
emission signals.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a confocal scanning
optical microscope comprising:
an optical scanning system;
means for simultaneously generating two or more input beams of optionally
different spectral composition and of differing orientations such that
after passage through the scanning system a specimen under test is scanned
with two or more distinct and separate elemental areas of illumination;
and
two or more detectors respectively for receiving two or more output beams
after de-scanning by the scanning means, each detector receiving an output
beam substantially restricted to output light derived from one of the
illuminated elemental areas.
The invention allows for two or more microscope channels having different
excitation wavebands but identical emission wavebands, in order to make
possible excitation ratio image measurements according to accepted
practice.
The invention also allows for two or more microscope channels having
identical excitation wavebands but different emission wavebands, in order
to make possible emission ratio image measurements according to accepted
practice.
The present invention is thus applicable to many kinds of scanning optical
microscopes. It provides a means by which two or more spectrally distinct
exciting spots or bars can be scanned together over the specimen during
each sweep of the scanning system. The emission from each spot is passed
individually and separately to a stationary confocal aperture leading to a
detector, there being at least one aperture and detector for each spot.
The emitted beam from each spot, due to specimen fluorescence or
reflection, may be filtered spectrally or subdivided between detectors in
accordance with established practice, or may be passed unselectively to
the detectors. It is thus possible to obtain, within a single scanning
cycle, two or more complete images, each of which may differ in both
excitation and emission waveband from the other images.
The invention may be considered as "a multiplexed optical system" because
it involves two or more sets of independent but near-parallel beam paths
passing through the same scanning system and objective lens, the optical
paths being multiplexed in the literal sense of being folded together.
BRIEF DESCRIPTION OF THE DRAWINGS
Further features and advantages of the invention will be apparent from the
following description of embodiments, making reference to the accompanying
drawings, in which
FIG. 1 is a schematic diagram of a confocal scanning microscope
incorporating the multiplexed optical system of the present invention;
FIG. 2 is a schematic diagram showing an alternative and preferred optical
arrangement for the upper part of FIG. 1; and
FIG. 3 is a schematic diagram showing an optical means by which several
beams of different spectral properties may be obtained from a single (e.g.
a multiline) laser, for use in the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring to the drawings, the present invention provides an optical
assembly which allows a number of independent optical channels to be used
simultaneously for excitation in a laser confocal scanning microscope with
an extended emission beam path, but is not restricted in application to
this kind of microscope. The invention can be applied to confocal
microscopes in which a bar or slit of light is scanned over the specimen
as well as to those in which a single spot is scanned.
In FIG. 1, to simplify the diagram, only two independent light paths are
shown, but there is no restriction on number in practice.
Light from two lasers, L1 and L2, with different spectral qualities, is
directed on to a beam splitter BS1. The two beams are at a slight angle to
each other, which angle is exaggerated in the diagram for the sake of
clarity. The two beams are reflected into a scanning system as shown,
which produces an angular scan of both beams simultaneously. The angular
separation of the beams is maintained throughout the scan, and results,
after passage through suitable microscope optics, typically an eyepiece E
and an objective O, in the formation of two distinct moving spots of light
S1 and S2 on the specimen.
Light is emitted from the specimen at S1 because of reflection or
fluorescence and a portion of this emitted light passes back through the
optical system, is descanned, i.e. reconverted into a stationary beam by
the scanning system, passes through the beam splitter BS1 and falls on a
confocal aperture Al leading to a detector D1. Light from S2 passes
through the optical system along a similar but distinct path and falls
upon detector D2. The preferred angular separation is the smallest
possible consistent with a satisfactory separation of the optical
channels. To allow image registration, the small difference in time
between the scanning of a given point in the specimen by the spots
corresponding to S1 and S2 may be compensated by suitable conventional
electronic means, for example by image processing software. It is not
essential to the functioning of the system that the two or more spots
should lie upon the same scan line.
In the preferred embodiment of FIG. 2, the scanning device and microscope
are not shown in the figure, but should be taken to be the same as in FIG.
1. Beams from lasers L1 and L2 again pass at a small angle on to the beam
splitter BS1. The returning beams, after passing through beam splitter
BS1, pass to a second beam splitter BS2, which has dichromatic properties,
so that most of the light in one of the beams, B2, passes through to
confocal aperture A1 and thus to detector D1, while the other beam B1 is
preferentially reflected to A2 and D2. This modification is preferred as
it allows the use of the second beam splitter BS2 to achieve a selection
of emission wavelengths, and also may be implemented by only slight
modification of existing instruments. The separation of the emitted beams
by wavelength may be improved by the addition of wavelength-selective
filters F1 and F2.
The aiming of the emission beams, each on to the appropriate aperture A1 or
A2, may conveniently be achieved by the use of mirrors (not shown)
interposed between BS2 and the detectors D1 or D2.
Additional mirrors and dichromatic reflectors may provide convenient means
of achieving an appropriate angle between the input beams L1 and L2. For
example, FIG. 3 illustrates one of many possible means by which light from
a single multiline laser L may be separated into beams of different
spectral composition and angle.
In this case, a parallel-sided block B of glass or other transparent
material is used to produce a small lateral separation of the beams
according to wavelength. The angle between the beams is then adjusted by
passing them through a prism P, where they undergo different angular
deviations because of the dispersing power of the prism. By appropriate
orientation of the prism, parallel beams, each corresponding to a single
wavelength, are generated, which converge towards the beam splitter BS1.
The angle of convergence is determined by the angle of the prism and its
refractive index and dispersive power. In the diagram, the solid line S
indicates a beam at a shorter wavelength, which is more strongly refracted
than the beam, shown by the dashed lines D, corresponding to light of a
longer wavelength.
Various modifications of the above-described and illustrated arrangements
are possible within the scope of the invention hereinbefore defined.
* * * * *
|
|
 |
|
 |
Description |
|
