Simultaneous single molecule atomic force and fluorescence lifetime imaging

Transcription

1 Simultaneous single molecule atomic force and fluorescence lifetime imaging Olaf Schulz 1, Felix Koberling 2, Deron Walters 3, Marcelle Koenig 2, Jacob Viani 3, Robert Ros 1* 1 Department of Physics, Arizona State University, Tempe, AZ , USA 2 PicoQuant GmbH, Rudower Chaussee 29, Berlin, Germany 3 Asylum Research, 6310 Hollister Ave., Santa Barbara, CA 93117, USA ABSTRACT The combination of atomic force microscopy (AFM) with single-molecule-sensitive confocal fluorescence microscopy enables a fascinating investigation into the structure, dynamics and interactions of single biomolecules or their assemblies. AFM reveals the structure of macromolecular complexes with nanometer resolution, while fluorescence can facilitate the identification of their constituent parts. In addition, nanophotonic effects, such as fluorescence quenching or enhancement due to the AFM tip, can be used to increase the optical resolution beyond the diffraction limit, thus enabling the identification of different fluorescence labels within a macromolecular complex. We present a novel setup consisting of two commercial, state-of-the-art microscopes. A sample scanning atomic force microscope is mounted onto an objective scanning confocal fluorescence lifetime microscope. The ability to move the sample and objective independently allows for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. Time correlated single photon counting (TCSPC) gives us the opportunity to measure single-molecule fluorescence lifetimes. We will be able to study molecular complexes in the vicinity of an AFM probe on a level that has yet to be achieved. With this setup we simultaneously obtained single molecule sensitivity in the AFM topography and fluorescence lifetime imaging of YOYO-1 stained lambda-dna samples and we showed silicon tip induced single molecule quenching on organic fluorophores. Keywords: AFM, FLIM, DNA, confocal, quenching 1. INTRODUCTION Advances in nanoscale science and technology, as well as progress in life sciences are closely related to the development of novel microscopy and spectroscopy techniques. Particularly the combination of complementary techniques, allowing the simultaneous acquisition of multi-parameter data, has the potential to give deeper insights into nanoscale systems. Over the last decades, tremendous progress has been made in scanning probe and optical microscopy, especially in imaging single molecules 1,2. For 3D noninvasive imaging of living cells, exciting progress has been achieved for sub-diffraction imaging with resolutions of 50 nm and below using different approaches of optical nanoscopy (reviewed in 3 ). Processes like FRET, fluorescence quenching and plasmon resonances between single dyes or nanoparticles can act as precise molecular rulers. This is frequently applied to study dynamics and conformational changes in biomolecules 4-7. Atomic force microscopy (AFM) has the capability to image surfaces with nanometer or even subnanometer resolution under ambient as well as physiological conditions 8. AFM is also used to manipulate single molecules with piconewton and sub-nanometer accuracy 9. Optical near field technologies are * robert.ros@asu.edu; phone: ; web: Single Molecule Spectroscopy and Imaging III, edited by Jörg Enderlein, Zygmunt Karol Gryczynski, Rainer Erdmann, Proc. of SPIE Vol. 7571, SPIE CCC code: /10/$18 doi: / Proc. of SPIE Vol

2 capable to combine topographical and optical information. The best optical resolutions are in the range of 10 nm. The basis for all high resolution near field applications is the emission behavior of fluorophores on surfaces in close proximity to a sharp tip. This behavior has been studied experimentally as well as theoretically The fluorescence properties of fluorophores (e.g. quantum yield, fluorescence lifetime, blinking, etc.) depend on their local environment. This is for instance used in fluorescence lifetime imaging microscopy (FLIM) 30. The measurement of the fluorescence lifetime can be realized by employing time correlated single photon counting (TCSPC). In TCSPC, pulsed