Note: Descriptions are shown in the official language in which they were submitted.
10~850~
_ Background of the Invention
This invention relates to radiation measuring
apparatus and more particularly to fluorescence spectro-
photometers of the type in which a sample is irradiated with
light of one wavelength and its emission spectrum is observed
through the use of a monochromator and a detection system. As
used herein and in the appended claims, the term "light"
includes not only visible light but also radiation having
wavelengths longer and shorter than the visible spectrum.
In the measurement of fluorescence and excitation
spectra it is customary to illuminate a sample with monochro-
matic light from an intense source and to observe the light
emitted by the sample with a monochromator and a photoelectric
detection system. Either the excitation or the emission
wavelength may be scanned to record the intensity of the spectrum
as a function of excitation or emission wavelength.
Heretofore, radiation measuring apparatus of the fore-
going type exhibited certain disadvantages. One of the more
significant problems was the comparatively low intensity of
the output signal particularly in measuring the spectra of
dilute materials. In the usual form of apparatus a magnified
image of the light source was focused on the entrance slit of
the excitation monochromator, and a reduced image of the exit
slit was focused on the sample by means of a first optical
system. Fluorescence from the sample was collected by a
second optical system and was focused on the entrance slit of
an emission monochromator such that the signal at the exit
slit of this latter monochromator was proportlonal to the
intensity of the light at the selected wavelength. Attempts
to increase the intensity of the signal commonly included a
reduction in height of the image of the excitation monochromator's
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iO68508
exit slit. These attempts were only partially successful,
however, and the measured intensity continued to be insufficient
to obtain readings of the desired accuracy for low intensity - `
samples.
Summary
One general object of this invention, therefore, is
to provide new and improved apparatus for measuring the
intensity of light emitted by a sample with respect to the
intensity of the light exciting the sample.
More specifically, it is an object of this invention
to provide radiation measuring apparatus which is effective -
to produce a high intensity fluorescence signal.
Another object of the invention is to provide a
fluorescence spectrophotometer utilizing comparatively simple
optical components which is economical to manufacture and
reliable in operation.
In a preferred embodiment of the invention, the
apparatus comprises a radiation source and an excitation mono-
chromator for isolating an excitation beam of monochromatic
radiation from the source. The excitation monochromator
includes first and second limiting apertures for the mono-
chromatic radiation which are respectively formed by the
excitation exit slit and the monochromator's dispersing means.
The radiation is received by a first optical system, and is
directed toward the sample being evaluated to cause the sample
to emit fluorescence. A second optical system collects
fluorescence from the sample and focuses a beam of the collected
radiation on the entrance slit of an emission monochromator to
produce a monochromatic emission beam at the monochromator's
exit slit. In a manner similar to that of the excitation
monochromator, the emission monochromator includes third and
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fourth limiting apertures which are formed by the emission
entrance slit and the dispersing means and are imaged adjacent
the sample. The emission beam from the exit slit is received
by a photoelectric detector to provide a signal proportional
to the intensity of the fluorescent light emitted by the
sample at the selected wavelength.
In accordance with one feature of the invention,
the longitudinal axes of the slit images adjacent the sample
lie in a single plane defined by the axial rays of the
excitation and fluorescence beams. In some cases this is
accomplished by an anamorphic mirror and lens arrangement in
each of the optical systems which orients the images at ninety
degree angles with respect to the exit and entrance slits of
the respective excitation and emission monochromators, while
in other embodiments the slits themselves are oriented parallel
to the plane. The arrangement is such that each point along
the entrance slit of the mission monochromator is filled with
light of an intensity corresponding to illumination of the
sample with light from all points along the length of the
excitation monochromator's exit slit, with the result that
a very substantial increase in the intensity of the output
signal is achieved.
In accordance with another feature of several
particularly advantageous embodiments of the invention, an
image of the first limiting aperture is formed adjacent a
first surface of the sample, and an image of the second
limiting aperture is formed adjacent a second surface of the
sample. Similarly, an image of the third limiting aperture is
formed adjacent a third surface of the sample, and an image of
the fourth limiting aperture is formed adjacent a fourth
surface of the sample.
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1{~68S08
The widths of the slits advantageously are of the same order -
of magnitude, and the magnification is chosen to make the
height of each radiation beam passing through the sample about
the same at each of the sample surfaces, to provide an addi- --
tional improvement in the output intensity. For a particular
instrument employing simple lenses the positions of the
various images relative to the sample will of course change
during variations in wavelength, but in these embodiments it
is important that a given image be located adjacent the
sample over at least a substantial portion of the wave-
length range for which the instrument is designed. As
used herein, the phrase "adjacent the sample" refers to the
location of the image either inside or outside the used volume
of the sample and in close proximity therewith over at least a
substantial portion of the wavelength range of the instrument.
In accordance with a further feature of certain
embodiments of the invention, the extreme rays between the
images of the two apertures in the excitation monochromator
illuminate a sample volume in the approximate shape of a
right rectangular prism, and the extreme rays between the
two images of the apertures in the emission monochromator are
illuminated from a sample volume which similarly is in the
shape of a right rectangular prism. The width of the beam
passing through the sample is comparatively uniform and is
maintained as small as practical, with the result that the
intensity of the output signal is further increased.
In accordance with another feature of an advantageous
embodiment of the invention, optical wedge shaped elements are
positioned adjacent the sample in the path of the light beams to
and from the respective monochromators to condense and concen-
trate the excitation beam at the axial intersection of the
respective beams and to pick up more emitted light to provide
an additional improvement in the output intensity.
