Anal. Chem. 2003, 75, 2147-2153
Detection and Identification of Single Molecules in Living Cells Using Spectrally Resolved Fluorescence Lifetime Imaging Microscopy Jens-Peter Knemeyer, Dirk-Peter Herten, and Markus Sauer*
Physikalisch-Chemisches Institut, Universita¨t Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany
The detection of single mRNA molecules tagged by microinjected, singly fluorescently labeled oligo(dT) 43-mer molecules in living cells in quasi-natural surrounding, that is, cell culture medium, is demonstrated. Single-stranded oligonucleotides were labeled at the 5′-end with a redabsorbing oxazine derivative (MR121) and excited by a pulsed laser diode emitting at 635 nm with a repetition rate of 64 MHz. Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) on untreated living 3T3 mouse fibroblast cells reveals autofluorescence signals found predominately in the cytoplasm with fluorescence lifetimes of ∼1.3 ns and emission maximums of ∼665670 nm. Hence, fluorescence signals of single MR121labeled oligonucleotide molecules that exhibit a fluorescence lifetime of 2.8 ns and a fluorescence emission maximum of 685 nm can be easily discriminated against autofluorescence. MR121-labeled oligonucleotides were microinjected into the cytoplasm or nucleus of living 3T3 mouse fibroblast cells using a micropipet. Since the micropipet exhibits an inner diameter of 500 ( 200 nm at the very end of the tipscomparable to the diameter of the detection volume appliedsthe number of molecules delivered into the cell via the micropipet can be counted. Furthermore, the presented technique enables the quantitative detection and time-resolved identification of single molecules in living cells as a result of their characteristic emission maximums and fluorescence lifetime. The results obtained from single-molecule studies demonstrate for the first time that 10-30% of the microinjected oligo(dT) 43-mer molecules cannot diffuse freely inside of the nucleus but, rather, are tethered to immobile elements of the transcriptional, splicing, or polyadenylation machinery. In the past few decades, in situ hybridization (ISH) protocols have been optimized such that they allow the microscopic detection of specific mRNA molecules employing fluorescently labeled nucleic acid probes. Essential steps in these protocols are the fixation of the biological sample, the subsequent permeabilization of the specimen to improve probe or antibody penetration, the hybridization of a probe to a target sequence, and finally, a * To whom correspondence should be addressed. Phone: +49-6221-548460. Fax: +49-6221-544255. E-mail:
[email protected]. 10.1021/ac026333r CCC: $25.00 Published on Web 03/25/2003
© 2003 American Chemical Society
proper washing and detection procedure.1,2 Although a lot of effort is still being put into optimizing RNA-ISH procedures on fixed cells and tissue materials, there is an increasing demand for techniques that allow the visualization of RNAs in living cells. The reason for this interest in living cell analysis is the concern whether the observed RNA localization patterns in fixed cells reflect the situation in living cells. Fixation and cell pretreatment may disturb localization patterns and to some extent deteriorate cell morphology.3 In addition, information about the dynamics of RNA or DNA synthesis and transport can be obtained best when these processes are studied in living cells. At present, little is known about the rate at which RNA or DNA molecules are synthesized and processed and about the trafficking of the different contributing molecules.4, 5 A way to learn more about the spatial localization and kinetics of these processes is to tag RNA, DNA, and associated enzymes with a fluorescent moiety, such as GFP, and to monitor them at different time intervals in living cells by real-time microscopy.6 A promising technique to reveal the structural interplay of different protein and mRNA molecules is the use of fluorescence resonance energy transfer (FRET) to measure their distances. FRET enables the determination of distances between donor- and acceptorlabeled molecules in the range of 2-8 nm, even between single molecules.7,8 Other high-resolution colocalization methods use different colors or fluorescence lifetimes of dyes to measure the distance between single biomolecules.9,10 The replication foci formed during the S phase of the cell cycle where DNA synthesis occurs, or the transcription factories, where DNA is transcribed into RNA, represent two examples of assemblies (molecular machines) where numerous nucleic acids and protein components are localized.11 However, little is known about how the components (1) Lawrence, J. B.; Singer, R. H.; Marselle, L. M. Cell 1989, 57, 493-502. (2) Dirks, R. W. Histochem. Cell. Biol. 1996, 106, 151-166. (3) Dirks, R. W.; Molenaar, C.; Tanke, H. J. Histochem. Cell. Biol. 2001, 115, 3-11. (4) Cardoso, M. C.; Leonhardt, H. J. Cell. Biochem. 1998, 70, 222-230. (5) Cardoso, M. C.; Leonhardt, H.; Nadal-Ginard, B. Cell 1993, 74, 979-992. (6) Tsien, R. Y. Annu. Rev. Biochem. 1998, 67, 509-544. (7) Weiss, S. Science 1998, 283, 1676-1683. (8) Kim, H. D.; Nienhaus, G. U.; Ha, T.; Orr, J. W.; Williamson, J. R.; Chu, S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4284-4289. (9) Lacoste, Th. D.; Michalet, X.; Pinaud, F.; Chemla, D. S.; Alivisatos, P.; Weiss, S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9461-9466. (10) Heilemann, M.; Herten, D. P.; Heintzmann, R.; Cremer, C.; Mu ¨ ller, C.; Tinnefeld, P.; Weston, K. D.; Wolfrum, J.; Sauer, M. Anal. Chem. 2002, 74, 3511-3517. (11) Cook, P. R. Science 1999, 284, 1790-1795.
