Anal. Chem. 1995,67, 139-144
Wavelength=ResolvedFluorescence Detection in Capillary Electrophoresis Aaron T. Timpennan, Karim Khatib, and Jonathan V. SweedleP Department of Chemistry, University of Illinois, Ubana, Illinois 61801
A multichannel laser-induced fluorescence detector for capillary electrophoresis is described. The detection system combines yoctomole limits of detection with the simultaneous acquisition of entire fluorescence emission spectra. An Ar/E mixed-gas ion laser provides great flexibility in excitation wavelengths, and a holographic grating and charge-coupled device detector combination allows a 500-nm spectral window to be acquired with 2-nm resolution. The limits of detection are 5 x M or 80 molecules for sulforhodamine 101 and 1.5 x M or 220 molecules for fluorescein in a 50 pm i.d. capillary. An electropherogram of a mixture of amino acids derivatized with both Bodipy 503/512 C3 and Bodipy 576/589 CB demonstrates that the analytes can be differentiated on the basis of both emission characteristics and migration limes. With the use of organic modiliers and other complex separation media, the spectral background increases as discrete spectral features appear; the wavelength-resolvedbackgrounds of a variety of common CE separation conditions are presented. Capillary electrophoresis (CE) has become one of the most powerful microseparation methods offering unparalleled separation efficiencies and sample thr~ughput.'-~CE has been applied to separationsof proteins, peptides, amino acids, inorganic ions, and nucleotides. In samplelimited applications such as in vivo sampling from a single cell, the analyte volume is typically in the picoliter to nanoliter volume although femtoliter injections have been ~ b t a i n e d . ~Analysis ,~ of even smaller regimes at the subcellular level has been unobtainable because the extremely small amounts of material are below the detection limits of present systems. These applications require improvements in detection sensitivity; ideally, such detectors more closely approach the ideal detector with the ability to detect and identify substances to the single molecule level. Since the introduction of the first laser-induced fluorescence (LIFJdetection system for CE by Zare,lothe limits of detection
O D s ) have improved rapidly so that the detection limits for most research grade instruments are in the high zeptomole range.'-4 Dovichi and co-workers have developed a more sensitive LIF method that uses a sheath flow cuvette as the sample cell and photomultiplier (PMT) detection.11J2 They reported that this arrangementreduces the scattered light and capillary background by more than l O @ f ~ l dleading , ~ ~ to large decreases in LODs. Recently, Dovichi reported detection limits of six molecules of sulforhodamine 101, the lowest limits of detection reported for capillary electrophoresis.14 Several previous approaches have been used for wavelengthresolved LIF detection in CE. A charge-coupled device (CCD) detector array and a grating spectrographwere first used for CE/ LIF detection by Cheng and co-worker~.'~Wavelength resolution of the fluorescence emission was demonstrated,but the CCD was used in a conventional camera mode with a shutter; the 3%openedshutter duty cycle was the primary cause of the relatively poor detection limit of 4 am01 for fluorescein. Zare et al. solved this problem by reading out the CCD in the time-delayed integration (TDI)mode to eliminate the need for a shutter and to reduce the number of readouts required to track the migration of labeled analytes in a 2-cm detection window. Using this system, fluorescence emission spectra were obtained of the components separated with CE while detection limits of 12 zmol were achieved for FITC.l6 Harris and colleagues demonstrated a CCD/LIF system with a 94% duty cycle and highquality spectral information; the concentration LOD for Joe-A oligonucleotide primer is 26 pM injected onto a gel-filled ~apillary.'~They also discussed the advantages of the wavelength-resolved information compared to multiple filters for DNA sequencing. Although most previous wavelength-resolved LIF systems have used CCDs, Karger et al. recently demonstrated excellent results with an intensified photodiode array system using two lasers to spectroscopically distinguish four different fluorophores used in DNA sequencing.ls The detection limits for the system are -1 zmol. Although
(1) Ewing, A G.; Wallingford, R A; Olefirowicz, T. M. Anal. Chem. 1989,61, 282A. (2)Monnig, C. A;Kennedy, R T. Anal. Chem. 1994,66,280R (3)Jandik, P. Bohn, G. Capillary Electrophoresis of Small Molecules and Ions; VCH Publishers: New York, 1993. (4)Landers, J. P.,Ed. Handbook of Capillary Electrophoresis; CRC Press: Boca Raton, FL, 1994. (5) Kennedy, R T.; Oases, M. D.; Cooper, B. R; Nickerson, B.; Jorgenson, J. W. Science 1989,246,57. (6)Ewing, A G.; Strein, T. G.; Yi, Y. Acc. Chem. Res. 1992,25,440. (7) Shippy, S. A; Jankowski, J. A; Sweedler, J. V. Anal. Chim. Acta, in press. (8) Olefirowicz, T. M.; Ewing, A G. Anal. Chem. 1990,62,1872. (9)Hogan, B. L.;Yeung, E. S. Anal. Chem. 1992,64,2841. (10)Gassman, E.; Kuo, J. E.; Zare, R N. Science 1985,230,813.