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The present invention, as well as further objects and
advantages thereof, will be understood more clearly and fully
from the following description of certain preferred embodi-
ments, when read with reference to the accompanying drawings.
Brief Description of the Drawings
Figure 1 is a simplified schematic pian view of a
fluorescence spectrophotometer in accordance with one illus-
trative embodiment of the invention. :~
Figure lA is an enlarged schematic plan view of the
light paths adjacent the sample holder of the spectrophotometer
shown in Figure 1.
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-: . ' ', ' ' . , . : ~ '
1~685013 i`
Figure lB is an enlarged fragmentary isometric
view of the sample holder and optical systems for the spectro- -
photometer of Figure 1.
Figure 2 is a simplified schematic elevational view
of a portion of the spectrophotometer shown in Figure 1, as
seen from the line 2-2 in Figure 1.
Figure 3 is a simplified schematic plan view of a
fluorescence spectrophotometer in accordance with another
illustrative embodiment of the invention.
Figure 4 is a simplified schematic elevational view
of a portion of the spectrophotometer of Figure 3, as seen from
the line 4-4 in Figure 3.
Figure 5 is an enlarged plan view of the sample
holder employed in the spectrophotometer of Figures 3 and 4.
Figure 5 is located on the sheet of drawings containing
Figure 2.
Figure 6 is a simplified schematic plan view of a
fluorescence spectrophotometer in accordance with one illus-
trative embodiment of the invention.
Figure 7 is an elevational view taken along line 2-2
of Figure 6 showing a light chopper used in conjunction with the
spectrophotometer of Figure 6.
Figure 8 is a simplified schematic plan view of a
fluorescence spectrophotometer in accordance with another illus-
trative embodiment of the invention.
Figure 9 is an elevational view taken along line 4-4
of Figure 8 showing a portion of the spectrophotometer illus-
trated in that Figure.
Figure 10 is an elevational view taken along line
5-5 of Figure 8 showing the spectrophotometer portion illus-
trated in Figure 9.
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8SV8
Figure 11 is a horizontal sectional view of a sample
holder useful in connection with the invention.
Figure 12 is a plan view of optical wedges and
associated components useful in connection with the invention
shown in Figure 6. Figure 12 is located on the sheet of
drawings including Figure 7.
Figure 13 is an elevational view taken along line
8-8 of Figure 12. Figure 13 is located on the sheet of
drawings including Figure 7.
Description of Certian Preerred Embodiments
Referring to Figure 1 of the drawings, there is shown
a schematic representation of a fluorescence spectrophotometer
having a xenon arc or other suitable source 10 of visible or
invisible light. Light from the source 10 is collected by an
ellipsoidal mirror 11 and is focused onto the entrance slit 12
of an excitation monochromator 13. The entrance slit 12 is of
rectangular configuration with its longitudinal axis extending
in a direction perpendicular to the plane of the drawing. The
monochromator 13 i5 of a conventional type and includes, in addition
to the entrance slit 12, a collimating mirror lS, a diffraction
grating 16, a telescope mirror 17 and an exit slit 18 which
likewise has its longitudinal axis extending parpendicular to
the plane of the drawing. The light entering the entrance
slit 12 is reflected by the mirror 15 to the grating 16 and
then from the mirror 17 to the exit slit 18. The periphery
of the grating 16 forms a limiting aperture 19, for purposes
that will become more fully apparent hereinafter.
The light emerging from the excitation slit 18
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is in the form of a monochromatic excitation beam. The
monochromatic beam is received by a first optical system
which comprises superimposed flat and spherical mirrors 20 ;~
and 21, a cylindrical lens 22 and a spherical lens 23. The
mirrors 20 and 21 are oriented at forty-five degree angles
with respect to the principal ray of the incident beam to
direct the light upwardly and then horizontally toward the
lenses 22 and 23. The mirrors 20 and 21 reflect the excitation
beam at right angles to its original direction.
The convex spherical lens 23 focuses the excitation
beam on a sample holder or cell indicated generally at 25. ;~
The sample cell 25 is of square configuration and includes
opposed pairs of flat surfaces 26 and 27, and 28 and 29. As
best shown in Figure lA, the lens 23 forms a real horizontal
image 30 of the aperture defined by the excitation exit slit 18.
The image 30 is located closely adjacent the surface 26 of the
sample cell 25.
In addition to the excitation exit slit image 30,
the first optical system is effective to form an image 31 of
the grating aperture 19. The image 31 is located in close
proximity with the surface 27 of the sample cell 25, that is,
the surface opposite that adjacent the image 30. The longi-
tudinal axis of each of the images 30 and 31 lies in a single
plane parallel to the plane of the drawing.
It will be noted that the flat angular mirror 20 and -
the spherical angular mirror 21 serve to orient the images 30
and 31 at right angles to the direction of the exit slit 18.
Thus, the mirrors 20 and 21 rotate the images through a ninety
degree angle such that their longitudinal dimensions are parallel
to the plane of the drawing. The mirrors 20 and 21, together
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~068508
with the lenses 22 and 23, form the excitation optical system
for the instrument and direct the excitation beam from the
exit slit 18 to the sample 25. The optical system is anamorphic,
and its magnification is such that the iength and width of
the exit slit image 30 are approximately equal to the length
and width of the aperture image 31, respectively. With this
arrangement, the extreme rays between the images 30 and 31
illuminate a sample volume in the approximate shape of a right
rectangular prism. The width of the beam passing through the
sample is comparatively uniform and is maintained as small as
practical, with the result that the intensity of the beam is ;-
substantially increased. `
To provide a further increase in the intensity of -
the light beam passing through the sample 25, a spherical
mirror 32 is located a short distance behind the sample adjacent
the sample surface 27 opposite that facing the excitation -~
monochromator 13. The mirror 32 directs the excitation beam
back through the sample for a second pass.
