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Biochemical applications of a synchronously pumped krypton ion dye laser fluorescence system
ANALYTICAL
BIOCHEMISTRY
Biochemical
97, 17-23 (1979)
Applications of a Synchronously Pumped Dye Laser Fluorescence System1~2~3
Krypton
Ion
J. H. RICHARDSON, L. L. STEINMETZ,~. B. DEUTSCHER, W. A. BOOKLESS,AND W. L. SCHMELZINGER Lawrence
Livermore
Laboratory,
University
of California,
Livermore,
California
94550
Received October 12, 1978 A synchronously pumped krypton ion dye laser fluorescence system is shown to provide tunable, polarized, subnanosecond pulses at high repetition rates, modest peak powers, and low energy. Such a source is uniquely suited to fluorescence investigations of biochemical mechanisms. Applications of this fluorescence excitation source to analysis, lifetime determination, and depolarization effects are discussed.
Conventional fluorescence analysis finds many applications in biochemistry because of its versatility and the wealth of information obtainable (1). The introduction of laser technology has greatly enhanced the power of fluorescence analysis by increasing the sensitivity (2) and temporal resolution (3). However, these two parameters, sensitivity and temporal resolution, have been improved separately and are not in general simultane-
ously available. Consequently, there is a requirement for a laser system which incorporates both high sensitivity and temporal resolution. High peak power or high repetition rate are required for sensitivity in the part-pertrillion range (4) but these two factors are mutually exclusive with current laser technology. Furthermore, many high peak power lasers are also high energy lasers, which may have unfortunate repercussions in sample degradation or nonlinear effects. * Work performed under the auspices of the U. S. Mode locking of lasers provides for subDepartment of Energy by the Lawrence Livermore Laboratory under Contract W-7405-ENG-48. This re- nanosecond temporal resolution (39, but port was prepared as an account of work sponsored by has been either limited in repetition rate or the United States Government. Neither the United tunability. Passive mode-locking techniques States nor the United States Energy Research & Deare limited by the spectral properties of velopment Administration, nor any of their employees, the saturable absorber and have been nor any of their contractors, subcontractors, or their employees, makes any warranty, express or implied, or primarily confined to the rhodamine dyes assumes any legal liability or responsibility for the and hence the orange-red portion of the accuracy, completeness or usefulness of any informavisible spectrum (6). Mode-locked solid state tion, apparatus, product or process disclosed, or lasers are usually high energy, single shot, represents that its use would not infringe privately or low repetition rate illumination sources, owned rights. * Taken in part from a presentation given at the 176th although they can be made tunable with American Chemical Society National Meeting, Miami ultrashort cavity dye lasers (7). A nearly Beach, Fla., September 1 l- 15, 1978. ideal source appears to be a synchronously ’ Reference to a company or product name does not imply approval or recommendation of the product by pumped dye laser (8), which has high repetithe University of California or the U. S. Department of tion rates, moderate peak power, low energy, Energy to the exclusion of others that may be suitable. and picosecond pulse widths. However, it 17
0003-2697/79/110017-07$02.00/0 Copyright All rights
8 1979 by Academic F’ress, Inc. of reproduction m any form reserved.
18
RICHARDSON
ET AL.
too has been limited to the orange-red portion of the spectrum because of the limitations of the argon ion pump laser. A krypton ion laser has many more lines than an argon ion laser; consequently a krypton ion laser can be used to pump various dyes and produce tunable cw lasting throughout the visible portion of the spectrum. In most applications the slightly lower power associated with the krypton lines is of little or no consequence. The near uv lines of the krypton ion laser have been recently mode-locked (9); thus, it is now possible to synchronously pump coumarin Light tight dyes and thereby achieve sensitivity and sample chamber temporal resolution in the blue-green portion of the visible spectrum. We have also mode-locked the other major lines in the XY recorder krypton ion laser; hence it is feasible to provide tunable, picosecond pulses of high FIG. I. Schematic of the synchronously pumped repetition rate, low energy, and moderate krypton ion dye laser fluorescence system. peak power (suitable for doubling) throughout the visible and near infrared spectrum sated goat anti-human IgA,4 IgG, and IgM with one single