Volume 52, Number 1, 1998 APPLIED SPECTROSCOPY 10003-7028 / 98 / 5201-0001$2.00 / 0q 1998 Society for Applied Spectroscopy

submitted papers

Single-Molecule Detection in the Near-IR UsingContinuous-Wave Diode Laser Excitation with anAvalanche Photon Detector

STEVEN A. SOPER* and BENJAMIN L. LEGENDRE, JR.Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1804

While single-molecule detection in ¯ owing sample streams has beenreported by a number of groups, the instrumentation can be some-what prohibitive for many applications due to the complexity andextensive expertise required to operate such a device. In this paperwe report on the construction of a single-molecule detection devicethat is rugged, compact, inexpensive, and easily operated by indi-viduals not well trained in optics and laser operations. The single-molecule detection apparatus consists of a semiconductor diode la-ser operating in a continuous-wave (CW) mode and a single photonavalanche diode transducer for converting the detected photons intotransistor± transistor logic (TTL) pulses for displaying the data. Inaddition, the sampling volume is produced by a single-componen tlens, to create a volume on the order of 1 pL, allowing the samplingof microliter volumes of material on reasonable time scales. Thedevice is targeted for operation in the near-IR region (700± 1000nm), where matrix interferences are minimal. Our data will dem-onstrate the detection of single molecules for the near-IR dyes IR-132 and IR-125, in methanol solvents in ¯ owing sample streams atsampling rates of 100± 250 samples/s. Detection ef® ciencies for theinvestigated near-IR dyes were found to be 98% for IR-132 and50% for IR-125. Previous attempts in our laboratory to detect sin-gle molecules of IR-125 using time-gated detection were unsuccess-ful because of the short upper-state lifetime of this ¯ uorophore (t f

5 472 ps).

Index Headings: Near-IR ¯ uorescence; Single molecule detection;Diode laser-induced ¯ uorescence.

INTRODUCTION

The ability to detect single chromophore molecules inliquids at room temperature has many interesting appli-cations in chemistry and biology, such as DNA sequenc-ing,1,2 monitoring of the photophysics of chromophoresin heterogeneous environments free from ensemble av-eraging,3 capillary separation techniques,4,5 and sorting ofindividual biopolymers.6,7 The ability to monitor individ-ual molecular events in solution at room temperature, es-pecially in ¯ owing sample streams, is fraught with anumber of potential dif® culties not typically encounteredin single-molecule experiments performed in the gasphase, at low temperatures, or when the molecules aresurface-immobilized with detection accomplished via mi-

Received 3 March 1997; accepted 17 June 1997.* Author to whom correspondence should be sent.

croscopic imaging techniques. Some of these challengesinclude (1) photochemical bleaching; (2) extensive scat-tering from the solvent matrix; (3) ¯ uorescence interfer-ences; and (4) the transitory nature of the molecule withinthe detection volume due to the ¯ owing stream.

In most single-molecule detection applications, theutility of single-molecule monitoring can be realized onlyif the detection ef® ciency approaches 100%. In order toaccomplish reasonable single-molecule detection ef® cien-cies, several dye and instrumental parameters must beoptimized. In terms of dye photophysics, the relevant pa-rameters are the absorption cross section ( s ), the ¯ uores-cence quantum ef® ciency ( F f), the ¯ uorescence lifetime( t f), and the photodestruction ef® ciency ( F d). In mostcases, the upper limit on the number of photons that canbe obtainable (analytical signal) per molecule (nf) is de-termined by the photochemical stability of the chromo-phore. When the molecular transit time through the laserbeam is in® nite (i.e., all molecules are photobleached), nf

can be simply determined from the ratio F f/ F d.8

The absorption cross section sets an upper limit on theamount of effective laser power one can use in the ex-periment before the onset of electronic saturation and foroptimal signal-to-noise ratio (SNR), the laser ¯ uencemust be set close to the saturation point.3 In the case ofcontinuous-wave (CW) excitation, the saturation pointoccurs when the absorption rate (ka) is approximatelyequal to 1/t f.

