Fluorescence Instrument Response Standards in Two-Photon

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Fluorescence Instrument Response Standards in Two-PhotonTime-Resolved Spectroscopy

RAFAL LUCHOWSKI,* MARIUSZ SZABELSKI, PABAK SARKAR, ELISA APICELLA,KRISHNA MIDDE, SANGRAM RAUT, JULIAN BOREJDO, ZYGMUNT GRYCZYNSKI, andIGNACY GRYCZYNSKICenter for Commercialization of Fluorescence Technologies (CCFT), Dept. of Molecular Biology & Immunology, UNTHSC, Fort Worth, Texas

76107 (R.L., P.S., E.A., K.M., S.R., J.B., Z.G., I.G.); Dept. of Biophysics, Institute of Physics, Maria Curie-Sklodowska University, 20-031 Lublin, Poland (R.L.); Dept. of Physics and Biophysics, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland (M.S.); and Dept. of Cell Biology and Anatomy, UNTHSC, Fort Worth, Texas 76107 (I.G.)

We studied the uorescence properties of several potential picosecondlifetime standards suitable for two-photon excitation from a Ti : sapphirefemtosecond laser. The uorescence emission of the selected uorophores(rose bengal, pyridine 1, and LDS 798) covered the visible to near-infraredwavelength range from 550 to 850 nm. We suggest that these compoundscan be used to measure the appropriate instrument response functionsneeded for accurate deconvolution of uorescence lifetime data. Lifetimemeasurements with multiphoton excitation that use scatterers as areference may fail to properly resolve uorescence intensity decays. Thisis because of the different sensitivities of photodetectors in different

spectral regions. Also, detectors often lose sensitivity in the near-infraredregion. We demonstrate that the proposed references allow a properreconvolution of measured lifetimes. We believe that picosecond lifetimestandards for two-photon excitation will nd broad applications inmultiphoton spectroscopy and in uorescence lifetime imaging microsco-py (FLIM).

Index Headings: Instrument response function; IRF; Two-photon excita-tion; Lifetime standards; Fluorescence lifetime imaging microscopy;FLIM.

INTRODUCTION

Time-resolved uorescence measurements require knowl-edge of the instrument response function (IRF) in order todeconvolute the decay parameters. In two-photon excitation(TPE) uorescence spectroscopy there are four major difcul-ties that can affect the IRF. If using scattering of the excitationlight, which is the most convenient way to record the IRF, thecalibration may fail because of (1) sensitivity of the detector;(2) nonlinearity of the TPE in comparison to the Rayleighscattering process, which inherently causes scatterers to beimproper for determination of IRF; (3) the microscopy set up:necessary realignment in order to remove the laser-blockingdichroic beam splitter; and (4) the color effect. Two-photonspectroscopy usually uses infrared (IR) light for excitation, sophoton detection in this wavelength region is a major problemfor almost all standard detectors (photomultiplier tubes(PMTs), avalanche photodiodes (APDs), and charge-coupleddevices (CCDs)). Detection of IR light with PMTs largelycauses emission of electrons not from the cathode but from therst dynode. This process may limit the use of scatterers asIRFs. The color effect for high-speed detectors, such as micro-channel plate photomultipliers (MCP-PMT) or streak cameras,is practically nonexistent. For these detectors, it is usuallysufcient to simply replace the sample with a scatteringmedium and record it as the IRF. However, for the most

popular detectors used in spectroscopy/microscopyPMTsand APDsthe different colors of the reference and uores-cence emission 13 may cause a signicant distortion in thereconvoluted lifetime. The idea of using reference uorophoresto avoid color effects is not a new one. Several research groupshave already proposed a number of useful uorophores to servethis purpose 46 in one-photon excitation spectroscopy. Recent-ly, we also described a few reference uorophores with short lifetimes emitting in the visible and near-infrared (NIR)

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for one-photon spectroscopy.The development of TPE has mostly been spurred bybiological applications 1012 to molecule detection at levels not reached by conventional (one-photon) excitation. The useful-ness of TPE arises from its different selection rules for absorption, higher values for anisotropy, and usually muchsmaller and more easily controllable confocal microscopevolume. In the case of TPE, however, there could be a largedifference between the excitation and emission wavelengths,and the need for reliable reference uorophores is even moredemanding than in single-photon spectroscopy/microscopy.According to our knowledge there are only a few reports onlifetime reference compounds for TPE 1316 that rely ondetection of hyper Rayleigh scattering (HRS) and second-harmonic generation (SHG). The advantage of using HRS andSHG in the lifetime deconvolution of the uorescence of typical uorophores has been shown in examples. We proposehere an alternative method that addresses the potential pitfallsmentioned above; the method relies on recording the IRF in theclose spectral region of emission of the studied uorophores.

