US 3715585 A
Apparatus for enhancing the sensitivity of fluorescence spectrophotometry is described. Enhanced sensitivity is obtained by a novel sample cell construction in the form of a flat plate providing multiple exposure of the sample to the exciting radiation to increase absorption, and providing concentration of the fluorescent radiation to a small aperture for more efficient collection. The novel cell also provides easy separation of the exciting and fluorescent radiations.
Claims available in
Description (OCR text may contain errors)
United States Patent [191 Harrick 51 Feb. 6, 1973 54] FLUORESCENCE SPECTROPHOTOMETRY USING MULTIPLE REFLECTIONS TO ENHANCE SAMPLE ABSORPTION AND FLUORESCENCE COLLECTION  Inventor: Nicolas J. Ha rrick, Croton Dam Road, Ossining, NY. 10562 221 Filed: June7, 1971 21 Appl.No.: 150,687
 US. Cl ..250/71 R, 250/77  Int. Cl ..G0ln 21/16,
 Field of Search ..250/71, 77, 80, 71 R  References Cited UNITED STATES PATENTS 3,604,927 Hirchfield ..356/74 3,491,366 1/1970 Harrick ..356/98 3,591,287 7/l97l Hannis ..356/51 3,470,261 9/1969 Roberts ...260/67l 2,971,429 2/1961 Howerton ..250/71 R Primary Examiner-.lames W. Lawrence Assistant Examiner-Harold A. Dixon Attorney-Jack Oisher  ABSTRACT Apparatus for enhancing the sensitivity of fluorescence spectrophotometry is described. Enhanced sensitivity is obtained by a novel sample cell construction in the form of a flat plate providing multiple exposure of the sample to the exciting radiation to increase absorption, and providing concentration of the fluorescent radiation to a small aperture -for more efficient collection. The novel cell also provides easy separation of the exciting and fluorescent radiations.
14 Claims, 12 Drawing Figures F'LUORESC ENCE PATENTEI] F E8 6 I973 SHEET 20F 2 Fig. 4
N. J. HARRICK hT'roRNEY EXCITATION FLUORESCENCE FLUORESCENCE SPECTROPIIOTOMETRY USING MULTIPLE REFLECTIONS TO ENHANCE SAMPLE ABSORPTION AND FLUORESCENCE COLLECTION This invention relatesto fluorescence spectrophotometry, and in particular to a novel sample cell for use therein to enhance sensitivity.
Fluorescence spectrophotometry apparatus comprises an excitation source generating a range of wavelengths for causing an unknown sample to fluoresce, an excitation monochromator to permit selection of the wavelength incident upon the sample housed in a suitable cell, an emission monochromator for selecting a particular wavelength of the spectral distribution of the fluorescent radiation emitted by the sample, and a photodetector, usually a photomultiplier tube, for converting the received radiation into an electrical signal proportional to the intensity of the received radiation. In the commercial instruments, the excitation and fluorescent beams are oriented90 with respect to each other in order to ensure adequate separation. After suitable amplification, the electrical signal is usually displayed on an XY recorder or stripchart recorder. The resultant curves give the fluorescent intensity as a function of wavelength and may be used for qualitative and'quantitative analysis of the sample. Such apparatus are noted for their high sensitivity, but modern science requires even higher sensitivity than that available from current instruments especially for very small samples, known as microsamples.
Canadian Spectroscopy, 10, 128 (1965) suggests one technique to increase the efficiency of excitation of film samples using multiple reflections of the exciting radiation in a thin transparent plate which supports the sample. The fluorescent radiation emitted from the sample is detected by a phototube located opposite the flat side of the plate and thus no fluorescence concentration results. Such a cell constructionis not usable with conventional double monochromator apparatus employing an emission monochromator because the fluorescent radiation extends over too large an area and therefore it is not readily focused for efficient use in the emission monochromator.
In a subsequent publicationin Applied Optics, 6, 715 (1967), an improvement is described which attempts to combine multiple reflection of the exciting'beam with collection of the emitted fluorescence by shaping the thin plate into a hemisphere, withthe sample on the curved surface and a photodetector ofa small diameter opposite the flat surface, the exciting radiation entering the free annulus surrounding the photodetector. The disadvantages of this construction are a complicated cell shape which is difficult to fabricate and maintain by repolishing, and insufficient collection of the emitted fluorescence, a substantial portion of which is lost via the annulus. A more important disadvantage is inadequate separation of the exciting beam from the emitted fluorescence, since much of the incident excitation radiation will also impinge upon the photodetector. As will be understood, the excitation intensity is usually much stronger than the fluorescent emission.
