Multiple Internal Reflection Fluorescence Spectrometry N. J. Harrick Harrick Scientific Corporation. Ossining, N. Y . 10562
George I. Loeb Ocean Sciences Division. Naval Research Laboratory, Washington. D. C. 20390
The multiple internal reflection technique is applied to fluorescence spectrometry. As with absorption spectrometry, multiple exposure of the sample to the incident light beam is achieved as exciting radiation propagates along a thin plate-like element. Emitted fluorescence from samples on large faces of the element is largely trapped and propagates to a narrow edge of the element, where it emerges concentrated into a narrow aperture. Both factors greatly enhance the sensitivity of fluorescence analysis, and emitted radiation may be separated from the exciting beam. Accessories for commercial equipment are described. The utility of this approach in the study of thin films is illustrated with adsorbed films of a dansyl conjugate of the protein plasma albumin. An adsorbed layer of monolayer thickness could be observed; the kinetics of adsorption were consistent with formation of a monolayer of essentially globular molecules.
The internal reflection technique has been very useful in enhancing the sensitivity of absorption spectrometry since its introduction more than a decade ago ( I , 2) to the extent that submonolayer spectra have been recorded (3, 4 ) . Fluorescence techniques have also been of great value in situations where the quantity or the nature of samples have required high sensitivity (5). The need for investigation of samples of very thin films, of highly self-absorbing materials where emitted radiation is rapidly attenuated, and of liquid-film interfaces (such as occur during adsorption from natural environmental and biological fluids) have led us to combine these techniques, and significant further increases of sensitivity have been achieved. In the recording of both absorption and fluorescence spectra, multiple internal reflection technique causes the incident radiation to impinge upon the sample many times. The resultant is essentially a sum of the signals produced at each impingement, so that the signal is from an apparently much thicker sample without as severe attenuation of emitted radiation as would occur in long optical paths in absorbing samples. Analysis of MIRF (Multiple Internal Reflection Fluorescence). When a light ray inside a transparent material approaches the interface between the material and a transparent surrounding medium, the ray is internally reflected, and trapped within the material, if it approaches the surface at an angle of incidence greater than the critical angle Oc. For any interface, Sin& = nz/nl
(1)
N. J. Harrick, "Internal Reflection Spectroscopy," Interscience, New York, N.Y., 1967. N. J. Harrick, Phys. Rev. Lett.. 4 , 224 (1960). L. H. Sharpe, Proc. Chern. Soc.. 1961, 461. G. I . Loeb, J. Colloid interface Sci.. 2 6 , 236 (1968). S. Udenfriend, "Fluorescence Assay in Biology & Medicine," Academic Press, New York, N.Y., 1962, p 3.
where nl is the refractive index of the material, n2 that of the surrounding medium, and nl > n2. If an optically absorbing sample is placed in contact with the surface of an internal reflection element (IRE) of transparent material, the light rays are attenuated a t each reflection. The reason for this is that there is penetration of the electromagnetic field associated with the light ray to a small depth beyond the reflection interface into the rarer medium, and this evanescent wave ( I ) can interact with the absorbing sample. The parameters affecting this interaction are the refractive index ratios, angle of incidence, and wavelength of the light, and are discussed in detail elsewhere ( I ) Excitation of Fluorescence via internal reflection is the classical method of demonstrating the presence of the evanescent field in total reflection (6). It has certain advantages over conventional external illumination. There may be stronger coupling of a thin film absorber to the exciting radiation, and this enhancement may be, e.g., a factor of four for a thin film on a substrate having the same refractive index ( I ) . Furthermore, by employing multiple pass sampling geometries ( I ) , the same sample region may be exposed to the exciting radiation a number of times; the light path in a double-pass cell is shown in Figure 1. The further obvious advantage is that the exciting radiation trapped within a thin internal reflection element propagates down the length and back and can be efficiently employed to excite large area weak absorbers. Some experimental work using MIR in excitation has been described by Hirschfeld (7). Collection of radiation emitted by fluorescent samples may be accomplished more efficiently by MIRF technique than is otherwise possible, since the fluorescent radiation is largely trapped within the IRE and exits via a small apperture. We will consider two cases of interest: first, when fluorescence is excited in the bulk of the MIRF element, as in Figure 2a and, second, when fluorescence is excited in a thin film in contact with the