In spite of photochemical bleaching, the primary lim-itation on ef® cient single-molecule detection has been thebackground produced by the solvent in the form of scat-tering or ¯ uorescent impurity photons. Researchers haveutilized several different instrumental strategies to mini-mize the background in single-molecule detection exper-iments using ¯ uorescence detection. The common ap-proach is to reduce the sampling volume to decrease boththe scattering and the ¯ uorescence impurity contributionsto the background. Keller and co-workers have reducedthe sampling volume by using a tightly focused laserbeam and a sheath ¯ ow arrangement.9 Another approachthat has been implemented in order to alleviate the scat-tering contribution to the background involves the trap-ping of microdroplets in electrodynamic traps.10 Zare and

2 Volume 52, Number 1, 1998

FIG. 1. Block diagram of the CW, near-IR diode laser-based single-molecule detection apparatus. FO, focusing optic; MO, collection mi-croscope objective; BD, beam dump; SPAD, single photon avalanchediode.

co-workers have demonstrated the use of confocal mi-croscopy to further reduce the sampling volume and haveshown single-molecule detection of R6G in volumes ap-proaching several femtoliters.11,12

The dif® culty associated with signi® cant reductions inthe sampling volume concerns the analysis time. For ex-ample, if one requires the ability to analyze the contentsof a sample with a volume of 1.0 m L for a particularmolecule type, and if the sampling volume is 0.01 pLwith an integration time of 10 ms (time for abstractingall the photons from the molecule), the amount of timerequired to completely analyze this sample is 277 h (11.5days). If the sampling volume is increased to 1.0 pL, theanalysis time is reduced to 2.77 h. Clearly, for analyzingmodest-size samples exhaustively, the sampling volumemust be relatively large ( . 1.0 pL).

Another approach toward minimizing the backgroundarising from scattering photons has been to implementtime-gated detection.13 The advantage of such an ap-proach is that large sampling volumes can be used with-out sacri® cing single-molecule detection ef® ciency. Theef® ciency of time-gated detection is determined primarilyby the lifetime of the chromophore and the timing re-sponse of the instrumentation. The dif® culty with time-gated detection is that it does not attack the ¯ uorescenceimpurity contribution from the background. Indeed, it hasbeen found that, with the use of time-gated detection ( l ex

5 532 nm), the single-molecule detection ef® ciency islimited primarily by the purity of the background solvent,and great care must be taken to rigorously preserve thecleanliness of the solvent.14 In addition, time-gated de-tection can be instrumentally intensive, requiring sophis-ticated electronics, pulsed lasers, and expensive photo-detectors.

An attractive alternative to visible ¯ uorescence single-molecule detection is the use of near-IR excitation anddetection (700± 1000 nm). The major advantage associ-ated with near-IR monitoring is the fact that few com-pounds show intrinsic ¯ uorescence in this region of thespectrum, along with the signi® cantly smaller Ramanscattering cross sections one encounters in this region ofthe electromagnetic spectrum, resulting in large Raman-free ¯ uorescence observations windows. We have re-cently shown that time-gated detection in the near-infra-red (near-IR) can achieve detection limits superior tothose in the visible region of the electromagnetic spec-trum.15 The instrument developed for single-moleculemonitoring in the near-IR used a passively mode-lockedTi : sapphire laser pumped by an Ar-ion laser. The detec-tor was a single photon avalanche diode operated in aGeiger mode with the electronics con® gured in a con-ventional time-correlated single photon counting format.

While the aforementioned results are encouraging forhighly ef® cient single-molecule detection in the near-IR,the instrumentation used was complex and required ex-tensive operator expertise in order to perform the mea-surement. In this paper, we will discuss the use of CWexcitation with a diode laser for monitoring single mo-lecular events in ¯ owing sample streams to produce asensitive device that is both simple to operate and verycompact. In earlier work, Winefordner and co-workers,using similar instrumentation, were able to demonstratemolecular detection limits for the near-IR dye IR-140 ap-

proaching 3000 molecules.16 In their work, the samplingvolume was 250 pL and the ¯ uorescence was excitedwith a AlGaAs diode laser with a conventional PMT pho-ton transducer. The sample stream consisted of a liquidjet, producing residence times of molecules within thesampling volume of approximately 20 m s. The instrumentdiscussed in this paper possesses a sampling volume of1 pL and has the ability to sample single molecules in¯ ow streams at high rates ( , 10 ms/sample) using simple,all-solid-state instrumentation.

