New Type of Spectrofluorometer with a Tunable Laser Source and Unique Optical System D. C. Harrington and H. V. Malmstadt’ School of Chemical Sciences, Department of Chemistry, University of Illinois, Urbana, Ill. 6 780 7
A new type of spectrofluorometer has been developed which utilizes a single modified monochromator both as a tunable dye laser excitation source and as a scanning monochromator or polychromator to measure emission fluorescence spectra. This unique design which simultaneously employs one monochromator in the dual role of the wavelength-selectable, narrowband excitation source, and as the dispersive system for the fluorescent emission spectra, can be readily assembled from a commercial monochromator and a few attachments, including an easily constructed nitrogen laser. With this system, fluorescence spectra can be easily obtained from low concentrations of fluorescent species with minimal chance for photodecomposition. Also, quantitative results can be obtained by measurement of a selected wavelength segment of the fluorescent radiation from as little as one 10-nanosecond laser pulse or, with better precision, by averaging the fluorescence results from several laser pulses.
Fluorescence spectrometry has been utilized in a wide range of disciplines. As a result, many techniques and instruments have been developed in an effort to obtain better sensitivities as well as to improve the quality of the spectra (1,2). As a further development in obtaining high sensitivity and fluorescence spectra, a new type of spectrofluorometer has been developed and is presented here, one that incorporates a tunable dye laser as the excitation source and utilizes a unique optical system that helps reduce the complexity of the instrumentation. Spectrofluorometers generally utilize two monochromators to isolate the wavelengths of excitation and emission. In the design presented here, a single modified monochromator performs this dual function. That is, part of the monochromator is utilized as the tunable section of the dye laser excitation source and the other part is used as a polychromator or a monochromator to measure the emitted fluorescence. Sources of excitation for fluorometers can generally be classified into two categories, line and continuum sources. A representative of the first type is the mercury vapor lamp in which discrete high intensity resonance lines are isolated by filters. A high-intensity continuum such as the Xenon arc is also frequently used, where a small segment of the continuum band is isolated with a monochromator. This usually leads to a source intensity below that exhibited in the resonance line sources (3). In the design presented here, a tunable dye laser has been utilized as the source. The tunable dye laser provides an inherently narrow bandwidth of radiation, exhibits a very high intensity, and is continuously tunable over a wide spectral range. Thus the wavelength-selectable features of the continuum-monochromator source and the high intensity of a line source are available in the tunable dye laser. In addition, the laser Send request for reprints t o this author.
light is collimated, much more intense, and the use of a pulsed dye laser can circumvent, in some cases, the problems associated with continuous wave sources which cause photodecomposition and quenching ( 4 ) . The use of lasers in fluorometry has been demonstrated ( 5 - 7 ) , but in each case the laser output was composed of discrete lines, one of which was isolated for the fluorometric analysis, much the same as for the mercury vapor lamp. The tunable dye laser has been shown as an effective source of radiation for atomic fluorescence (8-10); however, the analytical applications for molecular fluorometry have been few (11-13). This is caused, in part, by the fact that the dye laser’s normal tuning range is from -360-640 nm [second harmonic generation can extend this to -260 nm ( 1 4 ) ] and many compounds require excitation below 360 nm. The literature of the past few years indicates, however, that a large number of compounds relevant to clinical and biological analysis require excitation within the dye laser’s normal tuning range (15-20). Especially important is the fact that many clinical analytical procedures use coupling reactions in which the formed fluorophors’ excitation maxima fall within the dye laser tuning range (21,22). INSTRUMENTATION We previously reported the construction of a digital scanning tunable dye laser (23). The dye laser was formed within the interior of a commercial programmable monochromator (GCA/McPherson EU-700) between the grating and a partially transparent output mirror mounted a t the rear of the monochromator (see Figure 1).A nitrogen laser was constructed and used as an optical pump for an organic dye placed within the formed cavity. The dye laser produces coherent, monochromatic, high intensity output pulses that can be tuned continuously from 358-641 nm by using eight different dye solutions. A 1-nm bandpass (HWFM) is typical for all the dyes used when using the standard grating for the monochromator. The construction of the dye laser within a monochromator was done