(11) Cheng, Y.F.; Dovichi, N. J. Science 1988,242,562. (12)Chen, D. Y.;Swerdlow, H. P.; Harke, H. R ; Zhang, J. 2.; Dovichi, N. J. J Chromatogr. 1991,559,237. (13)Swerdlow, H.; Zhang, J. Z.; Chen, D. Y.; Harke, H. R ; Grey, R; Wu,S.; Dovichi, N. J. Fuller, C. Anal. Chem. 1991,63,2835. (14)Chen, D. Y.; Aldelhelm, Cheng, X. L.; Dovichi, N. J. Analyst 1994, 119, 349. (15) Cheng, Y. F.; Piccard, R D.; Vo-Dinh, T. Appl. Spectrosc. 1990,44,755. (16)Sweedler, J. V.; Shear, J. B.; Fishman, H. A; Zare, R N.; Scheller, R H. Anal. Chem. 1991,63,496. (17) Karger, A E.; Harris, J. M.; Gesteland, R F. Nucleic Acids Res. 1991,19, 4955. (18)Carson, S.;Cohen, A S.; Belenkii, A; Ruin-Martinez, M. C.; Berka, B. L.; Karger, B. L.Anal. Chem. 1993,65,3219.
0003-2700/95/0367-0139$9.00/0 0 1994 American Chemical Society
Analytical Chernisrry, Vol. 67, No. 1, January 1, 1995 139
comparable to the best previously reported for CCD-based systems, the LODs are determined by use of a stream of labeled primer flowing by the detector. In addition, the dynamic range of the system is limited by the intensified photodiode array and capillary background to less than 3 orders of magnitude, much less than from CCD systems. The major goal of the current work has been the development of a LIF detector system that combines the high sensitivity of PMT-based LIF detection with the ability to obtain high-quality spectroscopic data. The LIF/CCD system described here achieves LODs of less than 100 molecules for sulforhodamine 101 in both 20 and 50 ,um i.d. capillaries. Ideally, the multichannel LIF system should provide flexibility in selecting excitation wavelengths and emission observation regions so that the instrumentation places few restraints on choosing derivatizing chemistries. The current system uses a mixed-gas Ar/Kr laser, which allows selection of an excitation wavelength ranging from 350 to 750 nm. The spectrographand CCD have high efficiency throughout the 300900-nm region, contributing to system flexibility. Recording a complete fluorescence emission spectrum with each readout of the detector provides much more information than a single-channel detector. This information can be utilized in several ways to improve the detection abilities. The most obvious ability is to spectroscopically distinguish analytes labeled with different fluorophores. In fact, the ability to use four different fluorophores with DNA sequencing has been the major driving force for much of the multichannel work described previously.15J7,18 For singlechannel systems,the observed wavelength region is usually selected by using the appropriate optical filters determined through examination of the emission properties of the fluorescent probe of interest. However, a much higher fluorescent background can be encountered with many common CE separation conditions. Using a multichannel system, the wavelengths containing background features can be weighted less heavily or completely discarded depending on the particular spectral background features in the current separation. Thus, less degradation in sensitivity is expected when organic modifiers or gel-filled capillaries are used. Other advantages may include the ability to correct laser power fluctuations by monitoring solvent Raman bands and the use of sophisticated algorithms to further increase sensitivity. EXPERIMENTAL SECTION Detection System. The system utilizes high-efficiency light collection, a spectrograph, and a CCD detector to provide low detecton limits and spectral resolution. As shown in Figure 1, the excitation from an Ar/Kr (Innova Spectrum 70, Coherent, Palo Alto, CA) laser is passed through a prism and an iris to remove laser tube background before it is focused onto the capillary. This particular laser is chosen because it has the ability to lase at many wavelengths. Many of these wavelengths match the absorption of common labeling agents: 352-356 nm, fluorescamine; 457 nm, NDA and CBQCA; 488 nm, fluorescein; 531 nm, tetramethylrhodamine; 568 nm, sulforhodamine; and 647 and 752 nm for new near-IR derivatizing chemistries. A 40-mm focal length cylindrical lens illuminates an -2-mm section of the capillary in the detection window. The fluorescence emission is collected at 90" with an f/1.2, 3.5x magnification collection optics assembly. The collection optics are a combination of a convex-concave lens (LPKO19 and LE084 glued together), an aphtic meniscus lens (LAM138), and 140 Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
ArlKr laser buffer vial cylindrical lens
f buffer vial grating
Figure 1, Schematic of the wavelength-resolved laser-induced fluorescence detection system.