In this manuscript, we report a study on TPE referencestandards for time-resolved spectroscopy/microscopy whoseuorescence emissions cover the visible to NIR spectral region.These uorophores can be used to determine the IRF in lifetimemeasurements using TPE as well as for evaluation of detectorsused in TPE spectroscopy/microscopy.

MATERIALS AND METHODS

4-Dicyanomethylene-2-methyl-6-p-dimethylamino-styryl-4H-pyran (DCM), 1-ethyl-2-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-quinolinium perchlorate (LDS 798), and 1-ethyl-4-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-pyridinium per-chlorate (Py1) were purchased from Exciton, Inc. and used asreceived. Rhodamine 800 (Rh800) was from Lambda Physik(Fort Lauderdale, FL); spectral grade (99.9 % ) ethanol (EtOH)and methanol (MeOH) were from Sigma Aldrich and also usedas received.

Absorption and emission spectra were obtained using Cary50 Bio and Cary Eclipse (Varian, Inc.) spectrophotometers.

Received 22 April 2010; accepted 12 May 2010.* Author to whom correspondence should be sent. E-mail: rafal.

[email protected].

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The steady-state quantum yields (QY) of all uorophores usedwere determined using cresyl violet (QY 0.54 in MeOH) as a reference. LDS 798, Py1, and cresyl violet were excited at thesame wavelength (600 nm) and recorded with identicalequipment parameters. Calculations were performed usingextinction coefcients of 4.55 3 104 M 1 cm 1 (LDS 798), 3.83 104 M 1 cm 1 (Py1), and 1.06 3 105 M 1 cm 1 (cresylviolet).

Intensity decays were measured in the time-domain using anFT200 (Picoquant, GmbH) equipped with MCP-PMT (Hama-matsu, Inc.) detector. Data were collected and processed usingthe PicoHarp300 time-correlated single-photon counting(TCSPC) module 17 based on detection of single photons. Apolarizer, monochromator, and proper combination of lterswere placed in the detection path. In order to reject thescattered excitation light we used two 750 nm short wave passlters in front of the monochromator. For all the measurementsthe excitation was obtained using a Ti : sapphire laser with a repetition rate of 80 MHz. Data analysis was performed usingFluoFit software (v. 4.2.1, PicoQuant GmbH) to reconvolutethe uorescence decays on a sum of exponentials:

I t Xi

a i exp t =s i 1

where a i and si are pre-exponential factors and fluorescencelifetimes.

Two-photon uorescence cross-sections were estimated bycomparing two-photon-induced uorescence of the dyes usedwith that of rhodamine B (RhB) in methanol. After detection of the two-photon uorescence signal ( I ), the cuvette with thesolution under study was replaced with a similar reference

without changing the excitation and detection geometry. Thetwo-photon cross-section r was calculated by comparison withthe cross-section of the reference ( r 0 ), rhodamine B (RhB, r 0 170 GM for 810 nm), 18 using the formula

r I U0 r 0 C0

I 0 U C 2

where I 0 is the two-photon fluorescence signal of the reference,C and C0 are the molar concentrations of the sample andreference, respectively, and U and U0 are their quantum yields.

The quadratic dependence of the uorescence intensity onthe excitation intensity was veried for each sample, indicatingthat the measurements were carried out in intensity regimes inwhich saturation or photo-degradation did not occur.

RESULTS AND DISCUSSION

The one-photon absorption (Fig. 1a) and emission (Fig. 1b)spectra of dyes as potential IRFs are presented in Fig. 1. Their characteristics are summarized in Table I together with thevalues of the TPE cross-sections for 810 nm excitation. Colorsand appearance presented in Fig. 1a correspond to the colorsand appearance of emission curves presented in Fig. 1b. As canbe seen (Fig. 1b), the emissions of the dyes used for two-photon excitation cover the part of the spectra (550850 nm)that is popularly recognized as a high signal-to-noise ratio

FIG. 1. (a ) Absorption and ( b) emission spectra of pyridine 1 (solid line), LDS798 (dotted line) and rose bengal (dashed line) in water. Rose bengal solutioncontained 5 M potassium iodide. Arrows indicate the observation wavelengths.