laser system. The output with the respective antigen controls (Cappel). provides an ideal excitation source for Fluorescamine was reacted with represensitive, time-resolved fluorescence anal- sentative amino acids and proteins according ysis. to the recommended procedure (11). Borate In this paper we report the characteristics buffer solutions were used at either pH 8.0 of the krypton ion synchronously pumped (peptides) or 9.0 (amino acids and proteins); coum~n dye laser adapted for fluorescence typical concentrations were 5 x fO+ AI. analysis. Examples of sensitivity, polarizaThe fluorescein-conjugated antibodies and tion, and lifetime studies are taken from the respective antigens (IgG, IgM, and IgA) fluorescent-labeled proteins and amino were reconstituted as directed and then acids. diluted 1: 50 in buffer (pH 7 .O). Consequently, the resultant total protein concentration MATERIALS AND METHODS was ostensibly 0.4, 0.6, and 0.4 m&ml fluorescein-conjugated anti-human IgG, IgM, Riboflavin (Eastman Kodak) was recrystal- and IgA, respectively, with the antibody lized twice from 1 M acetic acid (10). All protein somewhat less. The antigen solutions other materials were obtained from com- were 0.2 mg/ml. Antibody-antigen solutions mercial sources and used without further were mixed 1:4 and incubated for at least purification: fluorescamine (Roche Diag- 10 min at 37”C, but no attempt was made nostics), acetone (Burdick and Jackson), to assay the extent of binding. arginine, glycineglycine (Calbiochem), tyroFigure 1 is a schematic of the experimental sine, tryptophane (Kodak), human serum apparatus. A Spectra Physics 171K was albumin (Cutter) glycine, polyarginine (Sigma), ~l~henyl~~e (Pilot), fluorescein * Abbreviations used: Ig, immuno~obulin; HSA, human serum albumin; cw, continuous wave. (Kromex- Baker), and fluorescein-conju-
cl
SYNCHRONOUSLY
PUMPED
KRYPTON
mode-locked with the Spectra Physics 361A acoustic modulator. The strongest near uv line was at 413.1 nm, with 1.3-W cw and typically 300 mW average power when mode-locked. The mode-looked output at 413.1 nm is a useful excitation source in its own right and was used directly for exciting the fluorescamine-labeled samples. Typical pulsewidths were about 80 ps, with a fixed repetition rate of 88 MHz. The excitation rate was reduced to 40 kHz through the use of a Bragg cell. This acoustooptic device was positioned external to the laser cavity, selecting a variable number of pulses at a variable repetition rate determined by the pulse generator. Because the pulse generator and the modelocker are not synchronized, occasionally portions of the leading or trailing pulse were included in the picked-off portion. It is possible to synchronize the pulse picking with the train of pulses through the use of a programmable counter (4). For the applications discussed in this paper, however, that further refinement was not necessary, It would be necessary when dealing with longer fluorescence lifetimes and/or a more sophisticated time-correlated photon-~ounting system (the boxcar trigger level discriminated against smaller pre- and postpulses).
ION DYE LASER FLUORESCENCE
A portion of the down converted pulse train is used to trigger a Princeton Applied Research 162/163 boxcar integrator with a Tektronix A-l sampling head (350 ps risetime). The temporal resolution, approximately 1.5 + 0.2 ns, was determined primarily by the fall time of the photomultiplier tube (jitter in the boxcar timebase is less than 0.1 ns). The five-stage photomultiplier tube, RCA C3 1024, was wired for maximum speed (12) while maint~ning reasonable gain. Schott RV450 and 550 filters were used to separate the fluorescence from scattered light. A Spectra Physics 375 jet stream dye laser was used to obtain tunable output. The output coupler was positioned at the proper distance such that the dye laser cavity length equalled that of the krypton ion laser. The output coupler was a 1.58-m radius of curvature mirror, ca. 2% transmission coated for either the blue or green potion of the spectrum. Figure 2a illustrates the typical pulse train from the dye laser TIX56 avalanche photodiode, oscilloscope limited to ca. 1.5 ns FWHM, while Fig. 2b illustrates a typical single pulse (sampling oscilloscope, detector limited to approximately 80 ps FWHM). Photographs with a streak camera of an argon synchronously pumped dye laser suggest that the pulsewidth is less than 25 ps. Re!ative
I
a
I
I
i
I
Time
I.
19
SYSTEM
I
I
b
I
I
Amplitude
I
I_
I
I
/
Time
FIG. 2. Typical output from the laser excitation source (A = 503 nm, (P ) = 30 mW): (a) pulse train, pulsewidth limited by the oscilloscope; (b) single picked pulse, pulsewidth limited by the detector.
I
1
20
RICHARDSON I
I
ET AL.
I I
I
I
480
/\ 500
I
1.0
1.0 -
> C E
> .!Z ; 8
E 0.5
5
0
0.5-
0-, 480
a
500 Wavelength
FIG.