reflection element, as in Figure 2 b . In the first case, fluorescence radiation generated within a small region is emitted in all directions, as shown in Figure 2a. In three quadrants, the shaded portion of the figure shows the angular range in which emission from the small region of emission is trapped. Several typical rays are also shown in the fourth quadrant. Radiation excited in the element and approaching the surface with angles of incidence between 0 = 0 and 0 = *0, may pass into the surrounding medium, and is refracted so as to emerge over an angular range of *go". This angular spread severely reduces the energy density of the emerging radiation, The rest of the radiation is trapped within the plate by total internal reflection (as shown in Figure 2a). The same conditions hold a t all plane faces of an element; however with
(6) R. W. Wood, "Physical Optics" 3rd ed. Macmillan, New York. N.Y., 1934. ( 7 ) T. Hirschfeld, Can. Spectrosc.. 10, 128 (1965). A N A L Y T I C A L C H E M I S T R Y , V O L . 45, NO. 4, A P R I L 1973
0
687
Figure 1. Path of laser beam
in double-pass MIR
f
/
element
ESCAPMO RAYS
thermore, there is no increase in angular spread as it exits (compare the two edges in Figure Zc). The enhancement gained from trapping radiation where 0 > 0, and reducing its emergent spread by viewing an aperture such as the hemicylinder is 90"/0,. This represents a gain of 2 for quartz (n = 1.4, 8, = 46-1, 3 for AgCl (n = 2.0, 0, = 307, and 6 for Ge (n = 4, 8, = 15"). The largest enhancement obtained from observing the internal reflection mode results from the possibility of collecting fluorescent radiation from large areas. Normally only fluorescent radiation from a sample area comparable to the slit width of the emission monochromator is effectively utilized for analysis since there is no means of concentrating emission from large areas into instruments with reasonable f-numbers. For internal reflection, on the &her hand, the fluorescent radiation from large areas which is trapped within the element all propagates ednes townrda the ..~. ----- of the element: i.e.. radiation from iivalent positions down the length of the element is all ev SUI,erimposed a t the exit aperture giving rise to a large "11 tlr. cy6ca .Ana" ~ y.J ....-*",I:":"" L..C~OL.L*LL.6 e n 1 m u ~ c u . x 2~. ~---":&~:.:+.U C . ~ U V ~uD. except the entrance aperture, all the radiation emerges from the entrance aperture. Thin samples spread over an element of length 100 times greater than the instrument slit width may thus appear 100 times brighter when viewed through the aperture. For weak absorbers where the exciting radiation is not highly attenuated, this alone will thus give an enhancement of 100 in this case for a .~ . . single pass element and ZW for a double pass element. The analysis above applies 1to the case that the fluorescent radiation is generated within the reflection element or in a thin film on the ellement surface. The critical angle for reflection 0, is, respect.ively, that of the elementair interface or film-air interf,ace. The same arguments also apply to the liquid-film-ell ement systems except that the critical angle is then that of the film-liquid interface. The presence of a liquid will increase the critical angle and thus reduce the amount o f radiation trapped within tho e b r_______, n m n t . _______, hence thew will k>e some reduction in sensi"_._ ...-.tivity. For studying homogeneous large volume liquid or solid samples in contact with the element, internal reflection excitation can still he employed; in this case, however, the fluorescent radiation is not trapped within the element hut passes through the plate with partial reflection a t the interfaces. The large gains in collection as discussed above are therefore not obtained. Advantage may still he gained by observing the aperture of an optically transparent probe immersed in the medium since the probe now acts as a window permitting some of the fluorescent radiation to escape from deep within the hulk of the liquid. For small volume and special geometries, however, large enhancement can also he obtained from solids and liquids. In the case of solids, the internal reflection element may be made from the sample material itself, while for liquids the sample is placed in a cavity within an internal reflection element designed for this purp Optical Materials a n d Geome tical materials for internal reflect cence must have high transmission m m e spectral region of interest and must he free of fluorescing impurities. For the UV-Vis spectral region, UV grade quartz and sapphire are suitable. Other materials that should he considered include LiF, MgFz, CaF2, NaF, SrFz, and BaFz. Materials of low refractive index (Comparable to that of the film) are preferable for thin films while materials of high refractive index are required for studying liquid interfaces. Various geometries may he employed for internal reflection fluorescence in the study of thin films in contact with ~~
FILM FLUORESCEWE
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t Figure 2. Emission patterns m miernai 8.
