Fluorophores for single molecule microscopy

JOURNALOF

LUMINESCENCE ELSEMER

Journal

of Luminescence

Fluorophores

72-74 (1997) 18-21

for single molecule microscopy

G.J. Schlitz, H.J. Gruber, H. Schindler, Th. Schmidt* Institute ,for Biophysics.

Unioersi~v of Lin:, Alfenherger

Str. 69, 4040 Linz. Austria

Abstract The fluorescence photoncount distributions, photobleaching characteristics, and saturation intensities of the commonly used fluorescence tags tetramethylrhodamine and phycoerythrin were measured on the level of individual molecules. Although, the fluorescence properties of phycoerythrin seem to be superior in view of single molecule detection, the increased photostability of tetramethylrhodamine coupled both, to a lipid or an antibody, makes it the molecule of choice for imaging applications.

Keywords: Single molecule detection; Photophysics; Photostability;

1. Introduction The increased sensitivity of state-of-the art optical detectors in combination with carefully designed optical systems allow for detection and imaging at the ultimate limit: the single fluorophore. Such single molecule techniques have been developed in the last years bearing numerous applications from highest-resolution spectroscopy [1,2] over DNA-sequencing [3,4] to control of biological evolution [S]. Detection of single molecular interactions [6] and time-resolved imaging of individual molecules in biological membranes [7,8] has been shown, expanding the applications of single molecule technology to native systems. In particular, the applicability to complex, heterogeneous systems as biomembranes seems to be one of the exciting directions of research in this field. For this, a thorough understanding of the photophysics

*Corresponding author. Fax: ( + 43) 732 2468 822; e-mail: [email protected]. 0022-2313/97/$17.00 ;q 1997 Elsevier Science B.V. All rights reserved PII SOO22-23 13(97)00090-2

Biological membrane

of the fluorophores used in the detection schemes is a prerequisite. In the current paper we have compared some photophysical parameter of typical fluorophores used in bioscience. The fluorescence photoncount distribution and fluorescence bleaching characteristics of individual tetramethylrhodamine molecules covalently linked to phospholipids and antibodies, and phycoerythrin linked to a biotin/ streptavidin complex were studied using a recently developed single molecules imaging technique [S].

2. Experimental 2.1. Sample preparation Samples were studied on supported phospholipid membranes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholin (POPC, Avanti) drawn from a monolayer trough using the Langmuir-Blodgett technique [7]. The membrane was held at ambient

19

G.J. Schiitz et al. 1 Journal of Luminescence 72-74 (1997) 18-21

from Rayleigh and Raman-shifted light by appropriate filter combinations (5 lSDRLPEXT02 dichroic, 570DF70 block, Omega, OG550 lowpass, Schott). Data were obtained at a rate of 29 images/s using a modified liquid nitrogen cooled CCD-camera system (AT200, Photometrix, equipped with a TK512CB-chip, Tektronix) and stored on a PC. An automatic analysis program determined the position of each fluorescence complex to within z 30 nm and its fluorescence signal to _ 20% accuracy by fitting the fluorescence photoncount profile to a two-dimensional Gaussian surface.

temperature and continuously flushed by aqueous buffer (150 mM NaCl, 10 mM NaH2P04, pH7.4). Concentrations of the tetramethylrhodamine labeled lipids (TRITC-DHPE, T1391, Molecular Probes, lop7 mol/(mol POPC)), the biotin labeled lipids (Biotin-X-DMPE, B-1616, Molecular Probes, 10e6 mol/mol), and the dinitrophenyl labeled lipids (DNP-X-DHPE, D-3798, Molecular Probes, 10e6 mol/mol) in the outer layer of the membrane were adjusted such, that the surface density of fluorescence labeled molecules did not labeled exceed 0.1 urn-‘. Tetramethylrhodamine antibody (aDNP-IgG-TMR, 2.9 anti-DNP A-5784, Molecular Probes, TMR/antibody, 50 nM), and phycoerythrin labeled biotin (RPhyXX-biotin, P-811, Molecular Probes, 50 nM) were applied in the aqueous subphase.

3. Results and discussion

2.2. Apparatus and data analysis The apparatus (Fig. l), data acquisition and automatic data analysis system were used as previously described in detail [7]. In brief, samples were observed while illuminated for 5 ms by (25 * 6) kW/cm’ of 528 nm circular polarized light from an Ar+-laser (C306, Coherent) using a x 100 objective (PlanNeofluar, NA = 1.3, Zeiss) in an epi-fluorescence microscope (Axiovert 135TV, Zeiss). The fluorescence was effectively separated

The fluorescence photoncount distributions obtained from individually fluorescence labeled molecules for the three systems studied are compared in Fig. 2. In all experiments, the samples were illuminated for 5 ms at a laser intensity of 25 kW/cm*. Each image of a single molecule seen as a fluorescence peak, well separated from the background, was analyzed using a non-linear least-squares fit procedure [9]. This analysis yielded values for the photoncounts, Fi, and the photoncount confidence intervals, dFi, on the basis of a single fluorophore. Subsequently, the photoncount distributions are

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G.J. Schiitz et al. /Journal of Luminescence 72-74 (1997) 18-21

20

DHPE.TMR

biotin:phycoerylhrin

0

200

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400

600

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intensity (cnts)

Fig. 2. Probability density (Eq. (1)) of the fluorescence photoncounts obtained for individual fluorescence labeled molecules illuminated for 5 ms at a laser intensity of 25 kW/cm’. The probability density of the background photoncounts is shown for comparison (dashed). The mean values are (F) = 170, 254 and 488 cnts for DHPE : TMR, antibody : TMR, and phycoerythrin, respectively. N = 116, 284 and 623 molecules, respectively, were analyzed.

