Quantum Coherent Modulation-Enhanced Single-Molecule Imaging

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Spectroscopy and Photochemistry; General Theory

Quantum Coherent Modulation-Enhanced Single-Molecule Imaging Microscopy Haitao Zhou, Chengbing Qin, Ruiyun Chen, Yaoming Liu, Wenjin Zhou, Guofeng Zhang, Yan Gao, Liantuan Xiao, and Suotang Jia J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b03606 • Publication Date (Web): 01 Jan 2019 Downloaded from http://pubs.acs.org on January 2, 2019

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The Journal of Physical Chemistry Letters

Quantum Coherent Modulation-enhanced Singlemolecule Imaging Microscopy Haitao Zhou1,2, Chengbing Qin1,2*, Ruiyun Chen1,2, Yaoming Liu3, Wenjin Zhou1,2, Guofeng Zhang1,2, Yan Gao1,2, Liantuan Xiao1,2*, and Suotang Jia1,2 1

State Key Laboratory of Quantum Optics and Quantum Optics Devices, Institute of Laser Spectroscopy, Shanxi University, Taiyuan, Shanxi 030006, China.

2

Collaborative Innovation Center of Extreme Optics, Shanxi University, Taiyuan, Shanxi 030006, China.

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Scientific Instrument Center, Shanxi University, Taiyuan, Shanxi 030006, China.

*Author to whom correspondence should be addressed. E-mail: [email protected] (C. Q); [email protected] (L. X.).

ACS Paragon Plus Environment

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ABSTRACT

In the fluorescence imaging and detection, undesired fluorescence interference (such as autofluorescence) often hampers the contrast of the image and even prevents the identification of structures of interest. Here, we develop a quantum coherent modulation-enhanced (QCME) singlemolecule imaging microscopy (SMIM) to substantially eliminate the strong fluorescence interference, based on the manipulation of the excited-state population probability of a single molecule. By periodically modulating the phase difference between the ultrashort pulse pairs and performing a Discrete Fourier Transform of the arrival time of emitted photons, the decimation of single molecules from strong interference in QCME-SMIM have been clearly determined, where the signal-to-interference ratio is enhanced by more than two orders of magnitude. This technique, confirmed to be universal to organic dyes and that linked with biomacromolecules, paves the way to high-contrast bioimaging under unfavorable conditions.

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Single-molecule fluorescence imaging and detection is a powerful method to understand the behavior of single biomolecules in their nano-environments as well as their roles in cellular processes.1-3 However, the sensitivity of conventional fluorescence imaging is still limited by the low fluorescence intensity of probes and the presence of strong background or interference, which often hampers imaging contrast and even prevents the identification of interested structures. It is an enormous challenge to separate fluorescence signals from interference (such as autofluorescence in biological tissues4,5) in fluorescence-based biological detection and biomedical diagnosis. To address such demands, hyperspectral imaging5,6 and fluorescence lifetime imaging7,8 have been developed to reduce the influence of interference. Hyperspectral imaging presents an excellent effect when the emission spectra of interference are sufficiently different from those of interest, and so as lifetime imaging. However, both methods cannot distinguish fluorescence spectra and lifetimes that are very similar, which limits their further applications.8-10 Recently, single molecule imaging microscopy (SMIM) based on fluorescence modulation has been exploited to circumvent this limitation and to enhance the sensitivity and selectivity of fluorescence imaging. Among fluorescence modulations, photoswitchable fluorophores11-13 or fluorescent proteins (PS-FPs)14 are frequently used. The fluorescence of PS-FPs can be optically switched either from one color to another15 or from on to off11,16 by a secondary excitation, while the interference cannot be simultaneously modulated. Thus, PS-PFs can be discriminated from the strong interference even with identical spectra or lifetime15-27. Up to now, fluorescence modulation techniques, such as dual-laser modulated synchronously amplified fluorescence image recovery (DM-SAFIRe),28-32 and that combined with out-of-phase imaging after optical modulation (OPIOM),33 have been well developed, which are used to reveal structures hidden below the ambient interference by enhancing the imaging contrast.34-37 However, these methods are merely