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From bulk Sample
b. From film Sample c. From edges of hemicylindrical-edgeelement
an element shaped in the form of a thin plate, these conditions result in the trapped radiation propagating toward the thin edges of the element where most of the energy may he emitted. Similar considerations hold in the case of fluorescence generated in a thin film in contact with the large faces of an element. In this case, illustrated in Figure 26, the radiation is trapped if it propagates toward the film-medium interface a t greater than the critical angle defined by the ratio of refractive indices of the film and the medium; trapped radiation may enter the element, (with refraction at the film-element interface), and remain trapped: the element and film together now constitute the volume in which the radiation is confined, with the refractive index of the film material defining the condition for trapping by total reflection. The shapes of the thin edges of a plate-like element determine the amount and angular spread of the trapped radiation emerging. By shaping the edge of the plate as a hemicylinder, all of the radiation not emitted through large surfaces can he extracted from the plate and, fur688
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ANALYTICAL CHEMISTRY, VOL. 45, NO 4. APRIL 1973
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sample emission
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a. 45' b. 60"
0
the internal reflection element. The curved surface of the double-pass variable angle multiple reflection element (shown in Figure 2c) can be used to introduce the exciting radiation and to extract the fluorescent radiation at various angles of incidence. Plates with simple bevels may also be used. The radiation distribution from plates with 60' and 45" bevels are shown in Figure 3. Note that the fluorescent radiation is emitted through the bevel and a broad face preferentially in the direction of the tip of the bevel for the 60" plate. An excellent geometry for observing fluorescence of transparent solids is a plate with a 45" bevel where the exciting and fluorescent radiation are readily separated as shown in Figure 4a. An element of similar geometry but with a cavity (Figure 4b) can be applied with advantage in the study of liquids. In this case, the refractive index of the plate and that of the liquid should preferably be closely matched. The optimum size of the cavity is dictated by the absorption coefficient of the liquid. In those cases where sensitivity is sufficient, and overlap of exciting and emitted wavelengths causes difficulty, one may reduce interference from the excitation beam by using anti-reflective coatings on thin edges instead of metallizing them. (We are grateful to Bruce Morrissey of National Bureau of Standards for this suggestion.)
EXPERIMENTAL Application of the concepts of the MIRF technique are illustrated by several experiments which were performed with a Model MPF-2A spectrophotofluorimeter (Perkin-Elmer Corp.). Preliminary experiments have shown that the hemicylinder type of element, the liquid cavity cell, and the 60" bevel element may all be used to confer enhancement of orders of magnitude on fluorescence signals, although the 45" bevel element was used for the series of experiments to be described below. The elements used were obtained from Harrick Scientific Corp., Ossining, N.Y. The elements are 50 mm long, 16 mm wide, and 1.5 mm thick if fused quartz, and 1.0 mm thick if sapphire. Elements of the fixed bevel type may be quite reproducibly aligned and held in the cell compartment so that the exciting beam enters the element normal to the bevelled face if a support allowing adjustment as in Figure 5 , is used. This support replaces the standard cell turret, is suitable for dry self-adhering samples, and allows horizontal adjustment of the element in the holder while constraining the element to pivot about the focus of the excitation beam. In the first experiments, sapphire elements which did not exhibit any detectable impurity fluorescence, when examined in the conventional direct reflection mode as in Figure 5b, were used with exciting radiation of 430 nm. When the MIRF mode was applied as in Figure 5a, it rapidly became apparent that there were low levels of impurities. (Figure 4a illustrates another method of excitation of transparent samples in which surface fluorescence is de-emphasized; both methods yielded similar spectra.) The conventional direct surface reflection and MIRF spectra are compared in Figure 6. The impurity is clearly detectable by MIRF as sharp emission peaks in the red (Figure 6a) although no such pattern may be observed by direct reflection (Figure 6 b ) . Thus, the sensitivity of this method for observation and measurement of
COLLECTING LENS
b
Practical arrangements for use with fluorescence spectrophotometers
Figure 5 .