The excitation beam passing through the sample 25
excites the sample and causes it to emit fluorescence of a
wavelength different from that of the exciting light. This
fluorescence is emitted in all directions. A portion of the
emitted fluorescence is collected by a spherical lens 33 and
is directed thereby through a cylindrical lens 34 to a spherical
off-axis mirror 35 and a flat off-axis mirror 36. The lenses 33
and 34 and the mirrors 35 and 36 form an anamorphic emission
optical system which is identical with the excitation optical
system comprising the mirrors 20 and 21 and the lenses 22 and
23. In a manner similar to that of the mirrors 20 and 21, the
mirrors 35 and 36 are oriented at forty-five degree angles
with respect to the principal rays of the emission beam collected
.. . ..
~1068508
from the sample 25. To further increase the intensity of the
emission beam, a spherical mirror 37 is positioned a short
distance behind the sample 25 in facing relationship with the
sample surface 29. The mirror 37 collects additional fluor-
escence from the sample and directs it through the emission
optical system.
The fluorescent emission beam from the emission
optical system is directed by the spherical mirror 36 to the
entrance slit 39 of an emission monochromator 40. This entrance
slit is of rectangular configuration and has its longitudinal
axis extending in a direction perpendicular to the plane of the
drawing. The monochromator 40 is similar to the excitation
monochromator 13 and, in addition to the entrance slit 39,
includes a collimating mirror 42, a diffraction grating 43, a
telescope mirror 44 and an exit slit 45 parallel to the entrance
slit. The fluorescence enters the entrance slit 39, is re-
flected by the collimator 42 to the grating 43 and is then
focused by the telescope 44 on the exit slit 45. The periphery
of the grating 43 defines a limiting aperture 46.
The light emerging from the exit slit 45 comprises
a selected, highly monochromatic portion of the luminescent
emission from the sample 25. The emerging light is received
by a photoelectric detector 50 which is of conventional con-
struction and preferably is of a type which exhibits high
sensitivity at the particular wavelengths of interest. The
detector 50 produces an output signal proportional to the
intensity of the light from the exit slit 45.
The spherical lens 33 in the optica:L system for the
emission monochromator 40 forms an optical image 52 of the
aperture defined by the emission entrance slit 39. This image
is located in close juxtaposition with the surface 28 of the
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sample cell 25. Similarly, an optical image 53 of the grating
aperture 46 is formed adjacent the opposite surface 29 of the
sample cell. By reason of the off-axis angular orientation ~ -
of the mirrors 35 and 36, the longitudinal axes of the images 52
and 53 lie in a single plane parallel to the plane of the
drawing and at right angles to the longitudinal axis of the
entrance slit 39. The extreme rays between the images 52 and `
53 outline a sample volume in the approximate shape of a right
rectangular prism, and the width of the beam passing through
the sample is comparatively uniform and is as small as practical. `
The principal rays of the beam from the excitation
monochromator 13 and the beam approaching the emission mono-
chromator 40 intersect at the sample cell 25. The longitudinal
axis of each of the anamorphic aperture images 30, 31, 52 and
53 lies in a plane defined by these principal rays. The exit
slit 18 for the excitation monochromator 13 and the entrance
slit 39 for the emission monochromator 40, on the other hand,
extend in directions perpendicular to the plane defined by
the principal rays. The image 30 of the exit slit 18 is parallel
to the path of the emission beam, and the image 52 of the
entrance slit 39 is parallel to the path of the excitation
beam. The arrangement is such that each point along the
entrance slit 39 is filled with light of an intensity correspond-
ing to the irradiation of the sample with light from the entire
length of the exit slit 18.
The resulting increase in the amount of fluorescent
light collected by the entrance slit 39 in theory may be as
large as the length to width ratio of the image 30 of the
exit slit 18. In terms of the properties of the monochromators,
and with slit and grating images of equal length and equal
width, the ratio is equivalent to the square root of the ratio
of the length of the exit slit multiplied by the angular slit
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iO68508
aperture in a plane including the longitudinal axis of the
slit divided by the width of the slit multiplied by the
angular aperture at the slit in the transverse plane. Because
of varying slit widths and aberrations the predicted increase,
while still substantial, may not be realized particularly for
comparatively large length to width ratios. In cases in which
the actual height of the beam is approximately the same
adjacent the opposite surfaces of the sample, however, the
actual increase closely approaches the theoretical value, and
signal increases may be achieved which are approximately five
to ten times that realized by conventional fluorescence
instrumentation.
In the excitation and emission optical systems the
spherical mirrors introduce a degree of astigmatism in the
slit and grating images. This astigmatism is corrected by the
cylindrical lenses in the systems. The systems have anamorphic
properties that distort the slit and grating images in such a
way that they both have the same length to width ratio.
The mirrors 32 and 37 serve to direct the respective
excitation and emission beams back through the sample 25 for
a second pass. The mirrors 32 and 37 are spherically concave
with centers of curvatures at the center of the sample. With
this arrangement each of the mirrors forms an image of the
facing surface of the sample adjacent the opposite surface
and also forms an image of the opposite surface adjacent the
facing surface. The increase in intensity as a result of these
mirrors is almost four times the intensity of instruments in
which the mirrors are omitted.