520
540
(nm)
b
520
Wavelength
540
560
(nml
3. Tuning for coumarin 30, synchronously pumped: (a) blue optics; (b) green optics.
Tuning curves for coumarin 30 are shown in Fig. 3. The threshold for synchronous pumping is about 120 mW average power at 413.1 nm, with about 10% efficiency. Thus, typical operation is 30 to 40 mW average power, corresponding to approximately 0.5 nJ/pulse, ~2 PW average power incident on the sample, and >25 W peak power (depending on the pulsewidth). This is sufficient peak power for doubling. Finally, the tuning range available can be extended toward the blue with coumarin 102 and toward the green with coumarin 7 (13). Angle-tuned KDP crystals can be used to efficiently double (-5%) the dye output at wavelengths longer than ca. 520 nm (i.e., coumarin 7 and coumarin 30 with the green optics). An autocorrelator based on frequency doubling with coumarin 30 yielded a pulsewidth of approximately 15 ps (14). This pulsewidth could undoubtedly be further shortened by stabilizing the dye cavity length and using a piezoelectric rather than a micrometer for fine adjustment of the cavity length. Doubling shorter wavelengths is currently inefficient at best, either with temperature tuning (limited wavelength region) or angle tuning (PB5 or lithium formate). Intracavity doubling would be a more reasonable approach but requires high gain and special coatings for output coupling.
We have also mode locked the other major lines in the krypton ion laser which are even more powerful than 413.1; consequently, with doubling capability it should be possible to generate continuous tunable picosecond pulses from the uv to the near ir which are ideally suited for fluorescence analysis (low energy, high repetition rate, and peak power) with one single laser system. RESULTS
Riboflavin and fluorescein (pH 9.0, 1 x lop5 M) were used as lifetime standards, with measured values 4.4 and 4.8 + 0.2 ns, which agree well with literature values of 4.2 and 4.6 ns, respectively (15). The results for the lifetime measurements for several fluorescamine-labeled amino acids, peptides, and proteins are listed in Table 1. All standard deviations are kO.2 ns. As the experimental data after the first couple of nanoseconds were fitted by a straight line very well on a semilog plot all the lifetimes were corrected for the photomultiplier tube response by a convolute and compare procedure. Known exponential functions were numerically convoluted with the PMT response and compared graphically with the experimental data. Deviations from the true fluorescence lifetime were attributable to
SYNCHRONOUSLY
PUMPED
KRYPTON
ION
the photomultiplier tube response. The excitation pulse is essentially a 6 function (~80 ps) and the boxcar jitter and risetime is all much less than 1 ns, whereas the photomultiplier tube response is 1.5 ns (l/e decay, not FWHM). There was no correction of lifetimes greater than 4.5 ns, the correction smoothly increasing to 0.6 ns for a 2.0-ns decay. Lifetimes shorter than 1.0 ns cannot be measured by this technique, but would require either a time-correlated photon counting system or, more ideally, a synchronized streak camera. The advantages of boxcar detection for fluorescence lifetimes are the relatively low cost and rapidity of data acquisition. The observed lifetimes for the amino acids are generally in good agreement with those recently reported by Chen and co-workers (16). The value for the dipeptide Gly-Gly fits neatly between that of Gly and Gly-Gly-Gly. In both HSA and poly-Arg there is less interaction with polar quenchers than in a simple amino acid; consequently the lifetime increases, as seen previously with other proteins (17). The lifetime of fluorescamine-labeled tryptophan was too short to be determined accurately. We have previously demonstrated that a high repetition rate, low peak power laser with laser parameters similar to those in the synchronously pumped krypton ion dye laser system can compete favorably with a low repetition rate, high peak power laser for achieving part-per-trillion detection levels (4). Furthermore, laser-induced fluorescence of fluorescamine-labeled amino acids has been shown to have several parts-per-trillion detection limits (18). Consequently, the synchronously pumped krypton ion dye laser system offers improved temporal resolution with comparable sensitivity to a high peak power laser system. Finally, the analysis time is reduced by using analog instead of digital data acquisition with no loss in sensitivity or temporal resolution. The improved temporal and polarization properties of this laser system also permit the study of the binding of fluorophors to
DYE
LASER
FLUORESCENCE TABLE
SYSTEM
21
1
LIFETIMES OF FLUORESCAMINELABELED BIOCHEMICALS Lifetime Compound Arg GlY Trp Tyr Gly-Gly Poly-Arg HSA
Measured 4.6 3.9 1.8 4.6 6.2 5.0 6.7
(ns) Corrected 4.6 3.8 Cl.5 4.6 6.2 5.0 6.7
large molecules and the resulting depolarization effects. Figure 4a represents the temporal and polarization decay following irradiation of a Ludox sample (Rayleigh scattering). Figure 4b represents the same experimental arrangement but with a solution of fluorescein-labeled anti-human IgM/ human IgM. From this data the emission anisotropy function can be calculated, and hence the rotational correlation time. The short lifetime of fluorescein prevents good data from being taken for a molecule with either a single long correlation time or multiple correlation times. However, a calculation of the rotational correlation time for the anti-IgM/IgM solution shown in Fig. 4b yields a value of 70 ns which is compatible with values observed for IgG (15,19). There was noticeable change in lifetime between fluorescein, fluorescein-labeled antibody, and the subsequent antibodyantigen complex. No fluorescence was detected for either antigen solution (IgG or IgM). With IgG the corrected values for the fluorescence lifetime of labeled antibody and antibody-antigen complex were 3.0 and 3.9 ns, respectively; with IgM the corresponding values were 3.3 and 3.8 ns, respectively. Similar results were obtained with IgA, although the signal-to-noise and reproducibility were noticeably inferior. These results underline the importance of using
22
RICHARDSON
I
a
I
I
I
I
I
I
ET AL.