a . MlRF mode with dry film samples b. Conventional mode
bulk fluorescence in transparent material is very high. Although high refractive index materials for MIRF elements allow more efficient trapping of energy, and higher purity sapphire should be more acceptable in terms of spurious peaks, the lower refractive index of quartz is still sufficiently high that very useable spectra are obtained. T h e absorption study to be described was performed with a quartz 45" element, and utilized the very pronounced surface activity of proteins to form surface films. Bovine plasma albumin (Armour) was conjugated with dansyl (dimethylamino-1- napthalene sufonyl radical) chloride according to the procedure given by Laurence (8). The resulting preparation had an average of 1.1 dyes/molecule of albumin, since the optical density of a solution containing 1.4 mg/ml was 0.10 a t 325 nm, and the molar extinction of the dye is taken to be 4.3 X 106 cm/ mole. This solution was diluted by a factor of 100 for the absorption experiments described below. At this concentration, the kinetics of the absorption process allowed sampling a t convenient times with sufficient accuracy. Adsorbed films were produced by immersing the element into the solution of labelled protein (15 ml of solution in a Kel-F cell). The solution was vigorously stirred with a magnetic stirrer bar encased in Teflon (DuPont). The elements were immersed while wet with solvent (water). After the period of time allotted for the adsorption process, the element was withdrawn from the solution and, while visibly wet, immediately plunged into a fresh portion (8) D. J. R . Laurence, Methods Enzymol. 4, 174 (1957) A N A L Y T I C A L C H E M I S T R Y , V O L . 45, NO. 4 , A P R I L 1973 a 689
b
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Figure 6. Spectra of
Figure 8. Absorption of labelled protein on quartz corrected for prism blank. Numbers on curves are absorption time in minutes. Curve T i s spectrum of transferred film (see text)
impure sapphire
a Arranged as in Figure 4a b. Direct illumination as in Figure 5b
801
I . 5 MIN ADSORPTION OF LABELLED PROTEIN I
I
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I
(nm)
Spectrum of absorbed layer: 1.5-minuteexposure
of solvent. This rinsing process was repeated three more times; thus, the sample was never allowed to dry until after the final rinse, and the film which forms at the air-water interface of a protein solution was not permitted to contact the element. The spectrophotofluorimeter slits were set a t 10 nm band pass, and excitation was a t 345 nm. A UV-39 filter was used a t the emission monochromator entrance slit, and a UV D-25 filter in the excitation beam. The instrument was always set up in the ratio recording mode, with the reference sensitivity control at ratio two. Before each spectrum was taken, the reference signal was adjusted to 100%, and the sample sensitivity adjusted so that a rhodamine solution gave a standard response. The quartz MIRF element was cleaned immediately before each run in a manner very similar to that previously used in cleaning germanium elements for infrared MIR spectra (4): it was washed in the commercial detergent Tide (Procter and Gamble), rinsed with tap and then distilled water and with acetone. Acetone wetting the element was wiped away with lens tissue, the elements were air-dried and then were processed for 10 minutes with the Plasma Cleaner (Harrick Scientific Co.) before immersion into solvent and then into the protein solution. In two instances, monomolecular films were formed and transferred to the quartz element in a manner similar to the studies of 690
0
(nm)
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4 , APRIL 1973
Langmuir and Blodgett (9) using castor oil to maintain a surface pressure of 16.5 dynes/cm (Davies and Rideal) (10). Multiple internal reflection fluorescence and conventional direct illumination of the same sample were compared using a film formed by adsorption for 90 min on the quartz MIRF element. The arrangements were as in Figure 5 and, for direct illumination, the large faces of the element were inclined 30" from the exciting beam direction. An emission spectrum of the DNS-albumin solution was taken using the standard 1-cm cell and turret supplied with the instrument. We measured the emission of a film-covered element while immersed in water by sealing the edges of a Teflon (DuPont) cavity to the 45" element with purified paraffin wax, and allowing adsorption to occur from this cavity, whose volume was 2 ml. The cavity was rinsed with water 4 times and refilled with water to take the wet spectrum; it was then emptied and allowed to dry to take a dry spectrum of the same sample.
RESULTS AND DISCUSSION Immersion of the quartz element in dansyl albumin solution for 1.5 minutes and rinsing resulted in a signal well above the noise level, as shown in Figure 7 . Upon subtraction of the signal due to the clean element, a fluorescent signal characteristic of the dye was evident. The instrument was set at sample sensitivity 6 for this spectrum. As the signal strength increased with increasing immersion time, the sensitivity was decreased, so that the peak fluorescence was kept between 50 and 100 on the chart, reaching sensitivity 4 at longer immersion times. The relationship among sample sensitivity ranges 4, 5 , and 6 was obtained by observing signals from the same sample on two ranges in those instances where it was possible to do so. If the spectra are all normalized so as to be equivalent to the signal obtained at sample sensitivity 4, a family of spectra is obtained. The spectrum obtained with a clean element may be subtracted and the resulting spectra are shown in Figure 8. Also shown in this figure is the spectrum obtained from a monomolecular film spread upon water and transferred to the prism in the manner of Langmuir and Blodgett at 16.5 dynes/cm. It may be seen that this curve, corresponding to an amount of transferred material equivalent to a monomolecular layer, corresponds to aporoximately three minutes adsorption in the system used here. If the peak fluorescence signal for adsorbed dansylated protein is plotted against adsorption time, the curve as Blodgett, d. Amer. Chem. Soc.. 5 7 , 1007 (1935). Davies and E. K. Rideal, "Interfacial Phenomena," Znd, ed.. Academic Press, New Y o r k . N . Y . , 1963, p 34.