The embodiment illustrated in Figures 1 and 2 employs
the respective pairs of angular mirrors 20 and 21 and 35 and
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1068508
36 to orient each of the slit images 30 and 52 in a direction
parallel to the direction of travel of the light of the other
beam. This same result may be achieved through the use of
various other optical systems which eliminate the need for ~ -
angularly disposed mirrors. In the embodiment shown in
Figures 3 and 4, for example, the slits themselves are located -
such that they extend in directions parallel to the direction
of the opposite beam. The instrument of these latter figures
includes a xenon arc light source 60 and an ellipsoidal
mirror 61 which focuses the light onto the entrance slit 62
of an excitation monochromator 63. Contrary to the embodiment
illustrated in Figures 1 and 2, the entrance slit 62 has a
longitudinal axis which lies in the plane of the drawing. A
selected, monochromatic portion of the light from the entrance
slit 62 is reflected by a concave diffraction grating 65 onto
an exit slit 70 which likewise has a longitudinal axis lying
in the plane of the drawing. As in the case of the pxeviously -
described embodiment, the periphery of the grating 65 forms
a limiting aperture 71 for the monochromatic light.
The monochromatic excitation beam emerging from the
exit slit 70 is received by a first optical system which
includes a torroidal lens 72 and a beam splitter 74. The beam
splitter 74 illustratively is in the form of a flat quartz
plate. A known fraction of this light passes through the
splitter 34 and is directed by a concave spherical mirror 75
to a convex spherical lens 76.
The lens 76 focuses the excitation beam from the
mirror 75 on a sample cell 78. The configuration of the cell 78
is similar to that of the cell 25 (Figure 1) described heretofore
and includes pairs of opposed surfaces 80 and 81 and 82 and 83.
The lens is effective to form a real horizontal image of the
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1068508
aperture defined by the excitation exit slit 18, and this -
image is located between the lens and the sample surface 80.
Similarly, a real horizontal image of the grating aperture 71 ~`
is formed adjacent the opposite sample surface 81.
As best shown in Figure 5, the sample cell 78 is
supported adjacent the periphery of a rotatable table 85. ~ -
The table 85 is of circular configuration and includes three
additional sample cells 88, 89 and 90 which may contain
different fluorescent materials and likewise are provided with
the opposed pairs of surfaces 80 and 81 and 82 and 83. The
various sample cells are spaced at ninety degree intervals
on the table 85 such that the sample being evaluated may be
readily changed merely by pivoting the table through a corres-
ponding angle.
A pair of mirrors 95 and 96 is located adjacent
each of the sample cells 78, 88, 89 and 90 in spaced juxta-
position with the surfaces 81 and 83, respectively. The
mirrors 95 and 96 are optically transparent except for
spherically concave reflective surfaces 99 and 100 on their
rear faces. Contrary to the sample mirrors in the embodiment
of Figures 1 and 2, these surfaces are positioned at the
approximate locations of the corresponding grating images with
their centers of curvatures at the approximate locations of
the associated slit images. The slit images are reimaged
back on themselves to further increase the intensity of the
output signal.
Fluorescence from the sample 78 is collected by
a convex spherical lens 105 (Figure 3) in the emission optical
system for the instrument. The fluorescent emission beam
then passes through a lens 107 and is focused by a lens 108
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106t3508
on the entrance slit 109 of an emission monochromator 110.
The longitudinal axis of the entrance slit 109 lies in the
plane of the drawing and is in coplanar relationship with
that of the excitation exit slit 70.
The emission beam entering the exit slit 109 is
received by a concave diffraction grating 112 having a grating
aperture 113 and is directed to an exit slit 114. The longi-
tudinal axis of this latter slit is coplanar with that of the
remaining slits. The fluorescence emerging from the exit
slit 114 is received by a reflecting prism 115 and is directed
thereby to a photoelectric detector 116 to provide an output
signal proportional to the intensity of the light from the
exit slit.
The emission optical system between the sample 78
and the entrance slit 109 is optically the same as the
excitation optical system between the exit slit 70 and the
sample except for the use of the cylindrical lens 107 in place
of the spherical mirror 75. The emission optical system forms
images of the exit slit 109 and the grating aperture 113 in
respective juxtaposition with the surfaces 82 and 83 of the
sample.
The longitudinal axes of the excitation exit slit 70
and the emission entrance slit 109 lie in a single plane
defined by the principal rays of the beam from the excitation
monochromator 63 and the beam approaching the emission mono-
chromator 110. The images of the slits 70 and 109, together
with the images of the grating apertures 71 and 113, similarly
have longitudinal axes which lie in this plane. As in the
previously described embodiment, each point on the emission
entrance slit 109 is filled with light of an intensity
corresponding to the irradiation of the sample with light from
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1068508
the entire length of the excitation exit slit 70. The resulting
increase in intensity is further e ~ nced through the use of
the mirrors 95 and 96 adjacent the sample cell in the manner
described heretofore.
As has been explained, the beam splitter 74 serves
to pass a known fraction of the light from the excitation mono-
chromator 63 to the mirror 75, the lens 76 and the sample 78.