for several reasons. First, the monochromator possesses a precision grating drive which provides for the convenient control of the dye laser wavelength. Second, the monochromator displays a digital wavelength readout, and with the positioning of the optical axis of the dye laser cavity as shown in Figure l, the output wavelength of the dye laser corresponds directly to that of the digital readout. Also, remote control of this specific monochromator has been demonstrated ( 2 4 ) which allows for the automation of the dye laser wavelength selection by a minicomputer or process controller. In the present design, the dye cavity has been enclosed within a light-tight baffle (Figures 2 and 3). This structure extends from the top half of the grating to the rear of the monochromator and encloses the dye cell, output mirror, and folding mirrors. A side arm assembly directs the nitrogen pumping radiation onto the dye cell but shields it from the rest of the monochromator. The top half of the grating that is enclosed within the baffle structure is used to tune
ANALYTICAL CHEMISTRY, VOL. 47, NO. 2 , FEBRUARY 1975
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and select the desired wavelength from the dye laser. A felt-lined cover is mounted to the top half of the grating (not shown in Figure 2 ) . This cover is used to connect the light-baffle structure to the grating via a flexible felt tube. This flexible felt tube allows rotation of the grating while the main light baffle remains fixed, and the overall baffle system provides optical isolation of the dye laser radiation from the rest of the monochromator. The dye laser pulses are directed out of the monochromator by the folding mirrors. These dye laser pulses can then be used to excite the fluorescent species. Specifically, the dye laser pulses are directed back toward the entrance slit of the same monochromator (See Figure 3). A sample cell is positioned a t the entrance port so that the fluorescence radiation from the sample is collected a t a 90° angle from the incident laser radiation. The fluorescent radiation enters the monochromator and is directed by the folding and parabolic mirrors onto the grating. The lower half of the grating has been left exposed but isolated from the top half by the light baffle. The lower half of the grating then disperses the fluorescence radiation and directs it toward the second parabolic mirror. Since the dye laser section is optically isolated from the bottom half of the monochromator, the radiation present at the focal plane of the spectrometer is the fluorescence from the sample. In this manner a single monochromator has been used to isolate the excitation wavelength of the dye laser and the emission wavelengths of the sample fluorescence. Observation of Fluorescence Radiation at Standard Exit Slit. Normally the optical arrangement of a monochromator directs the wavelength selected by the grating through the exit slit. In the monochromator previously described, this would correspond to the dye laser excitation wavelength since the grating is first used to tune the dye laser; however, by rotating the parabolic mirrors slightly, the selected dye laser wavelength can be moved off center 272
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and higher wavelengths corresponding to the sample fluorescence can be directed through the exit slit. With the standard grating employed in this monochromator (1180 lines/mm), a dispersion of 2 nm/mm is observed. By using a ruled grating of fewer lines (590 lines/ mm, grating assembly EU-700-3 GCA/McPherson), the dispersion is decreased to 4 nm/mm, and since the parabolic mirrors are 5 cm wide, a total spectral range of 200 nm exists a t the imaged focal plane of the exit slit. By positioning the dye laser excitation wavelenth a t the,extreme edge of this focal plane, the higher wavelength fluorescence, up to 200 nm from the excitation wavelength, can be directed through the exit slit. To obtain a fluorescence spectrum through the exit slit, either the grating or the parabolic mirror must be rotated so as to position sequential segments of the fluorescence radiation a t the exit slit. Scanning the grating is not possible since it must remain fixed to select the excitation wavelength from the dye laser. The parabolic mirror can be rotated, but this would require significant modifications within the monochromator. Therefore, alternative methods for obtaining the emission spectra were developed that could be readily implemented. Photographic Fluorescence Spectra. By inserting a reflecting plane mirror within the monochromator, the exit folding mirror and slit are bypassed and the lower half of the optical system becomes a polychromator which is useful for obtaining fluorescence spectra. We utilized this optical arrangement to obtain spectra by using an accessory that is attached to the cover as shown in Figure 4. The accessory positions a folding mirror, 5 cm wide, between the parabolic mirror and the normal fo!ding mirror located in front of the exit slit. This additional mirror directs the entire 200-nm spectral band from the parabolic mirror onto a