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suitable for the fluorophores with photosensitive reactant and product, such as methoxyspiropyran (MSP) and methoxymerocyanine (MMC) or other PS-FPs.11,13,17 A universal technique that applies to all dye molecules is still lacking. Here, we develop a new fluorescence modulation technique based on the quantum coherent manipulation of single molecules by modulating the phase difference between an ultrashort pulse pair, which is used to pump and probe single molecules.38-41 The periodic phase modulation substantially varies the population probability of the excited state of single molecules and thus changes their fluorescence emission. In contrast, the unwanted interference cannot be modulated. After demodulating fluorescence photons, the contrast of fluorescence imaging in the frequency domain can be dramatically enhanced. The signal-to-interference ratio is improved from inundated (below the background) ~0.054 in the time domain to a satisfactory ratio of 27.79 in the frequency domain, with a 515-fold contrast enhancement. More importantly, the enhancement represents a power-law exponential increase over integration time, hinting that an unlimited contrast enhancement can be achieved in theory. Three types of organic dyes and that linked with biomolecule protein have been tested successfully, indicating that our quantum coherent modulation-enhanced (QCME) SMIM is general applicable to all dye molecules. Furthermore, the maintained high-contrast enhancement in the region of 1 Hz to 500 kHz allows this technique to be applied for both wide field and confocal imaging with arbitrary modulation speed. These outstanding features suggest that our technique has great potential applications in bioimaging with strong interference, as well as probing fast dynamics of biological processes. The principle of fluorescence modulation of single dye molecules by the ultrashort pulse pairs can be understood as the manipulation of its excited-state population probability. Considering the two-level approximation, the interaction between a single molecule and an ultrashort pulse pair,


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which is on resonance between its ground state ( 0 ) and excited state ( 1 ), will prepare a coherent state, as shown in Figure 1a. This state can be illustrated by the Bloch vector on the Bloch sphere, where the poles correspond to the eigenstates (‘south’, 0 , ‘north’, 1 ) and any other points indicate coherent superposition states between ground- and excited-state electronic wavefunctions.39,42,43 The key to prepare coherent state is to control the phase difference, Δφ, between the pulse pair. When the single molecule interacts with the first pulse, the process of preparing coherent state is corresponding to a rotation of the Bloch vector away from its ground state, as shown by the blue arrows in Figures 1b and 1c. Under ideal condition where the single molecule has no dephasing, the final coherent state is determined by Δφ. When Δφ is fixed to 0 rad, the second pulse will rotate the Bloch vector to the excited state, indicating the maximum excited-state population probability, as shown in Figure 1b. While the second pulse will rotate the Bloch vector to the ground state when Δφ is fixed to ±π rad, indicating the minimum excited-state population probability, as shown in Figure 1c. The excited-state population probability ρee with arbitrary Δφ can be expressed as follows:

ee  1 2  (1  cos 1 cos  2  sin 1 sin  2 cos  )

(1)，

where θ1 and θ2 are the dimensionless pulse areas.42 For a certain single molecule and fixed power density, they are constants (for detailed derivation see Supporting Information, Section S1). According to Equation (Eq.) 1, the population probability of the excited state can be manipulated effectively by controlling Δφ. Considering that the fluorescence is directly correlated with its population probability, the fluorescence intensity of single molecule can be modulated by phase difference. While for interference (auto-fluorescence in tissues) or background (ambient light or laser leaking), their intensities cannot be modulated effectively, due to the lack of coherent interaction with the ultrashort pulse pairs.


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Figure 1. Principle of phase-controlled quantum coherent state. (a) A tunable phase between the ultrashort pulse pairs realizes the control of coherent state between the ground state (|0⟩) and excited-state (|1⟩) with resonance excitations. (b) The maximum excited-state population probability is achieved with Δφ=0. (c) The minimum excited-state population probability is achieved with Δφ=±π.

Schematic diagram of QCME-SMIM has been presented in Figure 2a, and the detailed description has been supplied in Method. Briefly, the sub-picosecond pulse pair with tunable phase difference was generated by a Michelson interferometer structure, where an electro-optical modulator (EOM) was on one arm. While on the other arm, the laser passed through a quarterwave plate. Thus, two recombined beams have orthogonal polarizations, which can eliminate the intensity modulation due to their own interference (To eliminate the polarization modulation, the modulation on unpolarized colloidal quantum dots has been performed, which presented clearly modulation, see Figure S1 for details). In the experiment, the phase difference was periodically modulated between –π to π, by applying a periodic saw tooth voltage on EOM, as shown in Figure 2b. The modulation frequency was adopted to 1 kHz for most cases. According to Eq.1, the population probability of the excited state will be modulated with sinusoidal waveform as a function of phase difference, as shown in Figure 2c. Thus, in each cycle, the probability of fluorescence from single molecule will also emit as the sinusoidal wave. However, due to the photophysical properties of single molecules (such as photoblinking) and the limited collection


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efficiency of the optical system, only several photons can be detected in each cycle (in 1 ms with modulation frequency of 1 kHz). By recording the absolute arrival time of each photon and carrying out the statistics of all photons as the function of phase difference in an integration time, the fluorescence photons in the modulation period can be achieved, as shown in Figure 2d. Note that photons emitted from a single molecule present sinusoidal waveform as expected, while those from background or interference are almost constant in full period. Demodulating the arrival time of photon series by Discrete Fourier Transform (DFT), the single molecule shows significant DFT signal at 1 kHz, which are orders of magnitude larger than that of background, as shown in Figure 2e. Thus, we can expect that reconstruction of single molecule imaging by DFT at modulation frequency can dramatically suppress the background/interference and significantly enhance the image contrast.