The remaining fraction is reflected by the splitter 75 through
successive lenses 122 and 123 to the reflection prism 115 and
then to the photoelectric cell 116. The remaining fraction
is used as a reference beam and is periodically interrupted
by a continuously rotating chopper 120 between the lens 123
and the photocell 116. The chopper 120 is oriented between
the lenses 107 and 108 in position to also periodically
interrupt the fluorescent emission beam. As will be understood,
the chopper is provided with suitable cut-outs (not visible in
the drawings) to simultaneously admit fluorescence to the
photocell and block the reference beam and to thereafter block
the fluorescent beam and pass the reference beam to the photo-
cell.
The photoelectric cell 116 is thus alternatelyilluminated by light from the luminescent sample 78 and by
reference light from the excitation monochromator 63. The
light detected by the photocell is alternately representative
of the unknown luminescent from the sample and the intensity
of the reference beam.
Through the use of conventional electrical circuitry,
the output signals from the photocell may be translated into a
net output signal corresponding to the ratio of the net sample
signal to the net reference signal.
1068S08
i~eferring to Figure 6, there is shown a schematic
representation of a fluorescence spectrophotometer having a
xenon arc or other suitable source 210 of visible or invisible
light. Light from the source 210 is collected by a convex
mirror 211 and is focused onto the adjustable entrance slit 212
of an excitation monochromator 213. The aperture defined by
the entrance slit is of rectangular configuration with its
longitudinal axis extending parallel to the plane of the
drawing. The monochromator 213 is of a conventional type and
includes, in addition to the entrance slit 212, a concave
diffraction grating 216 and an adjustable exit slit 218 which `~
likewise defines an aperture having its longitudinal axis
extending parallel to the plane of the drawing. The light
entering the entrance slit 212 is reflected by the grating 216
to the exit slit 218. The periphery of the grating 216 forms
a first limiting aperture 219, for purposes that will become
more fully apparent hereinafter.
The light emerging from the excitation exit slit 218
is in the form of a monochromatic excitation beam. This mono-
~hromatic beam is received by a first optical system 215 which
comprises a filter 220 and two concave parabolic mirrors 221
and 222. The mirrors 221 and 222 are preferably oriented at
forty-five degree angles with respect to the principal ray of
the incident beam to direct the light toward a sample holder
or cell 225. The sample cell 225 is of square configuration
and includes opposed pairs of flat surfaces 226 and 227, and
228 and 229. The optical system 215 forms a real horizontal
image of the aperture defined by the excitation exit slit 218
closely adjacent the surface 226 of the sample cell 225.
In addition to the excitation exit slit image the
first optical system is effective to form an image of the
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1068508
grating aperture 219. This latter image is located in close
proximity with the surface 227 of the sample cell 225, that is, -
the surface opposite the surface 226 and the exit slit image.
The longitudinal axis of each of the images lies in a single -
plane parallel to the plane of the drawing.
The filter 220 removes light of undesired wavelengths
from the excitation beam leaving the slit 218 and transmits
the rest of the excitation beam to the first concave mirror 221
which is set at an angle of about 45 to the optical axis of
the excitation beam. The beam is reflected to the mirror 222
which is positioned at right angles to mirror 221. Mirrors 221
and 222 form the excitation optical system 215 for the
instrument and direct the excitation beam from the exit
slit 218 to sample holder 225.
The excitation beam passing through sample 225
excites the sample and causes it to emit fluorescence of a
wavelength different from that of the exciting light. This
fluorescence is emitted in all directions. A portion of the
emitted fluorescence is collected by a concave mirror 233 and
is directed thereby to a second concave mirror 234 and then
to a filter 235. The mirrors 233 and 234 form an emission
optical system 31 which is identical with the excitation
optical system 215. In a manner similar to that of the
mirrors 221 and 222, the mirrors 233 and 234 are oriented at
forty-five degree angles with respect to the principal rays
of the emission beam collected from the sample 225. These
mirrors have the same differential focal properties in the
horizontal and vertical directions and form distorted images
of the illuminated part of the sample at the entrance slit 239
and grating 246 of an emission monochromator 240.
1~685~)8
The entrance slit 239 is of rectangular configuration
and has its longitudinal axis extending in a direction parallel
to the plane of the drawing. The monochromator 240 is similar
to the excitation monochromator 213 and, in addition to the
entrance slit 239, includes a concave diffraction grating 243
and an exit slit 245 parallel to the entrance slit. The
fluorescence enters the entrance slit 239, and is reflected
by grating 243 toward exit slit 245. The periphery of the
grating 243 defines a limiting aperture 246.
The light emerging from exit slit 245 comprises a
selected, highly monochromatic portion of the luminescent
emission from sample 225. The emerging light is received by
a concave mirror 248 which focuses the light beam on a photo-
electric detector 250 which is of conventional construction
and preferably is of a type which exhibits high sensitivity
at the particular wavelengths of interest. The detector 250
produces an output signal proportional to the intensity of
the light from the exit slit 245.
The mirrors 233, 234 in the optical system for the
emission monochromator 240 form an optical image of the aperture
defined by the emission entrance slit 239. This image is
located in close juxtaposition with the surface 228 of the
sample cell 225. Similarly, a reduced optical image of the
grating aperture 246 is formed adjacent the opposite surface 229
of the sample cell. The extreme rays between the images
outline a sample volume in the approximate shape of a right ~-
rectangular prism, and the width of the beam passing through
the sample is comparatively uniform and is as small as
practical.