two achromatic doublets (LA0138, all from Melles Griot, Irvine, CA), which are designed to provide a high-quality image on the spectrograph entrance slit from a several millimeter capillary length. The light is spectrally filtered to remove most of the Rayleigh scattering and then focused on the spectrograph slit. Careful alignment of the capillary image on the spectrograph entrance slit is crucial to maximize the signal and minimize background scattering. The ~72.2CP140 imaging spectrograph (Instruments SA, Edison, NJ) has high throughput and adequate dispersion with its 405 grooves/" holographic grating. The EEVlSllCCD (EEV, Essex, England) is mounted in a liquid nitrogencooled CH270 camera head and is controlled by a CE2OOA camera electronics unit and an AT200 controller card (Photometrics Ltd., Tucson, AZ). The CCD system has excellent figures of merit: a negligible dark current, a root-mean-square read noise of 5 e-, and a quantum efficiency of 235%in the 510850-nm range. To obtain the minimum dark current without a reduction in charge transfer efficiency the CCD is cooled to - 125 "C. The capillary is imaged axially along the 256 pixel elements in the parallel CCD dimension, and the grating diffracts the signal along the 1024 pixel elements in the serial CCD dimension. With a spectral distribution of -0.5 nmlpixel, a 500-nm interval is imaged simultaneously and is adjustable in the 300-1000-nm region. A 486 IBM ATcompatible 3?-MHz computer is used to read out the CCD and store data. To ensure a precise timing interval, the CCD parallel shifts are triggered directly with an AT-MIO16F-5 (National Instruments, Austin, 'I'X), a multifunction analog, digital, and timing I/O board with Sps timing precision. PMIS image processing software (Photometrics Ltd.) is used to align the system by acquiring images of the capillary detection window that consist of wavelength, axial capillary position, and intensity data. With these images, the intensity of the major Raman band of wate: is maximized while the background in other spectral regions is minimized. To ensure that the image is not smeared and the integration time is constant, a shutter is used while the system is aligned. Use of the Raman band allows optimization of the optical alignment without contaminating the capillary with fluorophore. For CE data acquisition, the CCD is controlled with a C program written in-house by use of the Lab Windows programming environment (National Instruments). The program is capable of acquiring data from multiplewavelength intervals and saving the data in separate arrays. In this manner, the CCD can function as if it is has multiple tunable emission filters that can be used simultaneously to select multiplewavelength regions.