The chief object of the invention is a novel cell construction offering enhanced interaction of the exciting,
radiation with the sample, efficient collection of the emitted fluorescence, and substantially complete separation of theexcitation and fluorescent radiations.
A further object of the invention is a novel cell construction of relatively simple shape which is easy to fabricate, and well adapted for use in a double monochromator fluorescence spectrophotometer.
These and other objects as will appear hereinafter are achieved, in accordance with the invention, with a cell construction comprising a flat optically transparent plate having opposed polished major surfaces, preferably one reflecting short edge, preferably reflecting long edges, at least one flat polished major surface of the plate serving to receive a sample'to be analyzed, and means at the opposite short edge adapted to allow a substantial part of any fluorescent radiation present in the plate to exit from the plate at a different angle from any excitation radiation introduced into the plate whereby the two radiations can be substantially separated from one another. In accordance with the invention, the excitation radiation is caused to multiply reflect within the plate between the polished major surfaces increasing interaction with the sample, and any fluorescent radiation emitted from the sample and entering the plate is brought by multiple reflections to one short edge' of the plate and thus concentrated to a small aperture and there caused to exit toward the emission optics.
The invention will now be described in greater detail with reference to the accompanying drawing, wherein FIG. '1 is a schematic view of a conventional double monochromator fluorescence spectrophotometer;
FIGS. 2 and 3 are plan and side views of one embodiment of my cell construction;
FIGS. 4, 5 and 6 are plan side and cross-sectional views of another embodiment of my invention;
FIG. 7 is a plan view of still another embodiment of my invention, with FIGS. 8 and 9 illustrating two modifications;
FIGS. 10 and 11 show two prism constructions for introducing the excitation beam into my novel cell; and
FIG. 12 shows my novel cell substituted for the conventional cell of a spectrophotometer.
FIG. 1 illustrates a typical conventional double monochromator fluorescence spectrophotometer. It
, comprises an excitation source 10 generating a range of wavelengths 11 which are passed through an excitation monochromator l2, 13, 14 for selecting one or more wavelengths for exciting a sample into fluorescence.
The monochromator is shown schematically as a mirror I2, a grating 13 and a mirror 14. The selected wavelengths are passed through an exit slit 15 usually forming a narrow vertical aperture and are focussed onto a portion of a sample 17 contained within a sample cell 16. The sample 17 is often a liquid to be analyzed. Fluorescence from the excited sample portion which exits 18 from the cellat right angles to the excitation radiation, is collected through an entrance slit 19 of the emission optics. The entrance slit 19 also usually forms a narrow vertical aperture. The fluorescent radiation is then passed through an emission monochromator 20, 21, 22 for analysis, comprising mirrors 20, 22 and grating 21, and the selected wavelengths then impinge upon a photodetector 23 which is usually a photomultiplier tube (PMT). As will be appreciated from the geometry illustrated, the extent of interaction of the excitation beam with the sample is limited thus reducing the absorption possibilities, the extent of collection of the fluorescent emissions is of the radiations, but none so far have been successful in combining increased interactions, increased collection'efficiency, reduction of the collected fluorescence to a small aperture, and good separation of the exciting and fluorescent radiations. In my invention I use as the sample cell to be substituted for the prior art cell 16 a flat plate of optically transparent material. Such a plate is illustrated in a top view in FIG. 2 and side view in' FIG. 3. The plate, designated-25, has major flat opposed parallel surfaces 26 and 27, both of which are polished to provide loss-less total reflection and one or both of which may receive a film sample 30 to be analyzed. One short edge surface'31 of the plate has metallization 32 to form a reflecting surface or mirror. The two long edge surfaces33 and 34 also have metallizations 35' thereon to form reflecting surfaces or mirrors. The opposite shortedge surface 36 has no metallization and is polished flat andremains transparent a'ndservesas a window to allow radiation to enter-and exit fromthe plate 25. All edge surfaces are perpendicular to the'r najor surfaces 26,27. Contacting the transparent short edge 36 at its flat side is a half round optically transparent 1 member 40, that is, a hemicylinder. The half round 40 serves to introduce and extract radiation from the plate 40. It will also be appreciated that the half round 40 contacting the short edge can i be replaced by its optical equivalent two quarter rounds, one sitting on surface 26 adjacent the short edge 36, and the other sitting on surface 27 ad.-
jacent the same short edge, with their curved surfaces facing outward.