-
Time
FIG. 4. Time resolved polarization spectra, excitation source vertically polarized; horizontally polarized spectra increased by 2.5 X: (a) scattering from Ludox solution; (b) fluorescence from incubated solution of fluorescein-conjugated anti-IgMiIgM.
the fluorophor lifetime when it is bound rather than the free fluorophor lifetime in polarization studies (15). (Our measured value for free fluorescein was 4.8 ns.) In summary, a synchronously pumped krypton ion dye laser system is a unique fluorescence excitation source. This one system provides polarized subnanosecond light pulses for time-resolved fluorescence polarization studies of the dynamics of biochemical mechanisms. The high repetition rate, low energy pulse train permits sufficient sensitivity to study reactions at in viva concentration levels without sample decomposition or nonlinear effects (the absence of intensity loss with time and agreement with literature lifetime values suggests that there is negligible decomposition or stimulated emission, although specific samples would have to be considered individually). Sufficient peak power is present to permit frequency doubling, consequently the uv down to ca. 260 nm as well as the entire visible spectrum can be accessed with one system.
ACKNOWLEDGMENTS We would like to thank B. W. Wallin for technical assistance in the early stages of this work, M. A. Revelli for assistance with the computer calculations, and M. A. Laskaris for some of the amino acids.
REFERENCES 1. Chen, R. F., and Edelhoch, H. (eds.) (1975) Biochemical Fluorescence: Concepts, Dekker, New York. 2. Richardson, J. H., and Ando, M. E. (1977) Anal. Chem. 49, 955-959. 3. Cramer, L. E., and Spears, K. G. (1978) J. Amer. Chem. Sm. 100,221-227. 4. Richardson, J. H.. and George, S. M. (1978)Anal. Chem. 50, 616-620. 5. Fleming, G. R., Knight, A. E. W., Morris, J. M., Morrison, R. J. S., and Robinson, G. W. (1977) J. Amer. Chem. Sot. 99, 4306-4311. Shank, C. V., and Ippen, E. P. (1974) Appl. Phys. Left. 24, 373-375. Cox, A. J., Scott, G. W., and Talley, L. D. (1977) Appl. Phys. Left. 31, 389-391. Harris, J. M., Gray, L. M., Pelletier, M. J., and Lytle, F. E. (1977) Mol. Photochem. 8,161- 174. Steinmetz, L. L., Richardson, J. H., and Wallin, B. W. (1978) Appl. Phys. Lett. 33, 163- 165.
SYNCHRONOUSLY
PUMPED KRYPTON
ION DYE LASER FLUORESCENCE
10. Smith, E. C., and Metzler, D. E. (1963)J. Amer. Chem. Sec. 85,3285-3288. 11. de Bemardo, S., Weigele, M., Toome, V., Manhart, K., Leimgrnber, W., Bohlen, P., Stein, S., and Udenfiiend, S. (1974) Arch. Biochem. Biophys. 163, 390-399. 12. Leskovar, B., and Lo, C. C. (1975) Nucl. Instr. Methods 123, 145- 160. 13. Steinmetz, L. L., Richardson, J. H., and Wallin, B. W. (1978) Presented at the Topical Meeting on Picosecond Phenomena, May 1978, Hilton Head, S. C.
SYSTEM
23
14. Steinmetz, L. L., and Bookless, W. A., manuscript in preparation. 15. Danliker, W. B., and de Saussure, V. A. (1970) Immunochemistry 7,799-828. 16. Chen, R. F., Smith, P. D., and Maly, M. (1978) Arch. Biochem. Biophys. 189, 241-250. 17. Chen, R. F. (1974) Anal. Left. 7, 65-77. 18. Richardson, J. H. (1977) Anal. Biochem. 83, 754-762. 19. Yguerabide, J., Epstein, H. F., and Stryer, L. (1970) J. Mol. Bioi. 51, 573-590.