The program also allows for control of serial and parallel binning and readout parameters, so the noise can be minimized and TDI used. The flexibility afforded by the CCD detector array provides precise optimization of the detection system in a way allowed by almost no other photon detector. However, adjustment of the readout parameters (e.g., readout rate, wavelength interval, serial binning, parallel binning, number of rows to read each time, etc.) is complex, and so we have chosen to optimize only a few crucial parameters. For the detection limit studies, the CCD readout rate that maximizes the signal-to-noiseratio (S/N) is 3 Hz with serial binning of 5 and parallel binning of 256. For the wavelengthresolved acquisitions, the readout rate is slowed to 1 Hz with a serial binning of 1 and a parallel binning of 64 to increase the dynamic range and reduce the amount of information obtained. Further details of the particular readout parameters are described in the sections detailing individual experiments. Nonoptimized parameters typically result in small to severalfold increases in baseline noise. Electrophoresis System. The CE system is built in-house and is similar to systems described previou~ly.~J~ Both the injection and elution ends are enclosed in separate plexiglass boxes with high-voltage interlocks while the optics and CCD are enclosed in aluminum light-tight boxes. The high-voltage power supply (Glassman, Whitehouse Station, NJ) is also controlled by the acquisition program to ensure that the separation and data acquisition are started simultaneously. Separation potentials of 25 and 30 kV are used for the detection limit and multiple fluorophore studies, respectively. The injection end of the capillary is at ground and the capillary outlet is held at negative potential, with connections made to the buffer vials with Pt electrodes. Both 50 and 20 pm i.d., 375 pm o.d., 64 cm long polyimide-coated capillaries (Polymicro Technologies, Phoenix, AZ) are used. The polyimide is removed from the detection window by burning it with a flame and removing the blackened polyimide with a stream of water to minimize scratching of the capillary, instead of removing the blackened polyimide with lens paper. Injections. All injections are l k m , 10-s gravity injectionswith a calculated volume of 2.4 nL. The injection volume is also determined experimentally by continuously injecting sample from the l k m height displacement and measuring the time required for the sample to reach the detector. The injection volume determined experimentally matches the 2.4 nL calculated using the Poiseuille equation.2O Immediately after an injection, the capillary injection end is dipped in a rinse vial (at the same height as the running buffer) to minimize the effects of spontaneous capillary displacement and ubiquitous injection.21 For the detection limit studies, blank injections are used to ensure the signal is not an artifact from a previous injection, and calibration curves over 4 orders of magnitude are made to ensure peak identity. Reagents. All solutions are prepared with ultrapure Milli-Q (Millipore, Bedford, MA) water to minimize fluorescent impurities, and all buffers are filtered with 0.2-pm syringe filters prior to use to remove particles. Borate buffer with a concentration of 50 mM at pH 9.1 is prepared by dissolving 0.6183 g of boric acid U. T. Baker, Phillipsburg, NJ) and 1.5828 g of sodium tetraborate (19) Tracht, S.; Toma, V.; Sweedler,J. V. Anal. Chem. 1994, 66, 2382. (20) Olechno,J. D.; Tso, J. M.Y.; Thayer, J.; Wainright, A. Am. Lab. 1990, 30. (21) Fishman, H. A; Amudi. N. M.; Lee, T.T.;Scheller, R H.; Zare, R N. Anal. Chem. 1994, 66, 2318.
decahydrate (Fischer, Fairlawn, NJ) in water to a final volume of 200 mL. Sodium dodecyl sulfate (SDS, lauryl sulfate), used to prepare the micelles, and 3- (cyclohexy1amino)-1-propanesulfonic acid (CAPS) for the buffer are from Sigma Chemical Co. (St. Louis, MO). All organic solvents, methanol, acetone, and acetonitrile, are spectroanalytical grade from Aldrich Chemical (Milwaukee, wr).