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are arranged in the assemblies and how they move during different cell cycle phases. One method to investigate the localization and transport pathways of specific RNA species inside living cells is to introduce fluorescently labeled RNA or complementary oligonucleotide molecules by microinjection and to follow their spatial and temporal distribution by fluorescence microscopy.12-14 Unfortunately, because of the limited sensitivity of common fluorescence microscopic imaging systems, an excess of fluorescently labeled molecules has to be introduced. This can significantly alter existing cellular RNA processing and transport pathways as a result of saturation and nonspecific binding of oligonucleotides to cell compartments. This underscores the need for sensitive imaging techniques for the monitoring of localization, organization, and the dynamic interplay of various molecules at the single-molecule level with high spatial and temporal resolution. While individual fluorescent molecules can be detected in solution,15-17 on surfaces or fixed cells,18-20 and in membranes of living cells21,22 with good signal-to-noise ratio (S/N), a key question is whether single molecule methodologies could be developed to study complex molecular processes in living cells. In contrast to clean and well-controlled conditions in vitro, the intracellular environment contains a broad collection of biological macromolecules and fluorescent materials, such as porphyrins and flavins. This complex environment is known to produce intense background fluorescence, commonly known as autofluorescence. Therefore, nanomolar or higher concentrations of fluorescently tagged proteins or oligonucleotides have been applied to measure intracellular and intranuclear diffusion by second-order fluorescence intensity correlation spectroscopy (FCS).23,24 Because relatively high probe concentrations were used, the detection of subpopulations, for example, small fractions of immobile molecules, is more difficult. Recently, Brock et al. showed the first signals of single diffusing EGFR-EGF fusion proteins in living cells using cells with very low expression levels.25 Confocal fluorescence microscopy has been applied, as well, to detect fluorescence bursts of rhodamine-labeled oligonucleotide molecules in living cells.26 Excitation was performed using the 488- or 514-nm line of argon ion lasers. To reduce the autofluorescence of the cell culture (12) Ainger, K.; Avossa, D.; Morgan, F.; Hill, S. J.; Barry, C.; Barbarese, E.; Carson, J. H. J. Cell. Biol. 1993, 123, 434-441. (13) Jacobson, M. R.; Pederson, T. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 50945099. (14) Kruse, C.; Willkomm, D. K.; Grunweller, A.; Vollbrandt, T.; Sommer, S.; Busch, S.; Pfeiffer, T.; Brinkmann, J.; Hartmann, R. K.; Muller, P. K. Biochem. J. 2000, 346, 107-115. (15) Mets, U ¨ .; Rigler, R. J. Fluoresc. 1994, 4, 259-264. (16) Nie, S.; Chiu, N. T.; Zare, R. N. Science 1994, 266, 1018-1022. (17) Ambrose, W. P.; Goodwin, P. M.; Jett, J. H.; van Orden, A.; Werner, H. J.; Keller, R. A. Chem. Rev. 1999, 99, 2929-2956. (18) Weston, K. D.; Carson, P. J.; Metiu, H.; Buratto, S. K. J. Chem. Phys. 1998, 109, 7474-7485. (19) Veerman, J. A.; Garcia-Parajo, M. F.; Kuipers, L.; van Hulst, N. K. Phys. Rev. Lett. 1999, 83, 2155-2157. (20) Fermino, A. M.; Fay, F. S.; Fogarty, K.; Singer, R. H. Science 1998, 280, 585-590. (21) Schu ¨ tz, G. J.; Kada, G.; Pastuhenko, V. P.; Schindler, H. EMBO J. 2000, 19, 892-897. (22) Sako, Y.; Minoguchi, S.; Yanagida, T. Nat. Cell Biol. 2000, 2, 168-172. (23) Politz, J. C.; Browne, E. S.; Wolf, D. E.; Pederson, T. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6043-6048. (24) Schwille, P.; Haupts, U.; Maiti, S.; Webb, W. W. Biophys. J. 1999, 77, 22512265. (25) Brock, R.; Va`mosi, G.; Vereb, G.; Jovin, T. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10123-10128. (26) Byassee, T. A.; Chan, W. C. W.; Nie, S. Anal. Chem. 2000, 72, 5606-5611.