BIOCHEMISTRY
Biochemical
97, 17-23 (1979)
Applications of a Synchronously Pumped Dye Laser Fluorescence System1~2~3
Krypton
Ion
J. H. RICHARDSON, L. L. STEINMETZ,~. B. DEUTSCHER, W. A. BOOKLESS,AND W. L. SCHMELZINGER Lawrence
Livermore
Laboratory,
University
of California,
Livermore,
California
94550
Received October 12, 1978 A synchronously pumped krypton ion dye laser fluorescence system is shown to provide tunable, polarized, subnanosecond pulses at high repetition rates, modest peak powers, and low energy. Such a source is uniquely suited to fluorescence investigations of biochemical mechanisms. Applications of this fluorescence excitation source to analysis, lifetime determination, and depolarization effects are discussed.
Conventional fluorescence analysis finds many applications in biochemistry because of its versatility and the wealth of information obtainable (1). The introduction of laser technology has greatly enhanced the power of fluorescence analysis by increasing the sensitivity (2) and temporal resolution (3). However, these two parameters, sensitivity and temporal resolution, have been improved separately and are not in general simultane-
ously available. Consequently, there is a requirement for a laser system which incorporates both high sensitivity and temporal resolution. High peak power or high repetition rate are required for sensitivity in the part-pertrillion range (4) but these two factors are mutually exclusive with current laser technology. Furthermore, many high peak power lasers are also high energy lasers, which may have unfortunate repercussions in sample degradation or nonlinear effects. * Work performed under the auspices of the U. S. Mode locking of lasers provides for subDepartment of Energy by the Lawrence Livermore Laboratory under Contract W-7405-ENG-48. This re- nanosecond temporal resolution (39, but port was prepared as an account of work sponsored by has been either limited in repetition rate or the United States Government. Neither the United tunability. Passive mode-locking techniques States nor the United States Energy Research & Deare limited by the spectral properties of velopment Administration, nor any of their employees, the saturable absorber and have been nor any of their contractors, subcontractors, or their employees, makes any warranty, express or implied, or primarily confined to the rhodamine dyes assumes any legal liability or responsibility for the and hence the orange-red portion of the accuracy, completeness or usefulness of any informavisible spectrum (6). Mode-locked solid state tion, apparatus, product or process disclosed, or lasers are usually high energy, single shot, represents that its use would not infringe privately or low repetition rate illumination sources, owned rights. * Taken in part from a presentation given at the 176th although they can be made tunable with American Chemical Society National Meeting, Miami ultrashort cavity dye lasers (7). A nearly Beach, Fla., September 1 l- 15, 1978. ideal source appears to be a synchronously ’ Reference to a company or product name does not imply approval or recommendation of the product by pumped dye laser (8), which has high repetithe University of California or the U. S. Department of tion rates, moderate peak power, low energy, Energy to the exclusion of others that may be suitable. and picosecond pulse widths. However, it 17
0003-2697/79/110017-07$02.00/0 Copyright All rights
8 1979 by Academic F’ress, Inc. of reproduction m any form reserved.
18
RICHARDSON
ET AL.
too has been limited to the orange-red portion of the spectrum because of the limitations of the argon ion pump laser. A krypton ion laser has many more lines than an argon ion laser; consequently a krypton ion laser can be used to pump various dyes and produce tunable cw lasting throughout the visible portion of the spectrum. In most applications the slightly lower power associated with the krypton lines is of little or no consequence. The near uv lines of the krypton ion laser have been recently mode-locked (9); thus, it is now possible to synchronously pump coumarin Light tight dyes and thereby achieve sensitivity and sample chamber temporal resolution in the blue-green portion of the visible spectrum. We have also mode-locked the other major lines in the XY recorder krypton ion laser; hence it is feasible to provide tunable, picosecond pulses of high FIG. I. Schematic of the synchronously pumped repetition rate, low energy, and moderate krypton ion dye laser fluorescence system. peak power (suitable for doubling) throughout the visible and near infrared spectrum sated goat anti-human IgA,4 IgG, and IgM with one single laser system. The output with the respective antigen controls (Cappel). provides