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Figure 2. (a) Experimental setup of QCME-SMIM. QWP, quarter-wave plate; BS, beam splitter; EOM, electro-optical modulator; DM, dichroic mirror; SPAD, single photon counting modulator; MCPET, multichannel picosecond event timer. (b) The periodic saw tooth voltage applied on EOM to change the phase difference of pulse pair from –π to π, (c) which produces the population probability of the excited state in sine form (waveform in grey). The blue lines indicate the possible photon emission in each cycle. (d) Statistics of fluorescence photons from a single molecule (SM. blue) and background (Back. gray) as a function of phase difference Δφ. (e) Discrete Fourier Transform (DFT) of arrival time of photon series from a single molecule and background, respectively.

To illustrate the contrast enhancement in the frequency domain based on QCME-SMIM, squaraine-derived rotaxanes (SR) molecules mixed with microspheres (based on melamine resin, marked with Nile Blue) were spin-coated on the substrate as a testing sample. Here, SR molecules with weak fluorescence emission (~104 counts/s) are used as the signal source, while the microspheres with strong emission (~106 counts/s) are used as the interference. Due to the strong interaction between closely packed dye molecules, the rapid decoherence of microspheres results in no response to quantum coherent modulation.39,44 SR and microspheres have overlapping spectrum, as shown in Figure S2. Thus, they cannot be distinguished by traditional fluorescence imaging. Figure 3 presents the fluorescence imaging in the time domain (fluorescence intensity for each pixel) and frequency domain (DFT magnitude for each pixel) with different integration times. As shown in Figure 3a, three bright patterns are originating from the emission of microspheres, while SR have been fully submerged in bright microspheres. Note that as the increasing of the integration time, no contrast improvement can be obtained in the time domain. On the contrary, in


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the frequency domain from the same data, the strong interference has been well suppressed and SR (highlighted by three dashed circles) can be clearly discerned, as shown in Figure 3b. More importantly, higher contrast can be achieved by increasing the integration time. This contrast enhancement can be explained as the non-modulation of microspheres under the periodic modulation of phase difference.

Figure 3. QCME-SMIM eliminates the strong interference. (a) and (b) are the time-domain and frequency-domain images of SR molecules mixed with microspheres recorded with various integration times. The three bright patterns in the time domain (a) are the strong fluorescence emission from three microspheres and the three emerging smaller circular patterns (highlighted by dashed circles) in the frequency domain (b) are the DFT signal from three single SR molecules. For clarity and comparability, all images are normalized by their maximum values. Scale bar: 1 μm. (c) and (d) are the fluorescence intensities and DFT magnitudes of labeled lines in (a) and (b) under four integration times, which are 0.05, 0.2, 0.8 and 2.0 s, respectively. Three letters (α, β, and γ) denote the beginning, the middle, and the end of the lines. S1 and S2 denote the signals from


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the two single molecules, respectively. B and I (highlighted by solid circles) denote the intensities from the general background and strong interference, respectively.

To further explore the contrast enhancement in the frequency domain, we plot both fluorescence intensities and DFT magnitudes of the selected lines (labeled in Figure 3a and 3b) varied as the integration time, as shown in Figure 3c and 3d, respectively. From the frequency domain, we can find that there are two single molecules in the selected line, marked as S1 and S2, where the general background and interference (strong emission from microsphere) are marked as B and I, respectively. Owing to the fluorescence intensities of all the four selected areas (labeled as color bars) in the time domain synchronously increasing with the integration time, thus no contrast improvement can be determined. While DFT magnitudes of single molecules increases much faster than that of interference/background, resulting in dramatic enhancement. This enhancement can be quantified by assuming average photons emitted per unit of signal and background/interference. The signal-to-background (S/B) ratio in the time domain can be represented as: RS/B (Fluo.)  kS  kB 1

(2),

where kS is photons emitted per second from a single molecule, and kB is photons emitted per second from background. The S/B ratio in the frequency domain can be described as:45-48 RS/B (DFT)    kS  kB 1/2  t1/2