lasers are used in combination with single photon detectors. By synchronizing photon arrival times with the laser repetition, fluorescence lifetimes can be measured. It has been shown that the combination of AFM and fluorescence microscopy offers new opportunities to gather multidimensional data from a given system 31-36, but it can also make use of fluorescence modulation effects which arise from the presence of the sharp AFM probe in the vicinity of fluorescent molecules. These effects include the enhancement 11-13,16 and quenching 17,18,20,22,23,37,38 of the fluorescence and a change in the fluorescence lifetime 23. Since both enhancement and quenching are highly localized phenomena, they can lead to much higher resolution than the diffraction limit dictates for optical imaging. Quake and Gerton have made use of enhancement of the fluorescence of semiconductor quantum dots 11,13,16, as well as organic molecules 12 to achieve resolutions below 10 nm using commercial silicon AFM probes 11-13,16 and carbon nanotubes 37. The fluorescence modulation in the vicinity of a nanostructure like an AFM tip depends on many factors such as the geometry of the tip, its material, and its distance to the fluorophore. Also, the orientation of the polarization of the incident light and its wavelength as well as the orientation of the molecule's polarizability play an important role especially for fluorescence enhancement 24,27, In principle, there are two inherently different modes of operation for the AFM which allow the exploration of both regimes of fluorescence modulation. In contact mode, the AFM tip is constantly gently pressed on the surface. When the tip comes very close to the fluorescent dye molecule, the latter can transfer the energy of its excited state to the AFM tip which leads to quenching of the fluorescence. In the other mode of operation, which is called tapping or intermittent mode, the AFM tip is oscillated above the surface. Since the distance to the surface continuously changes during the oscillation, the fluorescence modulation varies periodically. The information about the phase of the tip oscillation can then be correlated with the fluorescence intensity. For each cycle, the background is defined as the fluorescence intensity from distances where the influence of the tip is weakest. This background is then subtracted from the fluorescence intensity at times of maximal enhancement, thus leading to a high signal to background ratio 11,23,38,42. Here, we use contact mode AFM with commercial silicon AFM tips to induce quenching in organic fluorescent molecules. In bulk experiments, silicon has been shown to quench the fluorescence of a layer of organic dyes that is separated from a silicon wafer using Langmuir-Blodgett films 43. Several studies have investigated the quenching properties of metal coated tips 17-20,22,23 and silicon tips 22,23. So far though, metal tips have been proposed to yield better optical resolutions in combined AFM/fluorescence quenching experiments than silicon tips. We show here that silicon tips can yield resolutions on the order of what is achieved with metal coated tips. Our novel setup combines a commercial AFM with a commercial confocal laser scanning microscope (CLSM). It consists of a sample scanning AFM and a single molecule sensitive confocal fluorescence microscope. With this setup, the fluorescence dynamics can be followed on time scales from subnanoseconds to seconds. This setup is ideal for FLIM using time-tagged time-resolved (TTTR) single photon recording. This allows us to simultaneously record fluorescence intensity and lifetime information, both spectrally and spatially resolved, on a single photon basis. For the combined scans, the sample scanning AFM acts as master and the parallel TCSPC data acquisition serves as slave: start and stop scan line information from the AFM is transferred to the TCSPC electronics enabling us to achieve synchronized and matched AFM and fluorescence images with direct access to nanosecond time-resolved single photon data. Measurements on YOYO-1 labeled λ-dna demonstrate the synchronization of the two commercial instruments and prove the capability of our new combined setup. Further we demonstrate local silicon induced quenching of single fluorophores. Proc. of SPIE Vol