an ideal excitation source for Fluorescamine was reacted with represensitive, time-resolved fluorescence anal- sentative amino acids and proteins according ysis. to the recommended procedure (11). Borate In this paper we report the characteristics buffer solutions were used at either pH 8.0 of the krypton ion synchronously pumped (peptides) or 9.0 (amino acids and proteins); coum~n dye laser adapted for fluorescence typical concentrations were 5 x fO+ AI. analysis. Examples of sensitivity, polarizaThe fluorescein-conjugated antibodies and tion, and lifetime studies are taken from the respective antigens (IgG, IgM, and IgA) fluorescent-labeled proteins and amino were reconstituted as directed and then acids. diluted 1: 50 in buffer (pH 7 .O). Consequently, the resultant total protein concentration MATERIALS AND METHODS was ostensibly 0.4, 0.6, and 0.4 m&ml fluorescein-conjugated anti-human IgG, IgM, Riboflavin (Eastman Kodak) was recrystal- and IgA, respectively, with the antibody lized twice from 1 M acetic acid (10). All protein somewhat less. The antigen solutions other materials were obtained from com- were 0.2 mg/ml. Antibody-antigen solutions mercial sources and used without further were mixed 1:4 and incubated for at least purification: fluorescamine (Roche Diag- 10 min at 37”C, but no attempt was made nostics), acetone (Burdick and Jackson), to assay the extent of binding. arginine, glycineglycine (Calbiochem), tyroFigure 1 is a schematic of the experimental sine, tryptophane (Kodak), human serum apparatus. A Spectra Physics 171K was albumin (Cutter) glycine, polyarginine (Sigma), ~l~henyl~~e (Pilot), fluorescein * Abbreviations used: Ig, immuno~obulin; HSA, human serum albumin; cw, continuous wave. (Kromex- Baker), and fluorescein-conju-
cl
SYNCHRONOUSLY
PUMPED
KRYPTON
mode-locked with the Spectra Physics 361A acoustic modulator. The strongest near uv line was at 413.1 nm, with 1.3-W cw and typically 300 mW average power when mode-locked. The mode-looked output at 413.1 nm is a useful excitation source in its own right and was used directly for exciting the fluorescamine-labeled samples. Typical pulsewidths were about 80 ps, with a fixed repetition rate of 88 MHz. The excitation rate was reduced to 40 kHz through the use of a Bragg cell. This acoustooptic device was positioned external to the laser cavity, selecting a variable number of pulses at a variable repetition rate determined by the pulse generator. Because the pulse generator and the modelocker are not synchronized, occasionally portions of the leading or trailing pulse were included in the picked-off portion. It is possible to synchronize the pulse picking with the train of pulses through the use of a programmable counter (4). For the applications discussed in this paper, however, that further refinement was not necessary, It would be necessary when dealing with longer fluorescence lifetimes and/or a more sophisticated time-correlated photon-~ounting system (the boxcar trigger level discriminated against smaller pre- and postpulses).
ION DYE LASER FLUORESCENCE
A portion of the down converted pulse train is used to trigger a Princeton Applied Research 162/163 boxcar integrator with a Tektronix A-l sampling head (350 ps risetime). The temporal resolution, approximately 1.5 + 0.2 ns, was determined primarily by the fall time of the photomultiplier tube (jitter in the boxcar timebase is less than 0.1 ns). The five-stage photomultiplier tube, RCA C3 1024, was wired for maximum speed (12) while maint~ning reasonable gain. Schott RV450 and 550 filters were used to separate the fluorescence from scattered light. A Spectra Physics 375 jet stream dye laser was used to obtain tunable output. The output coupler was positioned at the proper distance such that the dye laser cavity length equalled that of the krypton ion laser. The output coupler was a 1.58-m radius of curvature mirror, ca. 2% transmission coated for either the blue or green potion of the spectrum. Figure 2a illustrates the typical pulse train from the dye laser TIX56 avalanche photodiode, oscilloscope limited to ca. 1.5 ns FWHM, while Fig. 2b illustrates a typical single pulse (sampling oscilloscope, detector limited to approximately 80 ps FWHM). Photographs with a streak camera of an argon synchronously pumped dye laser suggest that the pulsewidth is less than 25 ps. Re!ative
I
a
I
I
i
I
Time
I.
19
SYSTEM
I
I
b
I
I
Amplitude
I
I_
I
I
/
Time
FIG. 2. Typical output from the laser excitation source (A = 503 nm, (P ) = 30 mW): (a) pulse train, pulsewidth limited by the oscilloscope; (b) single picked pulse, pulsewidth limited by the detector.
I
1
20
RICHARDSON I
I
ET AL.
I I
I
I
480
/\ 500
I
1.0
1.0 -
> C E
> .!Z ; 8
E 0.5
5
0
0.5-
0-, 480
a
500 Wavelength
FIG.