EXPERIMENTAL

A block diagram of the CW diode-based single-mol-ecule detection apparatus is shown in Fig. 1. The exci-tation source was a GaAlAs diode laser that possessed aprincipal lasing line at 786 nm (26 8 C) with an outputpower of 17 mW (Melles Griot, Irvine, CA). The diodehead contained external optics (anamorphic prism pair)to produce a circular beam for tight focusing and oper-ated in a fundamental mode (pseudo-Gaussian). The laserwas driven by a constant-current source and the temper-ature controlled by a thermoelectric cooler to preventmode-hopping. The laser beam was focused onto a fused-silica capillary tube (Hewlett-Packard), which possessedan internal diameter of 50 m m and was expanded 3 3around the detection volume to avoid excessive scatteredlight from reaching the detector. The focusing of the laserbeam was accomplished with the use of a singlet lensand produced a beam waist (1/e) of 5.5 m m. The emissionwas collected with a 403 high-numerical-aperture (NA)microscope objective obtained from Nikon (NA 5 0.85).The ¯ uorescence was spectrally ® ltered with a Corninglong-pass ® lter (50% T at 810 nm) and then with aneight-cavity bandpass ® lter [center wavelength (CWL) 5835 nm; half bandwidth (HBW) 5 30 nm)], which wasobtained from Omega Optical (Brattleboro, VT). Theemission was ® nally ® ltered with a spatial ® lter (slit) thatwas placed slightly in front of the secondary focal planeof the collection microscope objective and set to a widthof 400 m m, producing a sampling volume (assuming acylindrical geometry) of ; 1 pL, which was determinedby the laser beam waist (1/e waist 5 5.5 m m) and the

APPLIED SPECTROSCOPY 3

FIG. 2. Chemical structures of the near-IR dyes IR-132 and IR-125.

TABLE I. Raman shifts for methanol with the use of visible and near-IR laser excitation.a

Excitation(mm)

OH stretch(3330 cm2 1)

CCO stretch(1033 cm2 1)

CH2 twist(1363 cm2 1)

d (CCO)(480 cm2 1)

Broad; weak Strong Weak Weak532 646 nm 560 nm 574 nm 543 nm786 1064 nm 856 nm 880 nm 816 nm

a Position of Raman bands taken from F. R. Dollish, W. G. Fateley, and F. F. Bentley, Characteristic Raman Frequencies of Organic Compounds(Wiley± Interscience, New York, 1974).

viewing distance along the propogation axis of the laserbeam, which was set by the slit width of the spatial ® lterand the magni® cation of the collection microscope ob-jective (slit width 5 0.4 mm; 40 3 microscope objective;viewing distance in capillary 5 10 m m). The probe vol-ume de® ned as such will give reasonable burst sizes forthose molecules that travel near the edges of this prede-® ned volume, since the laser intensity only drops to ap-proximately 37% of that intensity at the center of theGaussian beam. Taking into account the cross-sectionalarea of the capillary tube at the sampling point (17,671m m2) and that of the probe volume (110 m m2), the effec-tive sampling ef® ciency of all material passing throughthe capillary tube was 0.6%. Directly behind the spatial® lter was placed a single photon avalanche diode(SPAD), which was obtained from EG&G Electrooptics(Vaudreuil, Canada). The SPAD possessed a photoactivearea of 3.14 3 10 2 4 cm2 (diameter 5 200 m m) and was

operated in an actively quenched mode to allow high in-stantaneous counting rates before the onset of saturation.The transistor± transistor logic (TTL) pulses produced bythe output of the SPAD were directly fed into a multi-channel counter/timer board (PCA II, EG&G Ortec, OakRidge, TN) resident in a PC that contained the appropri-ate software for displaying the data. In all cases, thecounting interval was set at 2 ms, and the total data stringwas collected for 16 s with a dead time between countingintervals of approximately 1 m s.