' Assume that exciting radiation 39 is introduced into the half round 40?- at a 45 angle to theplane of the plate, andassume further that the critical angle c of tions. Thus all radiation at angles between Ocand 90 becomes trapped within the plate by the major surfaces 25, 27. However the radiation incident on the transparent edge 36 at angles between 0 and 00 (which equals between 90 and 90-6c relative to the major surfaces) will be emitted from the transparent edge. The radiation incident on the transparent short edge at angles relative to the major surfaces between 00 and 90-0c, in the absence of the .half round 40, remains permanently trapped within the plate and becomes reabsorbed. Also, in the absence of the half round 40, the radiation emitted from the transparent short edge fans out to 6 i 90. 7
The half round 40 is used to extract all of the energy trapped by the major surfaces within the plate, and thus all the energy incident on the edge surface 36 at angles relative to the major surfaces 26, 27 between 00 and 90, which relative to the edge 36 becomes 90 0c and 90, shown in FIG. 2 by the dashed lines designated 42.
As the emerging radiation suffers no refraction in its short edge surface 31 ensures that all the'energy trapped by the major surfaces is extracted through the transparent edge 36.
As will be observed, the exciting radiation has, due to I multiple reflections through theplate, increased opportheplate is below 45, Under these conditions, the exciting radiation will propagate by multiple total reflections down the length of the plate to the, right, reflect from theimirrored short edge 3l,-and propagate to the .left up the plate'and exit from the short edge 36into'the half round 40 still at 45 butopposite to ray 39. On the bottom surface of the element 40 is located a strip 38 of metallization serving as a. mirror. The excitation willtion will enter the plate 25. Of the reentering radiation,
that fraction which is incident on the major surfaces 26, 27 at an angle exceeding 0cwill undergo total reflection and propagate through the plate by multiple reflec-.
tunity to interact with the 1 sample and cause fluorescence, thus producing enhanced absorption. That fraction of the fluorescence entering the plate and trapped by total reflection by the major surfaces is efficiently collected within the plate and brought to the small aperture represented by the transparent short edge 36. By means of thehalf round 40, most of the trapped energy can be extracted" and directed toward the entrance slit 19 of the emission optics. As illustrated inFIG. 2, the collected radiation can extend between the lines 43, 44 and thus be adequately separated from the exciting radiation 39.-'Tlie structure illustrated in FIGS. 2 and 3 offers the further advantage that the angle of incidence for-the excitation beam can be variedcontinuously thereby changing the depth'of. penetration of the exciting beam into the sample.
The cell structure of FIGS. 2 and 3 is especially useful for analyzing this film liquid samples, which can be placed in contact with one or both of the polished major surfaces 26, 27. The film sample can have a refractive index n the same as, lower, or higher than that of the plate 25. Any optically transparent material can be used for the plate 25, but one with a low refractive index n is preferred, because 60 will be lower. Two
preferred materials are quartz (n 1.5, 0c 42) and radiation at a plate location different from the short edge surface from whence the fluorescence emerges and thus further increase their separation. For example, the exciting radiation can be introduced at one of the long edges, though this may also increase loss of the fluorescent energy. But the introduction of the excitation at a long edge offers the possibility of other structures for extracting the trapped fluorescence from the short edge and thus through a small aperture.
One such structure in accordance with the invention is a light funnel, which is illustrated in a top view in FIG. 4, a side view in FIG. 5, and a cross-sectional view in FIG. 6. The same reference numerals are used for corresponding elements. In this embodiment, one long edge surface and one short edge surface is metallized at 35 and 32. A part 45 of the opposite long edge surface is bevelled flat at a 45 angle. This angle is not critical and other angles can be used. The remainder of that long edge surface 46 is polished flat and metallized 47 similarly to the opposite long edge. The bevel 45 is used as a window to introduce the exciting radiation 41 at an angle to the major surfaces exceeding the critical angle to cause multiple reflections therefrom and also slightly oblique to the vertical to cause the beam to zig-zag across the width of the plate and thus propagate down the length of the plate 25 as shown. The emitted fluorescent radiation that enters and becomes trapped within the plate propagates along its length. The extraction means therefor is a funnel 50 with polished surfaces which can be optically contacted to the short edge of the plate as shown or be fabricated as an integral part of the plate. The fluorescent radiation crosses from the plate 25 into the funnel 50 through the short edge and as it propagates down the funnel towards the thinner-edge multiply reflects from its surfaces at increasing angles of incidence due to the tapering funnel. As critical angle is approached, the radiation emerges from the funnel at near grazing incidence, shown at 51, in the forward direction (towards the tip) and to the left in FIGS. 4 and 5, and thus can be directed into the emission optics.