The principal rays of the beam from the excitation
monochromator 213 and the beam approaching the emission
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1068S08
monochromator 240 intersect at the sample cell 225. The
longitudinal axis of each of the aperture images lies in a
plane defined by these principal rays. The image of the exit
slit 218 is parallel to the path of the emission beam, and
the image of the entrance slit 239 is parallel to the path
of the excitation beam. The arrangement is such that each
point along the entrance slit 239 is filled with light of an
intensity corresponding to the irradiation of the sample with
light from the entire length of the exit slit 218.
In the excitation and emission optical systems the
use of mirrors rather than lenses for focusing reduces the
amount of chromatic aberration in the system as compared to
an optical system relying primarily on lenses for focusing.
Preferably the mirrors of the optical systems have anamorphic
properties that distort the slit and grating images in such a
way that both images have about the same length to width ratio.
The aperture image produced at the sample cell is a reduced
and distorted image of the grating aperture.
The spectrophotometer of Figure 6 also includes
a beam splitter 260 which receives the monochromator excitation
beam reflected by mirror 221. The beam splitter illustratively
is in the form of a flat quartz plate or a partially reflecting
mirror. A known fraction of the received light is reflected
by the beam splitter and is directed through a plano-convex
lens 262 to a hollow prism 264 containing rhodamine B solution
or other so-called quantum detecting liquid that absorbs light
of all wavelengths incident on it and remits a fraction of
quanta in this light at a fixed wavelength. A double convex
lens 266 focuses the emitted light on the photoelectric
cell 250. This fraction is used as a reference beam and is
periodically interrupted by a continuously rotating chopper 268
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~068508
between the splitter 260 and the lens 262. The chopper is
oriented, as seen in Figure 6, to also periodically interrupt -
the monochromatic excitation beam between the splitter 260
and the mirror 222. The chopper is provided with an arcuate
cutout 269 (Figure 7) which simultaneously allows the mono~
chromatic beam to pass to the sample and blocks the reference
beam to the photocell and to thereafter block the monochromatic
beam and pass the reference beam to the photocell.
The photocell 250 is thus alternately illuminated
by monochromatic light from the luminescent sample in the
cell 225 and by reference light from the quantum counter 264.
The light detected by the photocell is alternately represen-
tative of the unknown luminescent intensity from the sample
and the intensity of the reference beam. By using conventional
electrical circuitry the output signals from the photocell
may be translated into a net output signal corresponding to
the ratio of the net sample signal to the net reference signal.
The light intensity to which the sample in the sample
cell 225 is subjected can be further increased in the
embodiment of Figure 6 by the use of two optical elements 270
and 271 (Figure 12). The elements 270 and 271 are shaped like
wedges taken from a sphere, having flat or beveled inner ends
272 and 273, respectively, and spherical outer surfaces 274
and 275 respectively. The center of curvature of the spherical
surfaces 274 and 275 is located near the axial center of their
bevels. The wedges are positioned adjacent the sample
holder 225 near the faces 226 and 228, respectively, in order
to be in the optical path of the monochromatic excitation
beam and the fluorescent emission beam produced by the sample.
In one embodiment of the invention, the apparatus
uses a wedge whose overall length from the maximum radius
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10~8508
thereof to its inner beveled surface is thirteen millimeters,
with the inner beveled surface spaced two millimeters from
the center of the sample cell 225, so that the overall distance
from the maximum radius to the center of the sample is fifteen
millimeters. These wedges have flat converging side surfaces
276 and 278 as seen in Figure 12, and serve to concentrate
more of the excitation light on a small sample at the axial
intersection of the optical axes of the light beams passing
through the wedges and the sample, a~d to pick up more emitted
light from the sample. Among their other advantages, the
wedges are substantially cheaper and easier to manufacture
than the two-dimensionally tapered systems used in cone or
pyramid optics, for example.
In use, an image of the exit slit 218 of the mono-
chromator 213 is projected into the wedge 270 to form an
illuminated band, illustratively six millimeters long, and
represented by the double headed arrow I in Figure 12. The
light rays which otherwise would go to the ends of the image
are intercepted by the polished wedge faces 276 and 278 and
are reflected through the bevel, as is the case in cone
optical systems. With the geometry shown and specified above,
the wedge will illuminate a length of two millimeters at the
bevel instead of the original six millimeters of the slit
image. The resultant illumination band expands to about three
millimeters at the axial intersection of the axes of the wedges
270 and 271. Thus, a three millimeter target at this inter-
section receives all of the light instead of only the half
of the light that it would have received if the wedge were
absent. The light distribution on the target is such that
the central two millimeter portion receives more than two
thirds of the light and possibly as much as three quarters.
10~8508
For example, in a sample which is two millimeters
long in the plane of the drawing (e.g., a rod two millimeters
in diameter standing perpendicular to the plane of the paper),
one third of the excitation light is intercepted without the
use of the wedge 270, while as much as three quarters of the
available light is intercepted when the wedge is used. This
represents a light increase of 2.25 times that which is
available without the wedge. Similarly, the wedge 271 picks
up 2.25 times more light from the rod sample than would be
collected without it. For a small sample the improvement in -
light intensity is cumulative for the excitation and emission
beams and amounts to almost a five times signal increase
projected to the photocell.
The spherical surfaces 274 and 275 on the wedges
270 and 271 contribute still another increase in signal by
reducing the original slit image to a smaller image than the
six millimeters assumed above. With the slit image near the
center of curvature, the reduction factor equals the index
of refraction, or approximately 1.5 if a silica material is
used to form the wedge. In the plane of the drawing, this
makes a further intensity increase that is again cumulative.