The NIR dyes used in the present single-molecule ex-perimentsÐ IR-132 and IR-125 (see Fig. 2 for chemicalstructures)Ð were prepared from stock solutions thatwere dissolved in methanol. The ® nal dye concentrationused for these experiments was 100 fM, giving a prob-ability for single-molecule occupancy (P0) of 0.06, whichwas calculated from

P0 5 C NAPV (1)

where C is the dye concentration, PV is the size of theprobe volume, and NA is Avogadro’s number. The samplewas hydrodynamically driven through the capillary tubewith a syringe pump (Harvard Instrumentation, SouthNatick, MA) and was set to produce a molecular transittime through the excitation beam of 9.0 and 4.5 ms,yielding linear ¯ ow rates of n 1 5 0.096 cm/s and n 2 50.19 cm/s, respectively, assuming a parabolic (laminar)¯ ow pro® le with the laser beam focused near the centerof the tube.

RESULTS AND DISCUSSION

The ability to detect single molecular events in solutionat room temperature with high ef® ciency depends uponthe ability to minimize the level of scattering and ¯ uo-rescence contributions to the background and, at the sametime, maximize the photon yield from each molecule. Inorder to minimize the scattering contribution to the back-ground, the ¯ uorescence observation wavelengths withinthe sampling volume must be signi® cantly free from Ra-man bands produced by the solvent, in this case metha-nol. In Table I is shown the Raman shifts for methanolproduced from either 532 or 786 nm excitation. If onerequires an approximate 10 nm shift from the excitationwavelength for ef® cient rejection of the Rayleigh scat-tered light and a 5 nm shift from the Raman band pos-sessing a strong relative intensity (large Raman cross sec-tion), the effective Raman-free observation window is 13nm (542± 555 nm) for 532 nm excitation and 55 nm (796±851 nm) for near-IR excitation. Clearly, the Raman-freeobservation window is larger in the case of near-IR ex-citation, allowing a higher collection ef® ciency of theemitted ¯ uorescence free from Raman scattering. In ad-dition, the Raman cross sections show 1/l 4 dependencies,

4 Volume 52, Number 1, 1998

FIG. 3. Photon bursts produced from the near-IR dye IR-132, whichwere ® ltered by using the weighted quadratic sum ® ltering algorithm.In this ® gure only 2 s of the 16 s data string is shown. The averagetransit time in the present case was calculated to be 4.5 ms. The exci-tation wavelength was set at 786 nm with 17 mW of laser power asmeasured at the ¯ ow cell. The probe volume was 1.0 pL, and the av-erage arrival rate of molecules traveling through the probe volume wasdetermined to be 12/s. The dye concentration was 100 fM, giving aprobability of single-molecule occupancy of 0.06.

FIG. 4. Photon bursts arising from IR-132 at two different linear ¯ owvelocities giving transit times of 9.0 (top) and 4.5 ms (bottom). Thediscriminator level was selected so as to set the error rate for falsepositives, as measured from the solvent blank, of 0. All other experi-mental conditions were similar to those in Fig. 3.

indicating that the intensities of these bands will be sig-ni® cantly reduced in the near-IR compared to visible ex-citation.

A block diagram of the diode-based single-moleculedetection apparatus using CW excitation is shown in Fig.1. The ® lter stack used in the device allowed highthroughput of the emission from IR-132, which has anemission maximum at approximately 845 nm in metha-nol, while still rejecting a substantial amount of the Ra-man bands produced from the solvent. A long-pass ® lterwas used to effectively block the Rayleigh and specularlyscattered radiation, and a bandpass ® lter was used toblock the Raman scattered light generated from the sol-vent (CWL 5 835 nm; HBW 5 30 nm). With excitationat 786 nm and 17 mW of laser power and a probe volumeof 1 pL, the background counting rate generated from themethanol solvent was found to be 2170 cps.