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medium arising upon excitation in the green wavelength range, most measurements were performed in PBS. More recently, Bra¨uchle and co-workers demonstrated the monitoring of the infection pathway of single viruses using real-time fluorescence imaging.27 To avoid the elimination of signals arising from single molecules due to background subtraction or application of a threshold, excitation in the red spectral range has been used. Above 600 nm, there are only a few naturally occurring substances that efficiently absorb and emit light.28,29 Here, we present a new technique for single-molecule imaging in living cells based on confocal spectrally resolved fluorescence lifetime imaging microscopy (SFLIM).30 The method takes advantage of fluorescent dyes that can be efficiently excited by a pulsed diode laser emitting at 635 nm but differ in their fluorescence lifetime and emission maximums from autofluorescence signals. To demonstrate the potential of the technique, we studied the localization of rhodamine 800 in live cells and the hybridization of fluorescently labeled oligo(dT) 43-mer molecules to poly(A) RNA molecules in the nucleus of living 3T3 mouse fibroblast cells. To ensure a controlled delivery of labeled oligonucleotides into the nucleus, a new microinjection technique has been developed. This technique allows the on-line fluorescence detection of each molecule passing the very end of the micropipet with an inner diameter of 500 ( 200 nm. MATERIALS AND METHODS Synthesis of Fluorescently Labeled Oligonucleotides. Rhodamine 800 was purchased from Lambda Physik (Go¨ttingen, Germany). The oxazine dye MR121 was kindly provided by K. H. Drexhage (Universita¨t-Gesamthochschule Siegen). The oligonucleotide (dT)43 carrying a C6 amino linker at the 5′ terminus was custom-synthesized by Carl Roth GmbH (Karlsruhe, Germany). The coupling reaction was carried out as follows: A 20nmol portion of the oligonucleotide was dissolved in 10 µL of sterile water, and the following reagents were added in order: 25 µL of O-(5-norbornene-2,3-dicarboximido)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (100 mg/mL dimethylformamide), 20 µL of the carboxyoxazine (MR121; 5 mg/mL dimethylformamide), and 5 µL of diisopropylethylamine. The solution was incubated for 4-8 h at room temperature in the dark. The labeled oligonucleotides were purified by reversed-phase (RP18-coluumn) HPLC (Aligent Technologies, Waldbronn, Germany) using a gradient of 0-75% acetonitrile in 0.1 M aqueous triethylammonium acetate. Cell Culture and Microinjection. 3T3 mouse fibroblast cells were cultured as exponentially growing monolayers in RPMI 1640 medium without phenol red supplemented with 10% (v/v) fetal calf serum (FCS) and 1 mM glutamine at 37 °C under 5% CO2. At ∼80% confluency, the cells were trypsinized and diluted in media solution. Before the experiments, the cells were washed twice with PBS, transferred into small cell culture dishes (PetriPERM 50 hydrophilic; Sartorius, Go¨ttingen, Germany) in the appropriate (27) Seisenberger, G.; Ried, M. U.; Endreβ, T.; Bu ¨ ning, H.; Hallek, M.; Bra¨uchle, C. Science 2001, 294, 1929-1933. (28) Sauer, M.; Zander, C.; Mu ¨ ller, R.; Ullrich, B.; Drexhage, K. H.; Kaul, S., Wolfrum, J. Appl. Phys. B 1997, 65, 427-433. (29) Neuweiler, H.; Schulz, A.; Vaiana, A. C.; Smith, J.; Kaul, S.; Wolfrum, J.; Sauer, M. Angew. Chem. Int. Ed. 2002, 114, 4769-4773. (30) Tinnefeld, P.; Herten, D. P.; Sauer, M. J. Phys. Chem. A 2001, 105, 79898003.
Figure 1. Scanning confocal fluorescence image (20 × 20 µm) of an untreated 3T3 mouse fibroblast cell in RPMI 1640 medium containing 10% (v/v) fetal calf serum, and 1 mM glutamine at room temperature (25 °C). The cell was scanned from top to bottom and from left to right with a resolution of 50 nm/pixel and an integration time of 6 ms/pixel at an excitation intensity of 5 kW/cm2. (A) Overall fluorescence intensity image detected at the short-wavelength detector I and long-wavelength detector II. (B) Fractional intensity image (F2 image) recorded at the longwavelength detector 2, F2 ) I2/(I1 + I2). (C) Fluorescence lifetime image (τ image) calculated from the overall photon counts detected at detectors I and II.
concentration, and left in medium at 37 °C under 5% CO2 for 48 h to fully attach to the surface. All live cell measurements were done at room temperature (25 °C). Rhodamine 800 was diluted to the appropriate concentration in the growth medium (10-8 M) and was introduced to the cell culture dishes to incubate for 2 h. Then cells were washed twice with PBS and left in medium at 37 °C under 5% CO2 for 4 h. For microinjection, 10-9 M aqueous solutions of the labeled oligo(dT) 43-mer were poured into micropipets (Femtotip I; Eppendorf-Nethler-Hinz, Hamburg, Germany) with an inner diameter of 500 ( 200 nm at the very end of the tip. Microinjection was performed with a FemtoJet (EppendorfNethler-Hinz, Hamburg, Germany), increasing the applied pressure of 5 hPa to 500 hPa over some seconds. Spectrally Resolved Fluorescence Lifetime Imaging Microscopy (SFLIM). Details of the setup for SFLIM are described elsewhere.30 For excitation, a pulsed diode laser with a center wavelength of 635 nm, a repetition rate of 64 MHz, and a pulse length of
