CA1068508A - Fluorescence spectrophotometer - Google Patents

Abstract

Abstract of the Disclosure A fluorescence spectrophotometer in which the optical system for the excitation monochromator includes an arrangement for forming an image of the exit slit of the monochromator adjacent a first surface of the sample being evaluated and for forming an aperture image adjacent a second surface of the sample. Fluorescence from the sample is directed to an emission monochromator which likewise has an arrangement for forming an image of the emission monochromator's entrance slit adjacent a third surface of the sample and for forming an aperture image adjacent a fourth surface. The optical components are arranged such that the images of the slits lie in a single plane defined by the axial rays of the excitation and fluorescence beams, and in several advantageous arrangements the slit images are oriented at ninety degrees from the slits themselves. In some cases the optical system includes aspherical mirrors arranged to give different magnifications of the images in the horizontal and vertical planes. The intensity of the output signal may be further increased by using wedge shaped optical elements adjacent the sample and by locating mirrors behind the sample holder to direct the light back through the sample for a second pass.

Description

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 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.

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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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.

~ -6-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 -6a-, 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 ~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 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 .

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 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 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 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 . .

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.

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 .

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 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 ~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 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.

\ -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 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 .- : . . . .

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 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 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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Claims (29)

What is claimed is:

1. Apparatus for measuring radiation from a sample, the apparatus comprising, in combination:
a source of radiation;
excitation monochromator means for isolating mono-chromatic radiation from said source, the excitation monochromator means receiving radiation from said source and having an excita-tion exit slit defining a first aperture and means including a second aperture for directing a part of the received radiation through said excitation exit slit in the form of a monochromatic excitation beam;
first means cooperating with the excitation mono-chromator means for directing the excitation beam to said sample and for forming an image of the excitation exit slit;
emission monochromator means for isolating radiation from said sample, the emission monochromator means having an emission entrance slit for receiving an emission beam of radia-tion from said sample;
second means cooperating with the emission monochrom-ator means for directing the emission beam to the emission entrance slit and for forming an image of said emission entrance slit;
said excitation beam and said emission beam inter-secting at said sample; and radiation detecting means for receiving monochromatic radiation from the emission monochromator means;
said first and second beam directing means including means for forming the respective slit images with their longitudinal axes lying in the plane defined by said inter-secting beams.

2. Apparatus as defined in Claim 1, in which the longitudinal axis of the excitation exit slit image extends in a direction parallel to the principal ray of the emission beam, and the longitudinal axis of the emission entrance slit image extends in a direction parallel to the principal ray of the excitation beam.

3. Apparatus as defined in Claim 1, in which each of said images is anamorphic.

4. Apparatus as defined in Claim 1, in which each of said first and second means comprises a pair of concave mirrors angularly related to each other.

5. Apparatus as defined in Claim 4, in which one of said mirrors in each pair is adjacent an associated slit in its associated monochromator and the other mirror is adjacent the sample; said one mirror having a longer focal length in the direction of the slit length than the focal length in the direction perpendicular to the slit length, and said other mirror having a shorter focal length in the direction of the slit image length than the focal length in the direction perpendicular thereto.

6. Apparatus as defined in Claim 5 wherein said mirror adjacent the sample is aspheric.

7. Apparatus as defined in Claim 1, in which each of said images has a length to width ratio smaller than the length to width ratio of the corresponding slit.

8. Apparatus as defined in Claim 7 wherein the exit slit of said excitation monochromator means and the entrance slit of said emission monochromator means are both horizontal.

9. Apparatus as defined in Claim 7 wherein the exit slit of said excitation monochromator and the entrance slit of said emission monochromator are both vertical, said first and second beam directing means including means for rotating said images ninety degrees.

10. Apparatus as defined in Claim 1, in which the image formed by said first means is located in close juxtaposition with a first surface of said sample, and the image formed by said second means is located in close juxta-position with a second surface of said sample.

11. Apparatus as defined in Claim 10, which further comprises, in combination:
reflective means in facing relationship with a third and a fourth surface of said sample in position to direct radiation from said excitation beam and said sample back toward said sample.

12. Apparatus as defined in Claim 10, in which each of said first and second means includes a pair of mirrors oriented at forty-five degree angles with respect to the principal ray of the radiation incident thereto.

13. Apparatus as defined in Claim 12, in which one of the mirrors in each said pair is spherically concave.

14. Apparatus as defined in Claim 1, in which said first means forms a first image adjacent a first surface of said sample and forms a second image adjacent a second surface of said sample, and in which said second means forms a third image adjacent a third surface of said sample and forms a fourth image adjacent a fourth surface of said sample.