3 2. SETUP Our setup is a combination of two commercial microscope systems: A MicroTime200 Fluorescence lifetime microscope from PicoQuant (Germany) 44 and a MFP-3D-Bio AFM from Asylum Research (California, USA). The mechanical link between the two systems is the AFM sample stage, which is mounted onto an Olympus IX-71 inverted microscope. The stage allows for coarse positioning of the sample with respect to the AFM probe as well as positioning of sample and AFM probe together with respect to the microscope objective. The link on the level of synchronization of the data is etablished in the PicoQuant TCSPC electronics (PicoHarp 300) with its router (PHR800), to which the AFM provides marker signals that indicate the start and stop of each scan line. Optical crosstalk between the fluorescence microscope and the AFM cantilever deflection detection is avoided by using an infrared laser with a wavelength of 860 nm in the AFM along with an infrared bandpass filter before the photodiode. The configuration of the fluorescence microscope is schematically shown in Figure 1. Figure 2 shows a photograph of the combined setup. Main Optical Unit (MOU) A AFM controller B Mirror Laser nm Dichroic Laser nm optical fiber Mirror Filter 1 Filter 4 Detector 1 Filter 3 Detector 2 Mirror Beamsplitter 2, switchable 0, 50, 100% Piezo controller Photodiode Beamsplitter 1 CCD Camera MOU TCSPC electronics Microscope Dichroic Filter 2 Pinhole Mirror Figure 1 (in color online): (A) Schematic of the main components of the setup. The position of the cantilever and the sample stage are controlled and monitored by the AFM controller. The objective (Olympus, 100x, NA 1.45, oil) can be moved in three dimensions using a P 733.2CL x,y-piezo scanner and a P 721.CLQ z-piezo objective positioner (Physik Instrumente, Germany) for lateral and axial movement, respectively. The AFM controller sends marker signals to the PicoQuant router, where they are embedded in the photon counting data stream. (B) The optical part of the combined setup consists of the laser unit, the main optical unit (MOU) and the microscope. As excitation sources the setup contains two diode lasers, LDH-D-C-470 and LDH-D-C-640 (470 nm and 640 nm wavelength, max. repetition rate 40 MHz, min. pulse width 73 ps). Excitation clean-up filters are mounted directly on the laser heads. The laser beams are combined using a dichroic mirror and coupled into a polarization maintaining single-mode fiber. Filter 1 can be used as clean-up filter for additional lasers. In the main optical unit, "beamsplitter 1" reflects a part of the laser light to a photodiode to estimate the laser power. The remaining light passes on to the dichroic mirror. For our experiments, we used a dual color dichroic (z467/638rpc, Chroma), that directs the laser light into the sideport of an Olympus IX-71 inverted microscope. Scattered light from the sample will be reflected by the dichroic, and "beamsplitter 1" sends the light to a CCD camera (FC25C, Ganz, Germany) which is used to monitor the laser beam during focusing as well as positioning of the AFM probe with respect to the focus. Fluorescence light passes through the dichroic and is spectrally and spatially filtered using "filter 2" and a 50 μm pinhole. Using "beamsplitter 2", which can be switched between open, a 50% mirror, and a 100% mirror, the fluorescence light can be directed onto detector 1 or 2 or both of them. The detectors are MPD single photon detectors from the PDM series. The signal from the detectors is fed into a PicoHarp 300 time correlated single photon counting (TCSPC) module. All the filters are exchangeable; in a normal experiment, we used a 500nm longpass filter or a 690/70 bandpass filter (HQ500lp and HQ690/70M, Chroma Technology Corp, Vermont). Proc. of SPIE Vol