(3),

where t is the integration time (in second), ξ is the slope factor of modulation, depending on the interaction strength between the single molecule and the pulse pair (for detailed derivation see Supporting Information, Section 4). Although the S/B ratio does not change in the time domain


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once the kS and kB are determined, as given in Eq.2, the RS/B(DFT) can be unlimitedly improved in theory by increasing the integration time, as given in Eq.3. To confirm the formulas, we plot the S/B ratio of time and frequency domains as the function of integration time, as shown in Figure 4a. The fluorescence intensities and DFT magnitudes of signal and background are extracted from marked areas in Figure 3c and 3d, S1 and B, respectively. Note that RS/B(Fluo.) are in the region of 8.8 to 9.8, consistent with Eq.2. The fluctuation mainly originates from the variation in the photon emission of single molecules (such as photoblinking). On the other hand, RS/B(DFT) is smaller at short integration time, while it is increased as a powerlaw function of integration time. The data can be fitted by the power law model, R=a×tb, yielding the exponent of b equaling to 0.536, very close to 0.5 (Eq.3), which is in good agreement with the theoretical predictions.48 In the case, RS/B(DFT) equals to RS/B(Fluo.) at the integration time of 0.32 s, and reaches to 24.13 at 2.0 s. In order to clearly reveal the contrast enhancement, we also present the ratio of RS/B(DFT) to RS/B(Fluo.). The enhancement is also increased in power law with an exponent of 0.503, indicating that the enhancement can be improved unlimitedly in theory by continuously increasing the integration time. This unlimited contrast enhancement is not only appropriate for S/B ratio, but also suitable for the ratio of signal-to-interference (S/I). To illustrate the applicability, the fluorescence intensities and DFT magnitudes of single molecule S2 and interference of I (Figure 3c and 3d) are selected. Note that the fluorescence intensity of S2 is fully inundated by microsphere, no signal can be found in Figure 3c. By assuming that the fluorescence intensity of S2 is equal to S1, the S/I ratios of time and frequency domains as the function of integration time are shown in Figure 4b. The QCME-SMIM dramatically improves the S/I ratio from undetectable levels of 0.054 to a favorable ratio of 3.1 even at the integration time of only 0.02 s. The fitting results proved that


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both the RS/I(DFT) and enhancement (the ratio of RS/I(DFT) to RS/I(Fluo.)) are increased in power law with exponents close to 0.5, coinciding with Eq.3. Due to the strong emission and nonmodulation feature of microspheres, the enhancement has reached to 515 at the integration time of 2.0 s. We also demonstrated the robustness of this technique for the case of a laser leaking/ambient light dominant background that drowns out the fluorescence of single molecules of interest. More than two orders of magnitude enhancement of S/B ratio has been determined (for details see Figure S4 and S5).

Figure 4. (a) Signal-to-Background ratio (S/B) and (b) Signal-to-Interference ratio (S/I) in the time domain (Fluo.) and frequency domain (DFT) as well as the corresponding enhancement (DFT/Fluo.) as a function of integration time. The signal and background are selected from Figure 3. S1 and B for S/B, S2 and I for S/I, respectively. All the ratios and enhancements are fitted by power law model, that is R=a×tb. The corresponding parameters have been shown in the figures.

Specifically, QCME-SMIM is a universal method, applying to all dyes with two-level approximation in theory, where the population probability of the excited state can be manipulated by the ultrashort pulse pairs. The huge contrast enhancement in the frequency domain has also been determined from two other dye molecules (Cy5 and DiD) and that bounded with biological proteins (streptavidin-bonded Cy5) (see Figure S6). Furthermore, by labeling SR molecules, the Escherichia Coli can be clearly distinguished from the large background. Figure 5 shows the


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comparison of the traditional wide-field fluorescence intensity imaging of coli bacillus cell and the corresponding QCME-SMIM. The fluorescence signal of coli bacillus cell is drowned in the large background, while it becomes clearly visible in modulated imaging, as shown in Figure 5b. These attempts illustrate not only the universal application of this technique, but also the potential applications for biological imaging.

Figure 5. QCME-SMIM retrieves single SR molecules signal in live cells. (a) Traditional fluorescence intensity imaging of coli bacillus cell in presence of large background. (b) The corresponding QCME-SMIM. Scale bars: 5μm.