(9) K. B. (10) J. T.
-
2 W
Figure 9.
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05
,
5
IO
' 15 210 TIME (MINUTES)
25
--&+--do
Time dependence of maximum fluorescence
,
450
ADSORBFD LAYER (I.5,HR)
500 AEM (NMI
,
550
Figure 10. Comparison of M I R F and direct illumination. DNSprotein film absorbed for 90 minutes
shown in Figure 9 results. There is a linear increase in signal to a value considerably greater than that corresponding to a monolayer of material before the plateau is reached. A linear relation is expected while stirring maintains a constant concentration in the bulk phase adjacent to the unstirred layer, if either (a) any change in the rate of adsorption due to surface coverage is balanced by an increase of concentration in the unstirred layer or (b) adsorption is faster than transport to the surface, as seems more likely (11). The extrapolation of the linear portion of the curve to zero signal does not pass through the origin. It is not yet certain whether the lag period may be interpreted as the time required for diffusion of dissolved material through the layer of solvent which is carried along by the wet prism as it is introduced into the protein solution or as due to other causes; in any case, the lag period amounts to less than one minute. The plateau or saturation value of the fluorescence is approximately three to four times that due to a transferred monolayer. The volume occupied by a monolayer of spheres in cubic close packing is 50% and in hexagonal packing it is 60% of their envelope, while the thickness of unfolded protein chains a t an interface is commonly held to be 8-9 A. Thus, a monolayer of albumin molecules (of diameter 60 A in the native conformation) may be consid(1 1) H. Sobotka and H.J. Trurnit, "Unimoiecular Layers in Protein Analysis," in "Laboratory Manual of Analytical Methods in Protein: Chemistry," Vol. H i , P. Alexander, and R. J. Block, Ed., Pergamon Press. Elmstord, N.Y., 1961. p 2111
EM
Figure 11. Comparison of M I R F
and solution fluorescence
ered to pack in a film whose material density per unit area is equivalent to a layer of material 30 to 36 i% thick, or 3 to 4 times the thickness of an unfolded monolayer, and the data are consistent with coverage of the material interface with an adsorbed monolayer of essentially spherical globular material, as has been suggested previously (12). We see then that the MIRF technique is suitable in sensitivity and precision to allow studies of monomolecular films. (The sensitivity of the MIRF and direct surface reflection methods for the same sample are compared directly in Figure 10, in which the same instrument settings were used for both curves.) If the fluorescence of a solution of dansylated protein is compared with that of the 1.5 minute absorption sample (Figure ll), only very minor differences are seen, and the band widths a t half the peak signal are equal. Thus, the signal has not been noticeably distorted by this method. A slight frequency shift may be due to the different environment of the chromophore. One very useful application of this technique using mixed films containing components with different labels will be the study of sequential processes or of exchange equilibria between absorbed and bulk material in dynamic systems, for which techniques for studying liquids are required. One may reasonably expect that the observed signal strength might be seriously degraded if the MIRF element is immersed in liquid, since the refractive index ratio nz/nl, will be lessened. In an experiment performed to test this hypothesis, a film absorbed over the course of 2 hr was observed in a water-filled cell, and subsequently after the water was removed and cell and element allowed to dry. The signal remained almost as strong for the wet as for the dry situation, encouraging us to refine our preliminary designs for liquid-holding accessories which will be described elsewhere. N o t e added in proof. The same techniques can be used with advantage in the study of photochemical reactions and Raman spectroscopy on thin films.
ACKNOWLEDGMENT We would like to thank Samuel Kaufman of the Naval Research Laboratory for a critical reading of the manuscript. Received for review May 10, 1972. Accepted November 24, 1972. Method and results were presented a t the Eastern Analytical Symposium, New York, N.Y., November 1971. (12) I . R. Miller, RecentProgr. SurfaceSci.. 4, (1972)
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