4 Figure 2 (in color online): Photograph of the combined AFM/FLIM setup including sound- and vibration isolation. 3. ALIGNMENT For simultaneous measurements of the surface topography and fluorescence properties of the sample, the AFM tip has to be aligned with the laser focus. In our setup, we use the AFM top-view camera and the beam diagnostic camera in the main optical unit to monitor the position of the AFM probe with respect to the laser beam. The AFM sample stage allows for coarse alignment by moving the sample and AFM head together with respect to the microscope objective (see Figure 3). For fine alignment, the objective piezo scanning stage is used to move the objective under the AFM tip until the tip appears in the center of the beam (see Figure 4). A B Figure 3 (in color online): Alignment of AFM tip and laser focus. The top-view camera image is showing the AFM cantilever from above. The bright spot on the end of the cantilever is the reflection of theafm laser. (A) The confocal laser spot can be seen next to the cantilever. (B) The diffuse light around the cantilever is the confocal laser spot. The diffuse spot at the right side of the cantilever is a reflection of the confocal laser. Proc. of SPIE Vol

5 A B C Figure 4: Beam diagnostic camera images. The left image (A) shows the pattern of the backreflected light from the sample surface with the AFM tip retracted (20 μm above the surface). The middle image (B) shows the beam profile with the AFM tip visible as a distortion of the pattern. For the right image (C), the objective was moved under the AFM tip to bring the distortion of the pattern in its center. For all images, the focus had been moved towards the AFM tip to show the distortion of the pattern more clearly. 4. MATERIALS AND METHODS λ-dna and YOYO-1 were purchased from Invitrogen Corporation, California. For fixation on mica, the DNA was dissolved 1:200 (v/v) in 10 mm HEPES buffer (ph 7.05) containing 1 mm NiCl 2. YOYO was added to yield a concentration of 1 molecule/4 base pairs. Atto647N was purchased from Sigma-Aldrich, Missouri and diluted in Millipore water (MilliQ Synthesis). Glass slides (VWR No 1, VWR International, Pennsylvania) were cleaned by subsequently sonicating in Acetone, (p.a.), Ethanol, (p.a.) and Millipore water (MilliQ Synthesis) and blowing dry with pure nitrogen. Two types of AFM probes were used: PointProbe-Plus PPP-NHC from Nanoworld AG, Switzerland, with a spring constant of N/m and a resonance frequency of about 350 khz and HYDRA 6R-100NG from Applied Nanostructures, Inc. (AppNano), California, with a spring constant of about N/m and a resonance frequency of about 66 khz. The PointProbe-Plus probes are made of silicon and do not have a reflex coating. The HYDRA probes consist of a silicon nitride cantilever with 100 nm gold reflex coating and a silicon tip. 5. COMBINED AFM / FLIM To show alignment of the two microscopes and the ability to collect topographic information from critically small structures with our combined system, we have imaged YOYO-1 stained λ-dna on mica. To prepare cover slides with a thin layer of mica, we made use of the technique described by Ma et al. in 12. DNA was immobilized and stretched out on the mica by letting 50 μl of aqueous solution containing λ-dna and Ni 2+ ions flow over the surface several times. Fluorescence was excited at 470 nm and the emitted light was filtered using a 500 nm longpass filter. The combined scan was taken at 1 line/2 seconds. The scanned area was (10 μm) 2. Figure 5 shows topographic and fluorescence data from a combined AFM/FLIM scan. In the AFM height image, the DNA is clearly visible. The image has been processed using the Fast Fourier Filter of the SPIP software (Image Metrology A/S, Denmark) to remove periodic noise which stems from oscillations in the microscope objective. The image also points out the superior resolution of AFM data over the optical image. Feature 1 in Figure 5 (C) shows tightly looped DNA, which appears as one bright spot in the fluorescence lifetime image, whereas in the AFM image, multiple parts of the DNA strand can be distinguished. Feature 2 in the same figure shows a region of the DNA, where the YOYO-1 has been bleached. Proc. of SPIE Vol