Fluorescein crystalline free acid and sulforhodamine 101 (Sigma) are dissolved initially at 1 mM in 10%(v/v) acetone in water. Bodipy 503/512 (excitation maximum, 503 nm/emission maximum, 512 nm) and Bodipy 576/589 (excitation maximum, 576 nm/emission maximum, 589 nm) C3 amine labeling kits (Catalog No. E2185 and E2226) are from Molecular Probes (Eugene, OR). The L-amino acids used for the derivatizations, arginine, phenylalanine, leucine, and alanine, are from Sigma.The Bodipy amine labeling kits also included the N-hydroxysulfosuccinimide (NHSS) sodium salt, l-ethylS-I3-(dimethylamino)propyl]carbodimide hydrochloride (EDAC), dry DMF on molecular sieves, and sodium tetraborate. Bodipy Derivatization. The Bodipy derivatives are prepared in a method similar to the Molecular Probes protocol. Solutions of 15 mM amino acids are dissolved in 0.7 mL of 50 mM borate buffer. For the Bodipy dyes, a carboxylic acid precursor is used to form a sulfosuccinimidylester, which is the derivatizing agent. Formation of the 3 mM Bodipy sulfosuccinimidylester is started by dissolving 2.5 mg of NHSS sodium salt in 30 mL of pure water and 1 mg of the BODIPY propionic acid in 50 mL of dry DMF. These two solutions are mixed and immediately 5 mg of EDAC dissolved in 50 mL of water is added. This reaction mixture is stirred at room temperature for 30 min. The reaction is then stopped with 10 mL of glacial acetic acid, and 32 mg of sodium tetraborate is added to the reaction mixture to absorb the excess acetic acid completing the preparation of the Bodipy sulfosuccinimidyl ester. The Bodipy sulfosuccinimidylester mixture of the active dye is then added to the amino acid solution and is stirred for 1 h at room temperature to form the Bodipy-amino acid conjugates. Each of the amino acids, arginine, phenylalanine, leucine, and alanine, is derivatized separately with each fluorophore. The eight individual amino acid derivatives are mixed and diluted for analysis to a concentration of 1 x M (assuming 100%yield). Detection Limit Studies. Both fluorescein and sulforhodamine 101 are used to determine the detection limits. The running buffer is 50 mM borate pH 9.1, and the separation voltage is 25 kV. For fluorescein, the 488-11, line at 18 mW is used for excitation, and two notch filters are used to block the Rayleigh scattering (488HNF Kaiser Optical, and a 488RB Omega Optical). A 50 pm i.d. separation capillary is used. The 503-560-nm region of the CCD is the optimum wavelength interval for data acquisition. For sulforhodamine 101, the 568nm line at 25 mW is used for excitation, and the Rayleigh filter is an absorptive glass highpass filter (OG 590, Melles Griot, Imine, CA). Both 50 and 20 pm i.d. capillaries are used. The optimum wavelength interval of the CCD for data acquisition is the region from 597 to 653 nm. Evaluation of signal-to-noise ratios is performed with a program written in Lab Windows. The LODs are determined using the 3a method. The baseline noise (a) is evaluated by performing a least-squares linear regression of the baseline and finding the standard deviation of the data points from this line. The signal is determined by finding the difference between the data point of Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
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time (min) Figure 2. (A, top) Electropherogram of a 2.4-nL injection of 2 x lO-I3 M sulforhodamine 101 with a SIN of 10. (B, bottom) Smoothed data having a S/N of 11.
maximum intensity and the linear regression of the baseline at the corresponding time. In addition, when reported as smoothed, a polynomial least-squares smoothing routine using nine-point quadratidcubic filter weights is applied to the data to remove the high-frequency noise.22 Wavelength-ResolvedAmino Acid Separations. Four amino acids are labeled with Bodipy 503/512 C3 and Bodipy 576/589 C3. The 514nm line is used for excitation, representing a compromise for excitation of both fluorophores. The separations are performed in a 50 pm i.d. capillary with a 3@kV separation voltage. The running buffer is 50 mM CAPS pH 10,100mM SDS, and 10%methanol. The CCD spectral acquisition region is from 510 to 640 nm. Spectral Background Analysis. In order to compare the spectral backgrounds under common CE separation conditions, capillaries are aligned and background spectra are acquired while the buffer is static. Backgrounds from 100%borate buffer and three solutions of 70%borate buffer, 30%methanol, acetone, and acetonitrile are analyzed. The background from a 50pm id., 375 pm 0.d. capillary with transparent polymer coating (polymicro, TSU-050375) is analyzed without removing the coating in the detection window. Lastly, two 75 pm i.d. polyacrylamide gelfilled capillaries are used: a 3%gel capillary with 7 M urea and TRIS buffer, and a 3%gel non-urea capillary with TRIS buffer U&W Scientitic, Folsom, CA). In all cases, a 1-s acquisition is performed using 25 mW of the 488nm line. For these studies, a 10@mm spectrograph slit is used to minimize the effects of slight differences in capillary/optical alignment. RESULTS AND DISCUSSION
LODs. Both sulforhodamine 101 and fluorescein are used as probes to characterize the detection limits of the LIF system. These dyes are chosen because they are the fluorescent moieties of common derivatizing agents, such as Texas Red, XRITC, FITC, and FSE.23 Figure 2 shows an electropherogram of a 2.4nL (22) Ratzlaff, K. R Introduction to ComputerAssisted Exfierimentation: John Wiley & Sons: New York, 1987; Chapter 11.3. (23) Haugland, R P. Handbook of Fluorescent Probes and Research Chemicals: Molecular Probes: Eugene, OR 1992.