The broad dynamic range of modulation frequency and potentially high imaging speed is another critical feature of this technique. We have performed the modulation in the frequency region of 1 Hz to 500 kHz, as shown in Figure S7. Apparently, the DFT magnitudes of both background and SR have no substantial change in almost six orders of magnitude. This broad region allows QCME-SMIM to be applied to both wide field imaging that can collect the information of numbers of individual molecules simultaneously, and confocal fluorescence imaging that allows investigating the real-time dynamics of a single molecule for a long period with high temporal resolution. In conclusion, we have developed a novel fluorescence modulation imaging technique with dramatic imaging contrast enhancement in the frequency domain by varying the phase difference


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between the ultrashort pulse pairs that is used for excitation, which can effectively manipulate the excited-state population probability of single molecules and therefore their fluorescence intensity. Imaging the single molecules signal by QCME-SMIM can efficiently suppress the background or interference, which will inevitably overwhelm the signal in traditional fluorescence microscopy. The S/I ratio has dramatically improved from below the background (0.054) to a satisfactory ratio of 27.79, with contrast enhancement of 515. More critically, this technique not only acts as a universal technique to the organic dyes and that linked with biomacromolecule, but also can be operated over the modulation frequency region from 1 Hz to 500 kHz. Thus, it offers great potential for discriminating single molecules from auto-fluorescence and strong interference, and simultaneously opens new opportunities for studying biological interactions and dynamics with improved speed and sensitivity.


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EXPERIMENTAL METHOD Experimental procedures. The experimental setup has been sketched in Figure 2a. The laser with pulse width of 389 fs at the wavelength of 635 nm was split by a beam splitter (BS). On one arm, the laser passed through the electro-optical modulator (EOM) used for phase modulation, while on the other arm, the laser passed through a quarter-wave plate (QWP) to convert the laser into vertical polarization. Thus, two beams recombing at BS have orthogonal polarizations, which can eliminate the intensity modulation due to their own interference. The phase difference between pulse pair was periodically modulated between –π to π, by applying a periodic saw tooth voltage on the EOM. The modulation frequency was adapted between 1 Hz to 500 kHz, and the modulation voltage was set as full wave voltage for better modulation depth. The delay of pulse pair was adjusted nearly to zero to address the coherent property of a two-level system. The re-combined light was reflected into the objective lens via a dichroic mirror (DM), then focused the light onto the prepared sample. The fluorescence photons from single molecules, background or interference, were all collected by the same objective, transmitted through DM and finally detected by a single photon detector (τ-SPAD,). The absolute arrival time of each photon was recorded by the timetagged, time-resolved and time-correlated single-photon counting (TTTR-TCSPC) technique with a multichannel picosecond event timer (MCPET). The temporal resolution of MCPET is 1 ps. Single-molecule fluorescence imaging in the frequency domain was obtained by demodulating the arrival time of photon series for each pixel through Discrete Fourier Transform (DFT). Sample preparation. In the experiment, the microspheres (based on melamine resin, nile blue-marked with 1μm @ Fluka Code 93887) were firstly dripped on the substrate (cover glass), and then irradiated with ultraviolet light for about 3 hours to reduce its fluorescence intensity. Hereafter, the colloidal quantum dots (QDs, CdSeTe/ZnS, purchased from NanoOptical Materials


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Inc.) and three organic dyes, including squaraine-derived rotaxanes (SR), 1,10-dioctadecyl3,3,30,30-tetramethylindodicarbo-cyanine49 (DiD) and Cy550, as well as streptavidin-bonded Cy5 (Cy5-SA) were prepared respectively, by spin-coated 20 μL solution (water for SR, Cy5 and Cy5SA, and toluene for DiD) containing 10-9-10-10 Mol. dye molecules. Then the poly(methyl methacrylate) (PMMA) film was spin-coated on the prepared dye molecules. Here PMMA film was used to prevent oxygen-induced fast photobleaching. The final sample was heated under vacuum to 373K for 3 h, and then held overnight under vacuum at room temperature to eliminate the remaining solvent and relax the stresses induced by the spin-coating process.

SUPPORTING INFORMATION AVAILABLE. Derivation of population probability under arbitrary phase difference; Fluorescence spectra of SR molecules and microspheres; Derivation of S/B ratio in time and frequency domains; Contrast enhancement in QCME-SMIM under strong background; Contrast enhancement in QCME-SMIM under ambient light; Universality of QCMESMIM; Applications of QCME-SMIM in living cells; Broad dynamic range of QCME-SMIM.

ACKNOWLEDGMENT The authors gratefully acknowledge support from National Key Research and Development Program of China (Grant No. 2017YFA0304203), National Natural Science Foundation of China (Grant No. 11434007), Natural Science Foundation of China (Nos. 61527824, 61605104, 11504216, 61675119 and U1510133), PCSIRT (No. IRT13076), and 1331KSC.


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Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5


Quantum Coherent Modulation-Enhanced Single-Molecule Imaging

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