ANALYTICAL CHEMISTRY, VOL. 47, N O . 2, FEBRUARY 1975
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photographic film. This additional mirror is located such that the radiation from the parabolic, mirror is directed on the photographic film a t the same distance and in the same way as to the exit slit, except that the entire spectral range of 200 nm is recorded on the film, whereas the exit slit selects only a small segment of the spectral range for observation. The dye laser excitation wavelength is directed to one edge of the film and the linearly dispersed fluorescence radiation is recorded across the film, providing a permanent record of the fluorescence spectra. Analysis of the exposed film on a densitometer can provide the qualitative structure of the fluorescence band as well as the establishment of a calibration curve (using the maximum emission wavelength) for quantitative analysis. Though this approach is useful, it does require additional equipment and time for obtaining fluorescence information; in particular. emulsion calibration would be plagued by the intermittancy effect and would require pulsed source calibration (25). A vidicon or array detector could, of course, be used instead of the photographic film; however, these detectors require considerable auxiliary equipment, and a t present the sensitivity is often insufficient (26, 27). Photometric Fluorescence Spectra. T o develop an attachment that could utilize the conventional P M tube, an alternate means of viewing the fluorescence spectra was devised. The existing folding mirror in front of the exit slit of the monochromator is readily removed by a single thumbscrew (see Figure 3). With the removal of the mirror, the focal plane is then in the plane of the front panel of the monochromator (28) which is also readily removed with four thumbscrews. Thus there is a straight optical path from the parabolic mirror to the front machined surface of the monochromator. By placing a detector on this front surface with suitable slit assemblies, it is possible to scan across the imaged focal plane and therefore scan the fluorescence. Again by positioning the dye laser excitation wavelength X,1 on the edge of the focal plane a spectral range (from Xd to A, the highest wavelength within the imaged focal plane) of 200 nm can be scanned. A new front panel is constructed to support a photomultiplier tube detect,or as shown in Figure 5. A slot is milled on the panel (l/z inch X 2% inches, positioned 1/2 inch from the right hand edge of the front surface) that corresponds to the imaged focal plane from the parabolic mirror. Two stainless steel rods are mounted on the panel to provide support and a track for the scanning photomultiplier tube housing. Two nylon bushings are attached to the housing and provide a smooth travel for the detector along the stainless steel tracks. One end of the housing is springloaded on the track, and a 2-inch micrometer is positioned on the opposite end to provide precise and smooth movement of the detector across the milled slot and focal plane
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of radiation. Apertures of various sizes can be mounted in front of the photomultiplier tube a t the focal plane so as to provide the desired resolution. I t is then possible to scan over a 200-nm range to obtain the fluorescence emission spectra. With the addition of a stepper motor, a digital drive can be added to the micrometer mount providing for an automated digital scan. The scan could be operated either independently of the excitation wavelength selection, providing an automated emission scan or, by using the same wavelength scan control unit for both the micrometer and grating drives, be synchronized with the grating rotation and provide the means to obtain excitation spectra. This synchronous scan would allow the emission maximum wavelength to be tracked and detected as it moves across the imaged focal plane when the grating rotates. Because of the large spectral range of the focal plane (200 nm), the entire excitation spectrum could be recorded as a function of the emission maximum wavelength intensity. The automated scan could be used to record the entire emission spectrum as a function of each excitation wavelength selected. This would eliminate any dependence upon previous knowledge of the emission maximum wavelength and its location in the focal plane, as well as record any shift in the emission maximum wavelength as the excitation wavelength is varied. Quantitative analysis is possible by locating the emission maximum of the fluorescence either through a qualitative scan or by available data. This selected wavelength region can then be used to obtain analytical curves and the sample data. Although these quantitative data would be obtained with the normal optical arrangement of the monochromator, using the folding mirror and the exit slit, it would then be necessary to rebuild the parabolic mirror mount and provide a calibrated drive. As stated earlier, this approach would be more difficult to implement, whereas with the scanning detector the implementation is concentrated outside the monochromator in an attachment that can be easily added or removed. Specific Apparatus. The dye laser excitation source and polychromator for obtaining fluorescence spectra was made by adding accessories to a GCA/McPherson Ell-700 programmable monochromator (GCA/McPherson, Acton, Mass.). The laser output mirror was obtained from Laser Optics (Laser Optics, Danbury, Conn.) and has an ultraviolet coating (LH 11) that enables lasing action to be produced down to 358 nm. A Sorenson power supply (Raytheon Company, Norwalk, Conn., model number 1020-30)