TABLE I. Photophysical data of chromophores used as references fortwo-photon excitation experiments. Cross-sections were calculated rela-tive to RhB.

Compound kmax [nm]a k max [nm]b Uc r [GM]d

RB KI in H2 O 550 570 0.003 50Py1 in H 2 O 430 670 0.03 0.35LDS 798 in H 2 O 500 780 0.002 2.4a One-photon absorption maxima.b One-photon emission maxima.c Fluorescence quantum yields determined relative to cresyl violet in MeOH.d Cross-sections calculated for 810 nm in GM (10 50 cm4 s photon 1 ) with

relative error 6 20% .

FIG. 2. Emission spectra of pyridine 1 in MeOH measured with ( a) one- and(b) two-photon excitation. Dashed lines represent the emission spectra with 2.3-fold attenuation of the excitation power. ( c) Loglog plot of the two-photon-induced uorescence intensity ( I ) versus different excitation laser powers ( P)(t calculation performed for 0.40.8 W laser power with regression coefcient equal to 0.9986).

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region for detectors. Fluorescence signals of the dyes weremeasured at wavelengths indicated by the arrows in Fig 1b.

The two-photon nature of excitation is usually proved bystudying the dependence of its uorescence intensity on theexcitation power. Figure 2 presents the results of such a studyfor Py1 in MeOH excited in both the one- and two-photonmodes at 500 nm and 810 nm, respectively. As can be seen, theemission spectra show a similar shape for one-photon (Fig. 2a)and two-photon (Fig. 2b) excitation processes. Next, weattenuated the excitation with a 0.4 neutral density lter, whichreduced the excitation power by 2.3 fold (Figs. 2a and 2b,dashed lines). The attenuation of the excitation power

proportionally reduced the one-photon-induced uorescenceby about 2.3 fold and the two-photon-induced uorescence byabout 5.3 fold. A more detailed study of the two-photon-induced uorescence of pyridine 1 as a function of theexcitation power is shown in Fig. 2c, which clearly indicatesthe two-photon process. We observed similar dependencies for other references studied, rose bengal (RB) and LDS 798.

Figure 3 presents the time responses of the silica scatterer Ludox and the proposed lifetime standards measured using theMCP-PMT detector. Excitation was at 810 nm from a Ti : sapphire laser (80 MHz repetition rate, 100 fs pulses) andthe observation was at various wavelengths, from 550 nm to

810 nm. All responses are very short, with full widths at half-maximum (FWHM) below 70 ps. The shortest FWHM of theIRF reported in the literature for the scatterer is 32 ps, 19 whichcorresponds well with the observations of our experiment (36ps, Fig. 3a). The time responses of uorophores are broader and depend on the lifetime of the dye. The relatively short lifetimes make those compounds attractive as IRF standards,allowing color-effect-free measurements in the emission regionof the studied specimens.

We measured the lifetimes of the proposed standards for time-resolved two-photon-induced uorescence using the same

FIG. 3. Temporal proles of ( a ) scattered 810 nm excitation light, and two-photon-induced uorescence of: rose bengal 5 M KI observed at (b) 550 nmand (c) 600 nm; pyridine 1 observed at ( d) 600 nm and ( e) 700 nm; and ( f ) LDS798 observed at 750 nm. Responses were measured using the ultra-fast-detector MCP-PMT.

FIG. 4. Two-photon-induced uorescence intensity decays of: ( a ) rose bengalin the presence of 5 M KI, ( b) pyridine 1 in water, and ( c) LDS 798 in water.The instrument response function was obtained from a Ludox scatterer (red).The recovered lifetimes are: 16 ps for rose bengal, 37 ps for pyridine 1, and 27ps for LDS 798. The excitation (810 nm) was from a femtosecond Ti: sapphirelaser.