520
540
(nm)
b
520
Wavelength
540
560
(nml
3. Tuning for coumarin 30, synchronously pumped: (a) blue optics; (b) green optics.
Tuning curves for coumarin 30 are shown in Fig. 3. The threshold for synchronous pumping is about 120 mW average power at 413.1 nm, with about 10% efficiency. Thus, typical operation is 30 to 40 mW average power, corresponding to approximately 0.5 nJ/pulse, ~2 PW average power incident on the sample, and >25 W peak power (depending on the pulsewidth). This is sufficient peak power for doubling. Finally, the tuning range available can be extended toward the blue with coumarin 102 and toward the green with coumarin 7 (13). Angle-tuned KDP crystals can be used to efficiently double (-5%) the dye output at wavelengths longer than ca. 520 nm (i.e., coumarin 7 and coumarin 30 with the green optics). An autocorrelator based on frequency doubling with coumarin 30 yielded a pulsewidth of approximately 15 ps (14). This pulsewidth could undoubtedly be further shortened by stabilizing the dye cavity length and using a piezoelectric rather than a micrometer for fine adjustment of the cavity length. Doubling shorter wavelengths is currently inefficient at best, either with temperature tuning (limited wavelength region) or angle tuning (PB5 or lithium formate). Intracavity doubling would be a more reasonable approach but requires high gain and special coatings for output coupling.
We have also mode locked the other major lines in the krypton ion laser which are even more powerful than 413.1; consequently, with doubling capability it should be possible to generate continuous tunable picosecond pulses from the uv to the near ir which are ideally suited for fluorescence analysis (low energy, high repetition rate, and peak power) with one single laser system. RESULTS
Riboflavin and fluorescein (pH 9.0, 1 x lop5 M) were used as lifetime standards, with measured values 4.4 and 4.8 + 0.2 ns, which agree well with literature values of 4.2 and 4.6 ns, respectively (15). The results for the lifetime measurements for several fluorescamine-labeled amino acids, peptides, and proteins are listed in Table 1. All standard deviations are kO.2 ns. As the experimental data after the first couple of nanoseconds were fitted by a straight line very well on a semilog plot all the lifetimes were corrected for the photomultiplier tube response by a convolute and compare procedure. Known exponential functions were numerically convoluted with the PMT response and compared graphically with the experimental data. Deviations from the true fluorescence lifetime were attributable to
SYNCHRONOUSLY
PUMPED
KRYPTON
ION
the photomultiplier tube response. The excitation pulse is essentially a 6 function (~80 ps) and the boxcar jitter and risetime is all much less than 1 ns, whereas the photomultiplier tube response is 1.5 ns (l/e decay, not FWHM). There was no correction of lifetimes greater than 4.5 ns, the correction smoothly increasing to 0.6 ns for a 2.0-ns decay. Lifetimes shorter than 1.0 ns cannot be measured by this technique, but would require either a time-correlated photon counting system or, more ideally, a synchronized streak camera. The advantages of boxcar detection for fluorescence lifetimes are the relatively low cost and rapidity of data acquisition. The observed lifetimes for the amino acids are generally in good agreement with those recently reported by Chen and co-workers (16). The value for the dipeptide Gly-Gly fits neatly between that of Gly and Gly-Gly-Gly. In both HSA and poly-Arg there is less interaction with polar quenchers than in a simple amino acid; consequently the lifetime increases, as seen previously with other proteins (17). The lifetime of fluorescamine-labeled tryptophan was too short to be determined accurately. We have previously demonstrated that a high repetition rate, low peak power laser with laser parameters similar to those in the synchronously pumped krypton ion dye laser system can compete favorably with a low repetition rate, high peak power laser for achieving part-per-trillion detection levels (4). Furthermore, laser-induced fluorescence of fluorescamine-labeled amino acids has been shown to have several parts-per-trillion detection limits (18). Consequently, the synchronously pumped krypton ion dye laser system offers improved temporal resolution with comparable sensitivity to a high peak power laser system. Finally, the analysis time is reduced by using analog instead of digital data acquisition with no loss in sensitivity or temporal resolution. The improved temporal and polarization properties of this laser system also permit the study of the binding of fluorophors to
DYE
LASER
FLUORESCENCE TABLE
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21
1
LIFETIMES OF FLUORESCAMINELABELED BIOCHEMICALS Lifetime Compound Arg GlY Trp Tyr Gly-Gly Poly-Arg HSA
Measured 4.6 3.9 1.8 4.6 6.2 5.0 6.7
(ns) Corrected 4.6 3.8 Cl.5 4.6 6.2 5.0 6.7
large molecules and the resulting depolarization effects. Figure 4a represents the temporal and polarization decay following irradiation of a Ludox sample (Rayleigh scattering). Figure 4b represents the same experimental arrangement but with a solution of fluorescein-labeled anti-human IgM/ human IgM. From this data the emission anisotropy function can be calculated, and hence the rotational correlation time. The short lifetime of fluorescein prevents good data from being taken for a molecule with either a single long correlation time or multiple correlation times. However, a calculation of the rotational correlation time for the anti-IgM/IgM solution shown in Fig. 4b yields a value of 70 ns which is compatible with values observed for IgG (15,19). There was noticeable change in lifetime between fluorescein, fluorescein-labeled antibody, and the subsequent antibodyantigen complex. No fluorescence was detected for either antigen solution (IgG or IgM). With IgG the corrected values for the fluorescence lifetime of labeled antibody and antibody-antigen complex were 3.0 and 3.9 ns, respectively; with IgM the corresponding values were 3.3 and 3.8 ns, respectively. Similar results were obtained with IgA, although the signal-to-noise and reproducibility were noticeably inferior. These results underline the importance of using
22
RICHARDSON
I
a
I
I
I
I
I
I
ET AL.