Another construction for extracting the trapped fluorescent energy is a simple bevel 55, which is illustrated in FIG. 7, at the short transparent edge. Here, however, instead of changing the angle of incidence gradually, as in the funnel extractor of FIGS. 4 and 5,.
the angle changes in one jump by one reflection from the bevel surface. However, by.a suitable choice of bevel angle, which also depends upon the refractive index of the plate, a substantial part of the trapped energy can be extracted so as to issue in the forward direction, i.e., away from the plate. The manner of choosing a suitable bevel angle will become apparent to those skilled in this art from a consideration of the cases of bevel angles of 60 and 45, illustrated in FIGS. 8 and 9, respectively.
FIG. 8 illustrates the case for a 60 bevel. The various solid line arrows show the different directions that can be taken by the emerging fluorescent radiation. Some of the radiation will be emitted from the bevel largely in the forward direction of its tip. The remainder will be totally reflected from the bevel surface and emerge ing in the backward direction will be lost. FIG. 9 illustrates the situation under the same conditions for a 45 bevel. In this case, part emerges as before from the bevel surface in the forward direction, but the part emitted from the lower major surface is generally in the backward direction and not usable. Thus the larger bevel angle is preferred. A suitable bevel angle range for most practical purposes is about 50- With the 60 bevel for example, at least 50 percent of the emitted radiation is in the forward direction. In general, the higher the refractive index of the plate and the smaller the bevel angle, the more energy will be emitted by the major surface 27. The light funnel of FIG. 4 may be regarded as a bevel with a very small angle, and thus nearly all of the energy is emitted from the major surfaces.
In the case of the FIG. 8 embodiment employing the bevel 55 as the extraction means, the exciting radiation can be introduced transversely at a bevelled portion along a long edge, as illustrated at 45 in FIG. 5, or it can be introduced into the extraction bevel as shown by the dashed arrow 61 in FIG. 8. Another way of introducing transversely the exciting radiation is by way of simple right angle prisms mounted on a major surface close to a long edge, in a position approximately corresponding to that of the bevel 45 of FIG. 5. This is illustrated in cross-sectional views in FIGS. 10 and 11, showing prisms 65, 66 arranged in two ways for introducing the excitation beam. When the excitation radiation is transverse to the fluorescent radiation, Raman Spectroscopy can be accomplished, since the latter requires excitation and observation at right angles.
As mentioned above, the half round extractor 40 can be used with thin film samples whose refractive index has any value relative to that of the plate. With the embodiments of FIGS. 4-9, the rule is the same for thin film samples.
My novel cell construction is also useful for analyzing liquids and solids, for example, immersing the cell plate in a liquid sample. However, in this case, critical reflection of the fluorescent radiation entering the plate cannot be obtained. However, that fluorescent radiation which has a direction toward the extraction short edge with an angle of incidence relative thereto between 6c and will be collected. The cell can be viewed as an extended window immersed in the liquid or solid sample and collecting fluorescence over 'a much deeper depth than would ordinarily be possible with a conventional cell, since the fluorescence emerging via the cell plate avoids being reabsorbed by the sample. In this case, the plates refractive index should be higher than but still close to that of thesample, since only the fluorescent radiation between 0 and 0c relative to the major surfaces can enter the plate. If the entire plate is immersed, the half round extractor of FIG. 2 is preferred as it permits also the exciting radiation to be introduced via the short edge.