15. Apparatus as defined in Claim 14, in which the excitation beam has extreme rays between said first and second images which illuminate a volume of said sample in the approximate shape of a right rectangular prism, and the emission beam has extreme rays between said third and fourth images which are illuminated by radiation from a volume of said sample in the approximate shape of a right rectangular prism.

16. Apparatus as defined in Claim 15, which further comprises a rotary chopper in position to periodically interrupt said emission beam.

17. Apparatus as defined in Claim 15, in which the longitudinal axis of said first and second images extend in directions parallel to the principal ray of the emission beam, and the longitudinal axes of said third and fourth images extend in directions parallel to the principal ray of the excitation beam.

18. Apparatus as defined in Claim 15, which further comprises, in combination:
a rotary support member for pivoting said sample into a position in which said first sample surface is in facing relationship with the excitation beam and said third sample surface is in facing relationship with the emission beam.

19. Apparatus as defined in Claim 15, in which the first optical system and the second optical system are optically equal to one another.

20. Apparatus as defined in Claim 15 wherein the images of said apertures adjacent said sample all have approximately the same length.

21. Apparatus as defined in Claim 1 wherein at least one of said first and second means includes an anamorphic optical element.

22. Apparatus for measuring radiation from a sample, the apparatus comprising, in combination:
a source of radiation;
excitation monochromator means for isolating an excitation beam of monochromatic radiation from said source, the excitation monochromator means having an excitation entrance slit for receiving radiation from said source, an excitation exit slit, and means for directing the excitation beam through said excitation exit slit;
a first optical system cooperating with the excitation monochromator means for directing the excitation beam to said sample and for forming an image of the excitation exit slit;
emission monochromator means for isolating radiation from said sample, the emission monochromator means having an emission entrance slit for receiving an emission beam of radiation from said sample, an emission exit slit, and means for directing monochromatic radiation from the emission beam through said emission slit;
a second optical system cooperating with the emission monochromator means for directing the emission beam to the emission entrance slit and for forming an image of said emission entrance slit;

at least one of said optical systems including a curved reflective surface;

said excitation beam and said emission beam having principal rays which intersect at said sample, and each of said images having a longitudinal axis which lies in the plane defined by said principal rays; and radiation detecting means for receiving the mono-chromatic radiation from the exit slit of the emission monochromator means.

23. Apparatus for measuring radiation from a sample, the apparatus comprising, in combination:
a source of radiation;
excitation monochromator means for isolating an excitation beam of monochromatic radiation from said source, the excitation monochromator means having an excitation entrance slit for receiving radiation from said source, an excitation exit slit, and means for directing the excitation beam through said excitation exit slit;
a first optical system cooperating with the excitation monochromator means for directing the excitation beam to said sample and for forming an image of the excitation exit slit;
emission monochromator means for isolating radiation from said sample, the emission monochromator means having an emission entrance slit for receiving an emission beam of radiation from said sample, an emission exit slit, and means for directing monochromatic radiation from the emission beam through said emission exit slit;
a second optical system cooperating with the emission monochromator means for directing the emission beam to the emission entrance slit and for forming an image of said emission entrance slit;
at least one of said optical systems including means for producing distortion in the corresponding beam;
said excitation beam and said emission beam having principal rays which intersect at said sample, and each of said images having a longitudinal axis which lies in the plane defined by said principal rays; and radiation detecting means for receiving the mono-chromatic radiation from the exit slit of the emission monochromator means.

24. Apparatus as defined in claim 23, in which the distortion producing means is anamorphic.

25. Apparatus as defined in claim 23, in which the distortion producing means comprises a mirror having a curved reflecting surface.

26. Apparatus for measuring radiation from a sample, the apparatus comprising, in combination:
a source of radiation;
excitation monochromator means for isolating an excitation beam of monochromatic radiation from said source, the excitation monochromator means having an excitation entrance slit for receiving radiation from said source, an excitation exit slit defining a first limiting aperture, and means for directing the excitation beam through said excitation exit slit, the excitation monochromator means including means defining a second limiting aperture for the excitation beam;
a first optical system cooperating with the excitation monochromator means for directing the excitation beam to said sample and for forming first and second images of the respective first and second limiting apertures;
emission monochromator means for isolating radiation from said sample, the emission monochromator means having an emission entrance slit defining a third limiting aperture for receiving an emission beam of radiation from said sample, an emission exit slit, and means for directing monochromatic radiation from the emission beam through said emission exit slit, the emission monochromator means including means defining a fourth limiting aperture for the emission beam;
a second optical system cooperating with the emission monochromator means for directing the emission beam to the emission entrance slit and for forming third and fourth images of the respective third and fourth limiting apertures;
at least one of said optical systems including reflective means for producing distortion in the corresponding beam;
said excitation beam and said emission beam having principal rays which intersect at said sample;
radiation detecting means for receiving the monochromatic radiation from the exit slit of the emission monochromator means.