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Figure 6. Photographic spectra of riboflavin (top) and sodium f l u e rescein (bonom) [4-methylumbeiliferrone (left) used as excltatiin dye-excitation wavelengths 474 and 469 nm, respectively]
was used to power the nitrogen laser pump. The additional electronics and operating parameters of the dye laser system have been reported elsewhere (23). The photographic detection system included the film mount and folding mirror, a Polaroid Land film holder (No. 500) and Polaroid type 47, 4 X 5 Land film. This film was used to obtain prints of the fluorescence band. Polaroid type 46 or 146L Land projection film (transparencies) are available if intensities are to he determined using a densitometer. The electronic detection system used a RCA-1P28 photomultiplier tube. with an operating voltage of 500-900 volts. The output of the photomultiplier tnhe was fed to a high-speed charge-to-count data domain converter developed by Woodruff and Malmstadt (29) which integrates and digitizes the incoming charge packets from the photomultiplier tube. Since the dye laser pulses and the flnorescence signals are on the order of 10 nanoseconds in duration, normal analog detection methods do not respond fast enough. As a result, detection methods such as the box-car integrator used by Fraser and Winefordner (10) or the charge-to-count converter (q-to-n) used in this work are necessary. The digitized information is then sent to a teletypewriter which produces a hard copy of the measured fluorescence intensity. General Procedure. For qualitative analysis using either the photographic or photometric detector, the dye laser excitation wavelength is tuned close to the absorption maximum of the sample, without overlapping the fluorescence hand spectra. The dye laser excitation wavelength is positioned at the extreme edge of the imaged focal plane, thereby allowing the higher wavelength fluorescence to be detected . The photographic detector requires only a few pulses of the dye laser to record the fluorescence radiation, and a single pulse of laser radiation is sufficient to expose the film. The dye laser excitation wavelength is recorded on the left portion of the photograph. Even though the entrance slit of the monochromator was very narrow (50 f i ) , the laser radiation is so intense that a broadened exposure spot results. When using the photometric detector, laser radiation scattered from the front surface of the sample is collected and positioned at the extreme edge of the observed focal plane of radiation, rather than the actual dye laser pulse. This is done since the dye laser pulse is several kilowatts peak power and direct exposure of the photomultiplier tube is harmful. A 1-mm aperture was used as the exit port in this study so as to provide a resolution of 4 nm. The in274
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Figure 7. Photographic spectra of anthracene (top) and quinine suC fate (bottm) [PED (left) used as excitation dye-excitation wave length 378 nm]
wavelength as determined by the position of the photomnltiplier attachment. The dye laser wavelength is located by scanning to the edge of the focal plane corresponding to the lower wavelengths. Then a scan of the high wavelength flnorescence is completed. This is done in increments ranging from 0.5-4.0 nm by step positioning the photomultiplier tube attachment. By knowing the wavelength of the dye laser (reference point) and the linear dispersion of the grating (4 nmlmm) one is able to indicate over what wavelengths the fluorescence occurs and locate the emission maximum. By locating the emission peak (or peaks) of a fluorescent sample, one is then able to perform quantitative analysis for a specific constituent. Using the scanning photomultiplier assembly, this is easily accomplished hy first scanning the fluorescence and then positioning the detector at the maximum emission wavelength. The detector movement is 0.0635 mmlrevolution of the micrometer, which is graduated into 25 divisions per revolution. Since the nominal reciprocal dispersion is 4 nmlmm with the 600 linelmm grating, one revolution of the micrometer is equivalent to a spectral displacement of 0.25 nm. Excitation spectra can he obtained by maintaining a constant wavelength of emission, which is usually a t the maximum emission (15).
RESULTS AND DISCUSSION
Photographic Fluorescence SDeetra. The Dhotogralihir attnchmenr waa w e d t u ohriiin the fluorescence Qmiislotl spertra o f Figurea C? and 7 . 'I'he Cprvtra in Figure 6 were o1)taint.d from solurions of acrdium tluorrscein (-10-'M in 0.034 Na?HPO,) and rtboilavin t-1U 544 ~n I