In the plane perpendicular to the drawing the increase is
not cumulative because it is assumed that the sample rod is
taller than the part of it that is to be illuminated. Thus,
the effect of the surfaces is to increase the signal by 3.37
times, which is 1.5 to the third power. As this is separate
from the previously described effect of the wedges, the
combined signal increase is 5 times 3.37 or almost 17 times.
Because of reflection losses, with internally reflecting
wedges, the actual signal increase is about 13.5; with
aluminized wedges it is about 12.7.
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\ -
1068508
If the same beam condensing wedges are used with a
set of sample optics in a spectrometer that forms slit images
oriented perpendicular to the plane of the paper, the effect
of the wedges is to increase intensity on the sample, but not
to recover any light that would otherwise be lost, with a
resultant gain of 3. Simultaneously, the effect of the
spherical wedge surfaces is to increase the signal by an
additional factor of 1.5, for a combined increase of about
4.5. Allowing for reflection losses with aluminized wedges
this becomes 3.3 times, which may be compared with 12.7 times
obtained under the conditions discussed above. The 3.8 fold f
difference between these factors arises primarily from the
light that is lost past the edges of the sample when the slit
images are not in the plane of the optic axis.
The embodiment of the invention illustrated in
Figure 8 includes a light source 280 and a source condensing
mirror 281 which are similar to those described previously.
An excitation monochromator 282 receives light from the source
280 through a vertical entrance slit 283, whence it passes to
a dollimating mirror 284 that converts its divergent beam into
an essentially parallel beam for illuminating a grating 285.
Part of the dispersed light from the grating falls on a
telescope mirror 286 which focuses a spectrum at a vertical
exit slit 287. The slit 287 selects a portion of the dispersed
spectrum and passes it as a nearly monochromatic beam to an
associated optical system including mirrors 288, 289, 290,
291 and 292.
This optical system serves several important
functions. First, it forms a reduced image of the exit slit
287 adjacent the facing surface of a sample holder 307 and a
reduced image of the limiting aperture from the grating 285
~068508
adjacent the opposite surface. Second, it is composed solely
of reflective optical elements causing the images to be
completely free from chromatic aberration. Third, it rotates
the beam of light ninety degrees around the direction of
propagation with the result that the length of the slit image
that is formed near the sample lies in the horizontal plane
instead of perpendicular thereto. Fourth, it distorts the
slit and grating images in the sense that the slit image is
shorter and wider than the actual slit and the grating image
longer and narrower than the actual grating. Fifth, it sets ~ `
the amount of reduction and distortion at those values that
make the slit and grating images approximately the same size
and shape. In the same way that was discussed above, the
light paths between the slit and grating images are all
included within a small right rectangular prism.
The mirror 288 receives the monochromatic excitation
beam from the exit slit 287 and directs the beam to the
mirror 289. In an illustrative embodiment the mirror 288 is
located 48 mm from the exit slit 287 and is of toroidal
configuration with radii of 116.5 in the horizontal direction
and 82.0 mm in the vertical direction. It forms a highly
astigmatic virtual image of the slit 287 back inside the
monochromator 282 and a highly astigmatic real image of the
grating between itself and the mirror 289. This latter mirror
is flat and is inclined upward forty-five degrees to direct
the reflected beam vertically upward.
From the mirror 289 the excitation beam proceeds to
a cylindrical mirror 290 and then to a flat mirror 291. The
mirror 290 illustratively is located 26 mm above the mirror 289
and again is inclined at forty-five degrees to the incident
ray, but in a plane ninety degrees away from the plane of the
mirror 289. The mirror 291 also is inclined at forty-five
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10~8S08
degrees to the incident ray but in still a third plane. As
best shown in Figures 9 and 10, one action of this group of
mirrors is first to reflect the light upward from the mirror
289, then horizontally from the mirror 290, and then backward
from the mirror 291 in the direction from whence it came but
offset from the origir.al line 26 mm upwardly and 20.7 mm
horizontally. In these reflections the vertical slit and
grating images are rotated to form horizontal images.
The mirror 288 (Figure 8) has concave radii in both
planes, such that when used at a forty-five degree angle of
incidence it has a shorter focal length, or stronger positive
focusing power, in the plane of the paper than in the plane
perpendicular to the paper. Thus, the virtual exit slit image
that it forms in the vertical plane is less magnified than and
nearer to the exit slit 287 than the virtual slit image in
the horizontal plane. Conversely, because the grating image
i5 real, the grating image in the vertical plane is more
magnified than and further from the mirror 288 than the grating
image in the horizontal plane.
The cylindrical mirror 290 illustratively has a convex
radius of 285 mm in the plane of incidence: i.e., in a
direction perpendicular to the length of the exit slit image.
The mirror 290 exhibits more negative focusing power in this
direction than in the direction of the slit length. This
amount of negative power at this location in the optical
system serves to correct the astigmatism introduced into both
the slit image and the grating image by the torroidal mirror
288. The distortion introduced into the two images by these
two mirrors, however, is not canceled. To achieve these two
results the mirror 288, which is nearer the slit, must form
virtual slit images and real grating aperture images and must
have more positive focusing power in the direction of the slit
.- : . . . .
1068S08
width than at right angles thereto. The mirror 290, on the
other hand, must have less positive focusing power (or more
negative focusing power) in the direction of the slit width
than in the perpendicular direction. In the plane perpendi-
cular to the slit length, the negative cylindrical power of ~-
the mirror 290 forms reduced virtual images of the slit and
grating images already formed by the mirror 288. Within the
accuracy necessary for the proper functioning of the system,
the image locations coincide with the locations of the -
corresponding images that are formed in the other plane by
the mirror.