-
Time
FIG. 4. Time resolved polarization spectra, excitation source vertically polarized; horizontally polarized spectra increased by 2.5 X: (a) scattering from Ludox solution; (b) fluorescence from incubated solution of fluorescein-conjugated anti-IgMiIgM.
the fluorophor lifetime when it is bound rather than the free fluorophor lifetime in polarization studies (15). (Our measured value for free fluorescein was 4.8 ns.) In summary, a synchronously pumped krypton ion dye laser system is a unique fluorescence excitation source. This one system provides polarized subnanosecond light pulses for time-resolved fluorescence polarization studies of the dynamics of biochemical mechanisms. The high repetition rate, low energy pulse train permits sufficient sensitivity to study reactions at in viva concentration levels without sample decomposition or nonlinear effects (the absence of intensity loss with time and agreement with literature lifetime values suggests that there is negligible decomposition or stimulated emission, although specific samples would have to be considered individually). Sufficient peak power is present to permit frequency doubling, consequently the uv down to ca. 260 nm as well as the entire visible spectrum can be accessed with one system.
ACKNOWLEDGMENTS We would like to thank B. W. Wallin for technical assistance in the early stages of this work, M. A. Revelli for assistance with the computer calculations, and M. A. Laskaris for some of the amino acids.
REFERENCES 1. Chen, R. F., and Edelhoch, H. (eds.) (1975) Biochemical Fluorescence: Concepts, Dekker, New York. 2. Richardson, J. H., and Ando, M. E. (1977) Anal. Chem. 49, 955-959. 3. Cramer, L. E., and Spears, K. G. (1978) J. Amer. Chem. Sm. 100,221-227. 4. Richardson, J. H.. and George, S. M. (1978)Anal. Chem. 50, 616-620. 5. Fleming, G. R., Knight, A. E. W., Morris, J. M., Morrison, R. J. S., and Robinson, G. W. (1977) J. Amer. Chem. Sot. 99, 4306-4311. Shank, C. V., and Ippen, E. P. (1974) Appl. Phys. Left. 24, 373-375. Cox, A. J., Scott, G. W., and Talley, L. D. (1977) Appl. Phys. Left. 31, 389-391. Harris, J. M., Gray, L. M., Pelletier, M. J., and Lytle, F. E. (1977) Mol. Photochem. 8,161- 174. Steinmetz, L. L., Richardson, J. H., and Wallin, B. W. (1978) Appl. Phys. Lett. 33, 163- 165.
SYNCHRONOUSLY
PUMPED KRYPTON
ION DYE LASER FLUORESCENCE
10. Smith, E. C., and Metzler, D. E. (1963)J. Amer. Chem. Sec. 85,3285-3288. 11. de Bemardo, S., Weigele, M., Toome, V., Manhart, K., Leimgrnber, W., Bohlen, P., Stein, S., and Udenfiiend, S. (1974) Arch. Biochem. Biophys. 163, 390-399. 12. Leskovar, B., and Lo, C. C. (1975) Nucl. Instr. Methods 123, 145- 160. 13. Steinmetz, L. L., Richardson, J. H., and Wallin, B. W. (1978) Presented at the Topical Meeting on Picosecond Phenomena, May 1978, Hilton Head, S. C.
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14. Steinmetz, L. L., and Bookless, W. A., manuscript in preparation. 15. Danliker, W. B., and de Saussure, V. A. (1970) Immunochemistry 7,799-828. 16. Chen, R. F., Smith, P. D., and Maly, M. (1978) Arch. Biochem. Biophys. 189, 241-250. 17. Chen, R. F. (1974) Anal. Left. 7, 65-77. 18. Richardson, J. H. (1977) Anal. Biochem. 83, 754-762. 19. Yguerabide, J., Epstein, H. F., and Stryer, L. (1970) J. Mol. Bioi. 51, 573-590.