In Fig. 3 is shown the photon bursts generated fromIR-132 using the diode-based single-molecule detectorfor only a 2 s interval of the 16 s data stream. The datain this case were subjected to a weighted quadratic sum® ltering algorithm, which is given by13

k2 1

2S(t) 5 w( t )(t 1 t ) (2)Ot 5 0

where k is set equal to the molecular transit time throughthe beam and w( t ) are weighting factors chosen to dis-criminate the analytical signal from the random back-ground. In the present case, these factors were chosen torepresent a Gaussian function due to the Gaussian inten-sity pro® le of the excitation beam and the fact that mostof the molecules are not photobleached before exiting thelaser beam (approximately 21% of the molecules are pho-tobleached prior to exiting the laser beam on the basis ofthe photobleaching lifetime and the linear ¯ ow velocity;

see below). As can be seen from Fig. 3, photon burstsare present that exceed the discriminator threshold, whichare absent in the case of the blank only. From the linear¯ ow velocity (n 5 0.19 cm/s) and the dye concentration(C 5 100 fM), the arrival rate (Nev) of molecules into thesampling zone can be calculated by using

2P v0N 5 (3)ev p v 0

where P0 is the probability of single molecule occupancy(0.06 for C 5 100 fM). Under these experimental con-ditions, the arrival rate was calculated to be ; 12 mole-cules per second. In the data shown in Fig. 3 over this 2s time interval, 21 events clearly exceed the discriminatorthreshold, while at this threshold level, no events werefound to exceed this level for the solvent blank (errorsdue to false positives are equal to 0). In this data set andall subsequent data, the discriminator threshold was setat a level such that, over the entire 16 s data stream, theerror rate due to false positives from the solvent blankwas equal to zero.

From the data of Fig. 3, it was found that the averagenumber of photoelectrons per molecule was 14. The av-erage photon yield per molecule ( ^ nf & ) can be calculatedfrom the experimental absorption rate (ka 5 5.6 3 107),the ¯ uorescence quantum yield ( F f 5 0.07), the instru-mental collection and conversion ef® ciency of photonsinto photoelectrons ( V 5 0.002), and the molecular tran-sit time ( t t 5 4.5 ms) from the relation

^ nf & 5 ka F f V t t (4)

and was determined to be 35 per molecule. The discrep-ancy between the calculated photon yield and that foundexperimentally results from the fact that, in the aboveexpression, the effects of photobleaching are not takeninto account, which would result in a lower photon yieldper molecule. In the present experiments, the averagebleaching lifetime is approximately 20 ms at this absorp-tion rate, and since the residence time is 4.5 ms, some of

APPLIED SPECTROSCOPY 5

TABLE II. Single-molecule detection ef® ciencies with the use of the CW diode-based single-molecule detector for IR-132 and IR-125 attwo residence times, 9.0 ms and 4.5 ms. In both cases, 17 mW of laser power was used, and the dyes were dissolved in methanol.

DyeNumber of observed

events (/16 s)Number of expected

events (/16 s)a

Detection ef® ciency(%)

IR-132 ( t t 5 9.0 ms)IR-132 ( t t 5 4.5 ms)IR-125 ( t t 5 9.0 ms)IR-125 ( t t 5 4.5 ms)

103 ( 6 6)209 ( 6 13)52 ( 6 7)

113 ( 6 11)

105211105211

981005054

a Calculate by using Eq. 3.

FIG. 5. Photon bursts from IR-125 with a transit time of 9.0 ms. SeeFig. 3 for experimental details.

the molecules are expected to be bleached (21%), low-ering the expected photon yield.