constructed in the form of probability-density tions, p(F) [IS]:

p(F) =&jjri;, exp =- ;Fi i

(F - Fi)2 ’ 2dF,’

func-

(1)

The mean fluorescence photoncounts, (F), and its respective standard deviation, CJ~,calculated from the square-root of the variance are determined from these distributions. For single tetramethylrhodamine molecules (TMR) covalently linked to single lipid molecules (DHPE : TMR) (F) = 170 f 67 cnts (N = 116 molecules analyzed) [7]. In comparison, the fluorescence photoncount distribution of individual antibodies tagged with tetramethylrhodamine molecules (antibody : TMR) is much broader and shifted to higher values. It is characterized by (F) = 254 k 132 cnts (N = 284). These higher values compared to DHPE : TMR are expected, since each antibody is labeled with 2.9 TMR molecules, on average [lo]. The three distinct peaks

and shoulders of the latter distribution at 93, 187, and 426 cnts might be attributed to antibodies labeled by 1, 2, and 4 fluorophores, respectively. With this interpretation, the fluorescence photoncount of a single tetramethylrhodamine molecule attached to a protein seems to be decreased by z 50% from 170 to 93 cnts compared to a TMR molecule attached to a lipid. For a more detailed analysis, however, taking into account the effect of fluorescence self-quenching more data have to be accumulated. The fluorescent protein phycoerythrin was the first molecule individually observed [ll]. Its advantage in ultrasensitive detection schemes is the very high extinction coefficient [12] and large Stokes shift as pointed out in Ref. [ 111. The fluorescence photoncount distribution of individually observed phycoerythrin molecules covalently bound to a single biotin molecule (biotin : phycoerythrin) is shown in Fig. 2. The mean fluorescence photoncount of (F) = 488 f 281 cnts (N = 623), is about three times that of a TMR attached to a lipid. The fluorescence photoncount distribution for biotin: phycoerythrin is very broad, even two times broader than that of the fluorescence labeled antibody. This extremely broad distribution can be explained by photobleaching. In our experiments we observed that photobleaching, the process of photo-induced destruction of the fluorophore, is much more rapid for phycoerythrin compared to tetramethylrhodamine. For the laser intensity of 25 kW/cm2 as used throughout this study, the average lifetime of tetramethylrhodamine for both, DHPE : TMR and antibody : TMR was 12 + 2 ms, whereas that of biotin : phycoerythrin was only 6 + 3 ms. The lifetime of biotin : phycoerythrin thus is comparable with the illumination time of 5 ms leading to significant photobleaching during illumination. This causes a broadening of the photoncount distribution to smaller values as being observed. Next to the fluorescence photoncounts, and the photobleaching characteristics of a molecule, the saturation of the fluorescence on laser intensity is an important photophysical parameter to be evaluated [ll]. Fluorescence saturation is explained considering a three-state model for the energy level structure of the fluorophore consisting of a ground,

G.J. Schiitz et ul. j Journal of Luminescence 72~74 (1997) 18-21

singlet excited, and triplet excited state. The triplet state is the bottleneck for efficient absorption-emission cycling. From this model, which neglects photobleaching, the dependence of the fluorescence photoncounts on the illumination intensity, IL, and the illumination time, till< is calculated: F = katiii/(l + 1,/I,). In this equation f, denotes the saturation intensity. We have performed experiments on individual DHPE: TMR [7] and molecules yielding k, = biotin:phycoerythrin 49 i 3, and 70 i lOcnts/ms, and I, = 7.6 ? 1.1, and 5 f 2 kW/cm2, respectively. As expected from the higher values for the absorption cross section and fluorescent quantum yield of biotin:phycoerythrin in comparison with DHPE : TMR, 1, is lower and k, higher for biotin : phycoerythrin. However, a significant part of the phycoerythrin molecules bleached within the illumination time, leading to a reduced value for k, and an increased width of the photoncount distribution (see Fig. 2).

mono-labeled proteins [S], seems impossible employing such statistically labeled ligands. Thus we believe, that in particular single-molecule singlelabel techniques applied to biosystems will allow for new and exciting research, from which a more detailed understanding of the structure/function relationship of biosystems is envisioned.

Acknowledgements

Financial support from the Austrian Research Fonds (project S06607-MED) is acknowledged.

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4. Conclusions In summary we have determined photophysical parameter of commonly used fluorescent tags. In view of the applicability to single molecule detection, the tetramethylrhodamine dye molecule seems superior to phycoerythrin. In particular, its higher photostability overcomes its disadvantage of lower absorption cross section and lower quantum yield, such that the total number of photons emitted from a single tetramethylrhodamine is even higher than that from a single phycoerythrin. There is an obvious advantage of proteins labeled with a few fluorescence markers in comparison to mono-labeled molecules because of their higher mean fluorescence photoncount for detection. However, due to the inherent photoncount distribution given by the statistical labeling of the protein, data analysis and interpretation becomes more difficult. For example, the determination of local stoichiometries by fluorescence photoncount correlation, as shown for

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[4] R. Rigler, J. Windengren and ii. Mets, in Fluorescence Spectroscopy, 0. Wolfbeis, Ed. W.B. Whitten, J.M. Ramsey, S. Arnold, B.V. Bronk; (Springer. Berlin. 1992) Anal. Chem. 63 (1991) 1027. [S] R. Rigler and M. Eigen, Proc. Natl. Acad. (1994) 5740. [6] Th. Schmidt, G.J. Schiitz, H.J. Gruber Anal. Chem. 68 (1996) 4397.

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R.P. Haugland, Handbook of Fluorescent Research Chemicals, Molecular Probes.

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Proc. and