6 Figure 5 (in color online): Simultaneous tapping mode AFM and CLSM images of YOYO-1 labeled λ-dna on mica. The AFM tip is aligned to the confocal volume of the CLSM. For imaging, the sample is scanned relative to the tip and confocal volume. The fast scan direction is up-down. (A) AFM topography (scale: 2nm). (B) confocal fluorescence lifetime image, and (C) overlay of the two images. The marked areas indicate features that show (1) the superior resolution of AFM and (2) its ability to image areas of unstained (bleached) DNA. 6. SINGLE MOLECULE QUENCHING In addition to the simultaneous collection of topographic as well as optical data from a sample, we have shown that the presence of the AFM tip has an influence on the optical properties of single Atto647N molecules. We used AFM in contact mode to achieve energy transfer from the molecule to the AFM tip which results in quenching of the fluorescence and hence in a dark spot in the optical image. Also, in some cases, we have seen enhancement of the emission and changes in the fluorescence lifetime. The samples were produced by drying a dilute solution of Atto647N on a clean glass surface. The concentration of the dye was adjusted to give a density of about one molecule per 1 μm2. The combined scans were performed at a scanning speed of about 1 line/2 seconds and the image size was 5 μm. Fluorescence was excited with the red laser (640 nm) at a power of about 3.2 μw. The polarization of the exiting laser beam is in plane with the surface, parallel to the fast scan axis. Experiments were performed using two different kinds of commercially available AFM silicon probes with high and low spring constant. Figure 6 (in color online): (A) and (B): Fluorescence intensity and fluorescence lifetime image of a combined scan of Atto647N molecules on glass with a PointProbe-Plus AFM probe. The color scale in (B) codes for the fluorescence lifetime from 0.5 ns (blue) to 4.5 ns (red). (C): Fluorescence intensity image of the same sample combined with a HYDRA 6R AFM probe. The inset shows the fluorescence intensity of the section through the indicated line. The FWHM of the drop in the intensity is 23 nm. The scan range for all images is 5 μm. Proc. of SPIE Vol

7 Figure 6 shows fluorescence intensity and lifetime images of two combined scans. The diffraction limited spots of the fluorophores show typical single molecule features like blinking and beaching. All bright fluorescence spots exhibit a dark area in their center that we attribute to energy transfer to the AFM tip. Typical dimensions for the full width at half maximum (FWHM) are nm. The section in Figure 6 (C) shows a FWHM of 23 nm. So far, in the combination of AFM and fluorescence imaging, metal coated tips have been preferred over silicon tips to induce quenching of the fluorescence in semiconductor quantum dots, since they lead to smaller values for the FWHM 22. Here, we show that commercial silicon AFM probes can be used to quench the fluorescence of an organic dye molecule and result in resolutions that are comparable to those with metal coated tips for quantum dots. For some of the molecules, an area of enhanced fluorescence that goes along with a change in fluorescence lifetime can be observed adjacent to the quenched spot (see Figure 7 and 6(B)). Previous studies have shown that fluorescence enhancement is strongest for molecules that have their dipole aligned with the tip axis 39, 41. Since we are using in-plane polarization, we would not expect to excite those molecules (see Figure 8). However, a molecule whose dipole vector has components in plane as well as out of plane might still be excited and exhibit fluorescence enhancement. We propose that the orientation of the enhanced area with respect to the quenched area corresponds to the orientation of the molecules dipole vector. Figure 7 (in color online): Fluorescence intensity of sections (lines in the insets) through two different Atto647N molecules, illustrating the respective orientation of areas of quenched and enhanced fluorescence. The color scale in the insets codes for the fluorescence lifetime from 0.5 ns (blue) to 4.5 ns (red). y x Figure 8 (in color online): Cartoon of the orientation of dipoles in an enhancement scheme. The arrows are (from bottom to top): polarization of the incident light, the orientation of the excited dipole (fluorescent molecule) and the orientation of a dipole at which fluorescence enhancement is maximal. Proc. of SPIE Vol