In Fig. 4 is shown the photon bursts for IR-132 at twodifferent linear ¯ ow velocities. A higher linear ¯ ow ratewould change the number of events processed during atypical experimental run (16 s) and reduce analysis time.However, at lower linear ¯ ow velocities, the transit timeis longer, allowing the ability to pump more photons outof the system using similar laser irradiances. In the ® rstcase (Fig. 4, top), the transit time was 9 ms, while in Fig.4, bottom, the average transit time was set at 4.5 ms. Inthis ® gure, only those events exceeding the preselecteddiscriminator threshold are plotted, which in this case waschosen to produce an error rate due to false positives of0 under both ¯ ow conditions. It should be noted that, atthe faster linear ¯ ow velocity, the quadratic ® lter wascarried out over a shorter time duration, resulting in alower required threshold level. As can be seen, the num-ber of processed events increased when the linear veloc-ity was increased. In Table II is shown the number ofprocessed events for the two different linear velocities aswell as the single-molecule detection ef® ciency for eachcase. The detection ef® ciency for both linear velocitieswas found to be near 100% for this particular dye mol-ecule. Even though the average molecular photon yieldwas found to be lower for the faster ¯ ow velocity due tothe shorter residence time in the excitation beam ( ^ nf & 514 for t t 5 4.5 ms compared to ^ nf & 5 31 for t t 5 9.0ms), the detection ef® ciency was still found to be near100%.

In Fig. 5 is shown the photon bursts arising from singleIR-125 dye molecules. The interesting photophysical

property associated with IR-125 is its short upper-statelifetime, which has been measured to be 472 ps in meth-anol.17 Due to its short ¯ uorescence lifetime, we havebeen unable to detect IR-125 in solution using time-gateddetection. The data in Fig. 5 clearly show that CW ex-citation can be used for the detection of molecules withshort excited state lifetimes. The transit time in this casewas set at 9.0 ms and the dye concentration was 100 fM.Over this 2 interval of time, 13 events are expected. Fromthe data, only seven events exceed the discriminatorthreshold. In Table II we show the single-molecule de-tection ef® ciencies for this dye under these experimentalconditions. As can be seen, the detection ef® ciency forthis particular dye is only 50%, signi® cantly poorer thanthat found for IR-132. This observation could result fromthe fact that the unbridged polymethine near-IR dyes,such as IR-125, have poorer photochemical stabilitiescompared to the bridged polymethine dyes, therefore pro-ducing lower numbers of photons per molecule.17

CONCLUSION

We have shown that a simple diode laser coupled to asingle photon avalanche diode can be used as the basicbuilding block for a single-molecule detector that canmonitor the ¯ uorescence signature from individual near-IR dyes in ¯ owing sample streams. In addition, the de-vice can be operated in a CW mode of operation, furtherreducing the complexity and simplifying the operation ofthe device. It is interesting to note that the device dis-cussed in this paper can be constructed for a minimalamount of money. For example, the present single-mol-ecule detection device was constructed for $8000, in-cluding the laser, photodetector, and miscellaneous opticsand electronics. In addition, the single-molecule detectiondevice possessed a sampling volume of 1 pL, making itamenable to sampling small volumes of materials quicklyand ef® ciently. In the present set of experiments, we dem-onstrated that the detection ef® ciency for IR-132 wasnear 100%, even for residence times of 4.5 ms. Therefore,with an approximate 5 ms sampling time and a probevolume of 1 pL, the time required to sample 1 m L ofsolution would be 5000 s or 83 min. However, it shouldbe pointed out that, due to the small size of the probevolume compared to the cross-sectional area of the cap-illary tube used in the present set of experiments, only0.6% of this total volume would be effectively sampled.If one needs to exhaustively sample this entire volume,the capillary tube must be made smaller in order to con-® ne the molecular trajectories through the excitationbeam to produce a sampling ef® ciency approaching100%. While the single-molecule detection ef® ciencywas found to be favorable in the present experiments, the

6 Volume 52, Number 1, 1998

signal-to-noise-ratio for each event was not optimal be-cause the absorption rate was not near saturation. For IR-132, the absorption rate was 5.6 3 107, nearly 253 belowsaturation of the electronic transition. Since the optimalsignal-to-noise ratio occurs near the saturation point,pumping with a higher laser power would produce a bet-ter SNR.

The very simple device that we have constructed will® nd applications in many areas where single-moleculemonitoring may be utilized, especially in biological ap-plications. In this area, a simple and rugged device is anecessity, since many potential users are not well trainedin optics and laser operations. In addition, the modestcost of the device will make it appropriate for smallerlaboratories with limited budgets.

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