One of the features of the invention is that my novel cell construction can be substituted for the conventional cell in known double monochromator fluorescence spectrophotometers with no change in the optics or at most with very little change. FIG. 12 shows this. The spectrophotometer geometry is the same as in FIG. 1, only the slits 15 and 19 being shown for simplicity. The cell 25 of FIG. '7 is positioned as in FIG. 8
such that the excitation 61 is normal to the bevel edge 55, and the fluorescence directed alongv the path 18 from the plate 1 which passes through the slit 19 is analyzed by the emission monochromator as previously described.
While I have described my invention in connection with specific embodiments and applications, other modifications thereof will be readily apparent to those skilled in this art without departing from the spirit and scope of the invention as defined in the appended claims. 7
What is claim is:
l. A fluorescence spectrophotometer comprising a sample plate of optically transparent material having major opposed planar parallel surfaces defining at least a first edge surface for admitting or transmitting an optical beam and at least a second edge surface remote from the first edge surface, both major surfaces being polished with at least one adapted to receive a sample to be analyzed, means for directing an excitation radiation beam into the plate at an angle exceeding the critical angle whereby the, excitation beam propagates through the plate by multiple reflections from at least the major surfaces, said excitation beam establishing at said one major surface at each reflection an evanescent wave capable of interacting with the sample and causing it to emit characteristic fluorescent radiation, some of which enters the plate and impinges on a surface at an angle exceeding the critical angle causing the fluorescent radiation to propagate through the plate by multiple reflections, and means located adjacent said first edge surface at such a position as to receive and detect substantially only that fluorescent radiation which propagates through the plate by multiple reflections and exits from the plate via said first edge surface, whereby both sample excitation and emission-collection are enhanced by multiple internal reflections in the plate.
2. A fluorescence spectrophotometer as set forth in claim 1 and further comprising a source of excitation radiation, and an excitation monochromator for receiving the excitation radiation and passing selected wavelengths thereof to the excitation beam directing means, said fluorescent radiation detecting means comprising an emission monochromator for receiving the exiting fluorescent radiation and passing selected wavelengths thereof to a photodetector.
'3. A fluorescence spectrophotometer as set forth in claim 2 wherein said sample plate has reflecting surfaces at all of its edge surfaces with the exception of the first edge surface, and the excitation radiation directing means is located adjacent the first edge surface for receiving and exiting aperture, and the excitation and emission monochromators each have slit systems whose size generally corresponds to that of the given aperture. 4 i
5. A fluorescence spectrophotometer as set forth in claim 1 wherein the fluorescence receiving means comprises a hemicylindrical structure located at the first edge surface.
. A fluorescence spectrophotometer as set forth ll'l claim 1 wherein the fluorescence receiving means comprises a light funnel whose large edge is located adjacent the first edge surface.
7. A fluorescence spectrophotometer as set forth in claim 1 wherein the first edge surface is beveled flat at an angle relative to the major surfaces.
8. A sample cell for use in fluorescence spectrophotometry comprising a flat plate of optically transparent material having major opposed planar parallel polished surfaces defining opposed short edge surfaces and opposed long edge surfaces, at least one of said long edge surfaces and one of said short edge surfaces being perpendicular to the major surfaces and being beam reflectors, at least one of said major surfaces serving to receive a sample to be analyzed, said other short edge surface being optically transparent for receiving and transmitting optical radiation, and means associated with said other short edge surface to permit fluorescent radiation trapped in the plate by multiple reflection by the major surfaces to exit from said other short edge surface at a different angle from that of excitation radiation introduced into the plate.
9. A sample cell as set forth in claim 8 wherein the reflecting surfaces formed on the one short edge and the one long edge surfaces are formed by metallizations thereon.
10. A sample cell as set forth in claim 9 wherein said other short edge surface is beveled flat at an angle between about 50 and relative to the major surfaces so as to permit a large fraction of fluorescent radiation in the plate to exit therefrom in a forward direction.
11. A sample cell as set forth in claim 9 wherein said other short edge surface extends perpendicular to the major surfaces, and a hemicylindrically shaped optically transparent member is located adjacent said other short edge surface.
12. A sample cell as set forth in claim 11 wherein a side portion of the hemicylindrically shaped member is metallized to reflect excitation radiation.
13. A sample cell as set forth in claim 9 wherein a light funnel is contacted via its long end to saidother short edge surface.
14. A sample cell as set forth in claim 8 and including means located adjacent part of a long edge surface for introducing excitation radiation into the plate substantially transverse to its longitudinal direction and at an angle relative to the major surfaces which exceeds the critical angle.