8 7. CONCLUSION In conclusion, we presented a novel setup consisting of two commercial microscopes, an Asylum MFP-3D atomic force microscope and a PicoQuant MicroTime200 confocal fluorescence lifetime microscope. We showed that with this setup we can achieve single molecule sensitivity in the topographic as well as the optical image. Aligning the AFM probe with the confocal laser focus allows us to simultaneously record data with both microscopes and synchronize them with one another. We presented topographic and optical images of YOYO-1 stained λ-dna that illustrate the setups capability to acquire both kinds of data at the same time. With optical images of single Atto647N molecules we demonstrate tip induced fluorescence quenching, which result in optical resolutions in the range of 20 nm. Additionally, these experiments prove the precise alignment of our system. REFERENCES [1] P. Hinterdorfer, and Y. F. Dufrene, "Detection and localization of single molecular recognition events using atomic force microscopy", Nature Methods 3, (2006). [2] W. E. Moerner, and D. P. Fromm, "Methods of single-molecule fluorescence spectroscopy and microscopy", Review of Scientific Instruments 74, (2003). [3] S. W. Hell, "Far-field optical nanoscopy", Science 316, (2007). [4] P. R. Selvin, "The renaissance of fluorescence resonance energy transfer", Nat.Struct.Biol. 7, (2000). [5] I. Rasnik, S. A. McKinney, and T. Ha, "Surfaces and orientations: Much to FRET about?", Accounts of Chemical Research 38, (2005). [6] A. R. Clapp, I. L. Medintz, B. R. Fisher, G. P. Anderson, and H. Mattoussi, "Can luminescent quantum dots be efficient energy acceptors with organic dye donors?", Journal of the American Chemical Society 127, (2005). [7] T. Pons, I. L. Medintz, K. E. Sapsford, S. Higashiya, A. F. Grimes, D. S. English, and H. Mattoussi, "On the quenching of semiconductor quantum dot photoluminescence by proximal gold nanoparticles", Nano Letters 7, (2007). [8] A. Engel, Y. Lyubchenko, and D. Muller, "Atomic force microscopy: a powerful tool to observe biomolecules at work", Trends in Cell Biology 9, (1999). [9] J. Zlatanova, S. M. Lindsay, and S. H. Leuba, "Single molecule force spectroscopy in biology using the atomic force microscope", Progress in Biophysics and Molecular Biology 74, (2000). [10] R. Hillenbrand, and F. Keilmann, "Material-specific mapping of metal/semiconductor/dielectric nanosystems at 10 nm resolution by backscattering near-field optical microscopy", Applied Physics Letters 80, (2002). [11] J. M. Gerton, L. A. Wade, G. A. Lessard, Z. Ma, and S. R. Quake, "Tip-enhanced fluorescence microscopy at 10 nanometer resolution", Physical Review Letters 93, (2004). [12] Z. Y. Ma, J. M. Gerton, L. A. Wade, and S. R. Quake, "Fluorescence near-field microscopy of DNA at sub-10 nm resolution", Physical Review Letters 97 (2006). [13] B. D. Mangum, C. Mu, and J. M. Gerton, "Resolving single fluorophores within dense ensembles: contrast limits of tip-enhanced fluorescence microscopy", Optics Express 16, (2008). [14] B. D. Mangum, E. Shafran, C. Mu, and J. M. Gerton, "Three-Dimensional Mapping of Near-Field Interactions via Single-Photon Tomography", Nano Letters 9, (2009). [15] V. V. Mkhitaryan, Y. Fang, J. M. Gerton, E. G. Mishchenko, and M. E. Raikh, "Scattering of Plasmons at the Intersection of Two Metallic Nanotubes: Implications for Tunneling", Physical Review Letters 101 (2008). [16] C. A. Xie, C. Mu, J. R. Cox, and J. M. Gerton, "Tip-enhanced fluorescence microscopy of highdensity samples", Applied Physics Letters 89 (2006). [17] R. Eckel, V. Walhorn, J. Martini, T. Nann, D. Anselmetti, and R. Ros, "Combined TIRF-AFM Setup: Controlled Quenching of Individual Quantum Dots", Proc. of SPIE 6092 (2006). [18] R. Eckel, V. Walhorn, C. Pelargus, J. Martini, J. Enderlein, T. Nann, D. Anselmetti, and R. Ros, "Fluorescence-emission control of single CdSe nanocrystals using gold-modified AFM tips", Small 3, (2007). Proc. of SPIE Vol

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