TABLE II. The measured lifetimes of recommended picosecond lifetimestandards in two-photon excitation. a

Compound Obs. [nm] IRF a s (ps) v2R

RB KI in H2 O 550 Ludox 1 14 2.0RB KI in H2 O 580 Ludox 1 16 1.9RB KI in H2 O 600 Ludox 1 17 1.4RB KI in H2 O 620 Ludox 1 17 1.9Py1 in H 2 O 620 Ludox 1 36 1.2Py1 in H 2 O 660 Ludox 1 36 1.6Py1 in H 2 O 700 Ludox 1 37 1.4LDS 798 in H 2 O 750 Ludox 1 27 1.9a The samples were excited at 810 nm.

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scatterer (Ludox) as the IRF. Figure 4 presents time-domainmeasurements for RB, Py1, and LDS 798 observed at 580 nm,700 nm, and 750 nm, respectively. The uorescence decays of all the molecules were tted with a single exponential modelwith lifetimes of 16 ps for RB, 27 ps for LDS 798, and 37 psfor Py1. We measured the lifetimes of these compounds for various observation wavelengths, from 550 nm to 750 nm.Good ts to the decays were found, with small values of v2R .All the results are summarized in Table II.

The resolution of decays on the order of a few or tens of nanoseconds using picoseconds standards is easy and intui-tively obvious. The question arises of whether subnanosecond

decays can be reasonably resolved using the proposedreferences. We selected uorophores with subnanosecondlifetimes and measured their intensity decays with both thescatterer and the reference IRFs. The recovered decayparameters are presented in Table III. There are only minimaldifferences in the lifetimes of the references and the scatterer.As an example, we present time-domain lifetime measurementsof the same sample of DCM in methanol resolved with thescatterer and the proposed references (Fig. 5). The reconvolu-tion resulted in the same lifetime, independent of IRF.

We believe that one can use either the scatterer or theproposed reference if detection is carried out with a micro-channel plate photomultiplier. In the case of photodiodes or photomultipliers, however, the reference standards offer detection at the same wavelengths as the emissions of thestudied uorophores. Also, these detectors are more sensitivein the uorescence region than in the NIR region.

Finally, we present the subnanosecond lifetime measure-ments in the NIR region. The two-photon-induced uorescenceintensity decay of LDS 798 in ethanol is presented in Fig. 6.The reconvolution of the decay with the IRF obtained fromLDS 798 in water resulted in a lifetime of 148 ps, just 4 psshorter than that recovered using the scatterer (Table III).

CONCLUSION

We believe that the proposed picosecond lifetime referencestandards will accurately deconvolute decays in two-photon-induced time-resolved uorescence measurements. The advan-tage of uorescence references over scattering is evident because decay measurements can be deconvoluted with the

TABLE III. Subnanosecond/nanosecond intensity decays of dyes with two-photon excitation measured versus different IRFs.

Compound Obs. [nm] IRF a 1 s1 [ps] a 2 s2 [ps] s [ps]a v2R

RB in MeOH 580 Ludox 1 516 - - 516 2.2RB in MeOH 580 RB KI 1 510 - - 510 2.3DCM in MeOH 620 Ludox 0.82 1299 0.18 225 1264 1.1DCM in MeOH 620 RB KI 0.82 1291 0.18 194 1259 1.0DCM in MeOH 620 LDS 798 in H 2 O 0.82 1292 0.18 197 1256 1.1DCM in MeOH 620 Py1 in H 2 O 0.84 1277 0.16 179 1248 1.1LDS 798 in EtOH 750 Ludox 1 152 - - 152 2.4

LDS 798 in EtOH 750 LDS 798 in H 2 O 1 148 - - 148 1.3a The samples were excited at 810 nm.b s X

i

a is i .

FIG. 5. Two-photon-induced uorescence intensity decay of DCM in MeOHreconvoluted versus: ( a) scatterer (Ludox), and using ultra-short uorescencestandards: ( b ) quenched rose bengal, ( c) LDS 798 in water, and ( d) pyridine 1in water. The excitation was from a Ti: sapphire laser (810 nm, 80 MHzrepetition rate).

FIG. 6. Two-photon-induced uorescence intensity decay of LDS 798 inethanol. A solution of LDS 798 in water was used for the IRF.

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Fluorescence Instrument Response Standards in Two-Photon