27. Apparatus for measuring radiation from a sample having pairs of opposed surfaces, the apparatus com-prising, in combination:
a source of radiation;
excitation monochromator means for isolating an excitation beam of monochromatic radiation from said source, the excitation monochromator means having an excitation entrance slit for receiving radiation from said source, an excitation exit slit defining a first limiting aperture, and means for directing the excitation beam through said excitation exit slit, the excitation monochromator means including means defining a second limiting aperture for the excitation beam;

means cooperating with the excitation monochromator means for forming a first image of said first limiting aperture adjacent said sample and a second image of said second limiting aperture adjacent said sample;
emission monochromator means for isolating radiation from said sample, the emission monochromator means having an emission entrance slit defining a third limiting aperture for receiving an emission beam of radiation from said sample, an emission exit slit, and means for directing monochromatic radiation from the emission beam through said emission exit slit, the emission monochromator means including means defining a fourth limiting aperture for the emission beam;
means cooperating with the emission monochromator means for forming a third image of said third limiting aperture adjacent a third surface of said sample and a fourth image of said fourth limiting aperture adjacent a fourth surface of said sample;
at least one of said images being located adjacent a surface of said sample over a substantial range of wavelengths of the measured radiation;
said excitation beam and said emission beam having principal rays which intersect at said sample, and each of said first and third images having a longitudinal axis which lies in the plane defined by said principal rays; and radiation detecting means for receiving the mono-chromatic radiation from the exit slit of the emission mono-chromator means.

28. Apparatus for measuring radiation from a sample having a plurality of surfaces, the apparatus comprising, in combination:

a source of radiation;
excitation monochromator means for isolating an excitation beam of monochromatic radiation from said source, the excitation monochromator means having an excitation entrance slit for receiving radiation from said source, an excitation exit slit, and means for directing the excitation beam through said excitation exit slit;
means cooperating with the excitation monochromator means for directing the excitation beam to said sample and for forming an image of the excitation exit slit;
emission monochromator means for isolating radiation from said sample, the emission monochromator means having an emission entrance slit for receiving an emission beam of radiation from said sample, an emission exit slit, and means for directing monochromatic radiation from the emission beam through said emission exit slit;
means cooperating with the emission monochromator means for directing the emission beam to the emission entrance slit and for forming an image of said emission entrance slit;
each of said images being located in close proximity with respective surfaces of said sample over a substantial range of wavelengths of the measured radiation;
said excitation beam and said emission beam having principal rays which intersect at said sample, and each of said images having a longitudinal axis which lies in the plane defined by said principal rays; and radiation detecting means for receiving the monochromatic radiation from the exit slit of the emission monochromator means.

29. Apparatus for measuring radiation from a sample having pairs of opposed surfaces, the apparatus comprising, in combination:
a source of radiation;
excitation monochromator means for isolating an excitation beam of monochromatic radiation from said source, the excitation monochromator means having an excitation entrance slit for receiving radiation from said source, an excitation exit slit defining a first limiting aperture, and means for directing the excitation beam through said excitation exit slit, the excitation monochromator means including means defining a second limiting aperture for the excitation beam;
means cooperating with the excitation monochromator means for forming a first image of said first limiting aperture adjacent said sample and a second image of said second limiting aperture adjacent said sample;
emission monochromator means for isolating radiation from said sample, the emission monochromator means having an emission entrance slit defining a third limiting aperture for receiving an emission beam of radiation from said sample, an emission exit slit, and means for directing monochromatic radiation from the emission beam through said emission exit slit, the emission monochromator means including means defining a fourth limiting aperture for the emission beam;
means cooperating with the emission monochromator means for forming a third image of said third limiting aperture adjacent a third surface of said sample and a fourth image of said fourth limiting aperture adjacent a fourth surface of said sample;
at least one of said images being located in close proximity to a surface of said sample over substantially the entire range of wavelengths of the measured radiation;

said excitation beam and said emission beam having principal rays which intersect at said sample, and each of said images having a longitudinal axis which lies in the plane defined by said principal rays; and radiation detecting means for receiving the mono-chromatic radiation from the exit slit of the emission mono-chromator means.