The mirror 291 reflects the excitation beam to a
curved mirror 292. This latter mirror has an ellipsoidal
form with major and minor image distances which illustratively
are 132.7 and 57.3 mm, respectively. The mirror 292 is located
51.0 mm from the center of the sample within the cell 307,
and it forms an accurate but distorted grating image just behind
the sample and a less accurate but also distorted exit slit
image just in front of the sample. The mirror 292 reduces
both of these images to approximately the same size.
Located in close juxtaposition with the rear surface
of the sample holder 307 is a concave meniscus mirror 293. The
mirror 293 serves as a retro mirror and has a radius of
curvature suitable to form a second image of the excitation
exit slit adjacent the front surface of the sample holder,
thus passing the excitation light twice through the same
volume of sample.
The excitation beam passing through the sample excites
the sample and causes it to emit fluorescence of a wavelength
different from that of the exciting light, as in the previous
embodiment. This fluorescence is emitted in all directions.
A portion of the emitted fluorescence is collected by a second
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1068508
optical system associated with the remaining opposed surfaces
of the sample cell 107 to form an emission beam. The second
optical system includes mirrors 297, 298, 299 and 300 which
perform the corresponding functions of imagery, rotation,
distortion and reduction in the emission beam that the group
of mirrors 288 through 292 serve in the excitation beam. The
mirrors 297 and 300 are spherically concave and may be identical
to their counterpart mirrors 292 and 288, respectively, with
their distances from the slit and sample and from each other
the same as the corresponding distances in the excitation
beam. In the illustrative embodiment of Figure 8 there is no
counterpart in the emission system for the flat mirror 291
because mechanical convenience does not require it, and indeed
in other embodiments the mirror 291 may not be needed depending
upon the physical location of the various system components.
Also, the negative cylindrical power in the excitation system
mirror 290 appears in the emission system in the mirror 299,
and because the mirror 299 is tilted in a different direction
from the mirror 290 its convex radius is 142.6 mm, exactly
half that of mirror 290. None of these differences signifi-
cantly affects the performance of the optical systems. Identical
stigmatic but distorted slit and grating images are formed
adjacent the opposed pairs of surfaces of the sample holder 307.
A concave retro mirror 320 is located in position to
reflect additional emitted light through the sample to the
mirror 297. Contrary to the retro mirror 293 in the excitation
systeml the mirror 320 is remote from the sample holder 307
and has its center of curvature close to the center of the
sample. The light path still traverses essentially the same
part of the sample twice, but the imagery is inverted between
the two. In each system the effec-t of the second traversal
through the sample is to almost double the intensity of
~068501~3
fluorescent light collected. In the excitation system, the
increase comes from doubling the excitation power density in
the sample; in the emission system the increase comes from
doubling the effective thickness of illuminated sample that
is observed.
An advantage of the concave retro mirrors 293 and
320 over, say, flat mirrors is that the imaging properties
of the concave mirrors preclude the possibility of divergent
light rays that might otherwise strike the walls of the
sample cell 307 on the second pass through the cell. This -
is a particularly important feature in measuring weak samples
whose fluorescence might otherwise be concealed by scattered
light from the walls.
Following the group of mirrors 297 through 300,
the emission beam is directed through the vertical entrance
slit 302 of an emission monochromator 301. The emission
monochromator 301 may be similar to the excitation mono-
chromator 282 and includes a collimator 303 which illuminates
a diffraction grating 304 with an essentially parallel beam
of light. Part of the diffracted beam is focused by a
telescope mirror 305 on and through a vertical exit slit 306.
Monochromatic light isolated thereby reaches a photomultiplier
detection system 355 in a manner similar to that described
heretofore.
In the embodiment of Figure 8 the sample cell 307
is supported adjacent the periphery of a rotatable table 311.
The table 311 is of circular configuration and includes three
additional sample cells 308, 309 and 310 which may contain
different fluorescent materials. The various sample cells
are spaced at ninety degree intervals on the table 311 such
that the sample being evaluated may be readily changed merely
- 28 -
1068508
by pivoting the table through a corresponding angle. Concave
meniscus mirrors 294, 295 and 296, each similar to the mirror
293, are located adjacent the inwardly facing surfaces of
the cells 308, 309 and 310, respectively, for directing the
exc:itation beam back for a second pass during the evaluation
of the corresponding samples.
Another advantageous form of turret and retro mirror
arrangement is illustrated in Figure 11. Four sample cells 356,
357, 358 and 359 are respectively mounted adjacent the four
corners of a square table 360 which is supported for pivotal
movement about a vertical axis 361. Each of the cells 356,
357, 358 and 359 has one corner facing the axis 310, instead
of a flat surface facing the axis as shown in Figure 8.
Behind the adjacent inner surfaces of each cell are concave
reflectors 362 and 363. In the Figure 11 embodiment each
of the reflectors 362 and 363 is in the form of a two-sided
first surface mirror, although in other arrangements the
reflectors may be the same as the mirrors 293, 294, 295 and
296 of Figure 8. The reflectors 362 and 363 serve as retro
mirrors for the excitation and emission optical systems,
respectively, in a manner similar to that described above.
The terms and expressions which have been employed
are used as terms of description and not of limitation, and
there is no intention in the use of such terms and expressions
of excluding any equivalents of the features shown and
described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed.
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~ . , . . ~ . . .. . .