142 Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
3.5
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time (min) Figure 3. (A, top) Electropherogram of a 90-pL injection of 2 x M sulforhodamine 101 with a S/N of 25. (B, bottom) Smoothed data having a SIN of 33.
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time (min) Figure 4. (A, top) Electropherogram of a 2.4-nL injection of 5 x lO-I3 M fluorescein with a S/N of 6. (B, bottom) Smoothed data having a S/N of 10.
injection of 2 x 10-13 M sulforhodamine 101 obtained using a 50 pm i.d. capillary. The S/N is 9.7, which corresponds to a detection limit of 6 x M or 90 molecules. After data smoothing, the detection limits decrease slightly to 5 x 10-14 M or 80 molecules (Figure 2B). These concentration detection limits are among the lowest published for on-column detection in capillary electrophoresis. Smaller internal diameter capillaries have been used to decrease mass detection limits for detectorsthat are concentration sensitive. Although we have used such a 20 pm i.d. capillary, our detector appears to be mass sensitive in this regime and a decrease in mass LODs proportional to the change in capillary cross-sectional area is not realized. We do observe a slight improvement in detection limits, presumably from better spatial atering of the Rayleigh scattering from the outside of the capillary. Figure 3A shows an electropherogramof a 9@pLinjection of 1 x lo-" M sulforhodamine 101, which yields a S/N of 25. The calculated detection limit is 50 molecules for the smoothed data (Figure 3B).
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Figure 5. (A, top) Wavelength-resolvedelectropherogramof a mixture of Bodipy 503/512 C3 and Bodipy 576/589 C3 amino acid conjugates. The emission from the Bodipy 503/512 C3 derivatives appears at shorter wavelengths than the emission from Bodipy 576/589 C3 derivatives. The continous feature at -583 nm is the major Raman band of water. (B, bottom) A contour plot of the electropherogramshowing the elution order: dye (*), alanine (A), leucine (L), phenylalanine (F), dye (*), arginine (R).
Although slightly poorer than sulforhodamine 101, excellent detection limits are also achieved with fluorescein. With the flexible design of the multichannel system, changing fluorophores is very simple; for fluorophores requiring a different excitation wavelength,the only hardware changes involve adjusting the laser and switching the Rayleigh filter. As shown in Figure 4, the limits of detection for fluorescein are 340 molecules or 2.5 x M for the raw data, and 220 molecules or 1.5 x M for the smoothed data. In previous LIF/CCD work, the TDI method had been used to increase sensitivity by allowing a 2-cm capillary section to be imaged.16 We have chosen not to use TDI readout because the nonuniform data acquisition rate and increasing baseline make the use of smoothing routines problematic. However, the much shorter observation zone in the current system allows the entire CCD to be read at each sampling interval yielding a column efficiency of -250 000 for the 20 ,umi.d. capillary. Wavelength-ResolvedDetection. The two-color separations demonstrate the ability of the detection system to discriminate between fluorophores having different emission characteristics. A separation of four amino acids labeled with two fluorophores having different emission spectra, Bodipy 503/512 C3 and Bodipy 576/589 C3, is shown in Figure 5. As can be seen, there is good spectral resolution between the amino acids labeled with the d ~ e r e n ttags, and all eight of the amino acid derivatives are resolved. Upon derivatization, the formation of an amide bond eliminates the charge on the primary amine as it links the neutral fluorophore to the amino acid. Therefore the Bodipy conjugates of alanine, phenylalanine, and leucine have a charge of -1, and
the arginine conjugate is neutral. Thus, micelles are used to enhance the separation because the amino acid derivatives are similar in charge, size, and shape. The Bodipy dyes are available with a range of excitation/ emission characteristics. Bodipy 503/512 C3 and Bodipy 5761 589 C3 have molar absorptivities of 74 000 and 84 000 respectively, quantum yields of
