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Mode-Selective Raman Imaging of DopamineHuman Dopamine Transporter Interaction in Live Cells Achut Prasad Silwal, and H. Peter Lu ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00301 • Publication Date (Web): 19 Jul 2018 Downloaded from http://pubs.acs.org on July 20, 2018

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Mode-Selective Raman Imaging of Dopamine-Human Dopamine Transporter Interaction in Live Cells Achut P. Silwal, and H. Peter Lu* Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University, Ohio 43403, United States. ABSTRACT: Dopamine (DA) is the catecholamine neurotransmitter which interacts with dopamine receptors (DARs) to generate dopaminergic signals in nervous system. Dopamine transporter (DAT) interacts with DA to maintain DA’s homeostasis in synaptic and perisynaptic space. DAT and DARs have great importance in central nervous system (CNS) since they are associated to the targeted binding of drugs. Interactions of DA, or its analogue with DARs, or DAT have been studied extensively to understand the mechanism of the dopaminergic signaling process and several neurodegenerative diseases including schizophrenia, Parkinson’s diseases, addiction, attention deficit hyperactivity disorder (ADHD), and bipolar disorder. However, there is still a lack of a risk free, label free, and minimally invasive imaging approach to probe the interaction between DA and DAT or DARs. Here, we have probed the dopamine (DA), human dopamine transporter (hDAT), and DA-hDAT interactions in live cells using combined approach of two-photon excited (2PE) fluorescence imaging and mode-selective Raman measurement. We utilized the signature Raman peak at 1287 cm-1 to probe the location of DA and 807 and 1076 cm-1 to probe the DA-hDAT interaction in live cells. We found that the combined approach of modeselective Raman imaging, 2PE fluorescence imaging, and computational methods is successful to probe and confirm the DA-hDAT interactions in living cells. The probing of the interactions of DARs or DAT with DA or other targeting drugs is crucial for the diagnosis and cure of several neurodegenerative diseases. Also, this analytical approach could be extended to probe other types of protein-ligand interactions. KEYWORDS: Dopamine (DA), Mode-selective Raman imaging, Human dopamine transporter (hDAT), HEK293 cell, DAhDAT interaction

INTRODUCTION Dopamine (DA) is the biogenic amino neurotransmitter that plays a significant role on the behavioral and psychological activities of human beings.1-6 Environmental stimulations generate the action potential which opens the voltage gated Ca+2 ion channel and allows Ca+2 flux to enter neuron cells. The elevation of Ca+2 ions inside the cell stimulates the fusion of synaptic membrane with plasma membrane of neuron cell and diffusion of DA into synaptic cleft.7, 8 The diffused DA interact with dopamine receptors (DARs) located in the post synaptic region of neighboring dendrites and generates dopaminergic signals.9-12 The prolonged or excessive interactions of DA with DARs cause the hyperstimulation of DARs and ruin the dopamine system, which is avoided by Na+ and Clions modulated DA reuptake mechanism of dopamine transporter (DAT).13-16 The interactions of DA with DAT and DARs are ongoing processes in the synaptic cleft which regulate functions of the dopaminergic system. The impairments of these interactions are associated to psychiatric and neurological disorders such as depression, schizophrenia, Parkinson’s disease, and Alzheimer diseases. The probing of DA, DARs, DAT and their interactions in the nervous system is highly required for the mapping

of neural circuits, revealing the pathophysiology of many neurodegenerative diseases, and gaining new visions to develop therapeutic treatments.17-20 Different types of experimental approaches such as fluorescence microscopy, positron emission tomography (PET), single photon electron tomography (SPET), X-ray crystallography, computational modeling, and molecular dynamics (MD) simulation have been applied to explore the facts on structures and functions of several signaling proteins.21-41 Booij and others used single photon emission computed tomography (SPECT) to observe the relationship between Parkinson’s diseases and loss of human dopamine transporters (hDAT) in the striatal region.27 Photo switchable fluorescence microscopy and fluorescence resonance energy transfer (FRET) microscopy have been used to study the modes of hDAT trafficking. Similarly, the total internal reflection fluorescence (TIRF) microscopy has been used to study the real-time trafficking of hDAT in the response of signaling molecules like DA and amphetamine.22, 23, 42 The label-free, risk free, and minimal invasive experimental approaches are highly desirable and recommended to probe the interactions of DAT or DARs with signaling molecules like DA and other psychoactive drugs. In

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the search of label-free imaging methods to probe neurotransmitters in living cells, Webb and coworkers first used the multiphoton fluorescence imaging approach to probe the serotonin neurotransmitters in rat basophilic leukemia cells.43 Similarly, Maiti and coworkers utilized two-photons fluorescence imaging of dopamine in living cells and tissues for the quantitative mapping of DA concentration.44 The aforementioned approaches including two photons excitation fluorescence imaging methods are typically used for the mapping of signaling proteins and quantitative measurement of the neurotransmitters content in cells or tissues. However, these techniques are not convenient for probing the atomic level interaction between neurotransmitters and signaling proteins. In this context, Gouaux and coworkers are the first who utilized X-ray crystallography based studies to resolve the atomic level interaction of DA with drosophila dopamine transporter (dDATmfc).36, 37 Computational modeling and X-ray crystallographic studies provide the useful information about the interaction of DA with dopamine transporter, but there is still a need of minimal invasive and effective technique to study DA-hDAT interaction in living system. Here, we explain how the combined approach of twophoton excited fluorescence (2PE) imaging, modeselective surface-enhanced Raman spectroscopy (SERS) imaging, and DFT Raman calculation works effectively to probe DA, hDAT, and DA-hDAT interaction in living cells. Two-photon excited (2PE) fluorescence microscopy allows the imaging of submillimeter thick biological samples like cells and tissues. In 2PE fluorescence microscopy, the wavelength of emission light is shorter than the wavelength of excitation light. This technique typically uses longer wavelength femtosecond laser for the excitation of molecules; for each excitation, two photons of excitation laser are utilized.45-49 The photon energy required for the 2PE fluorescence approach is approximately half of the energy required for the single photon excitation. This technique typically requires a high flux of excitation photons because simultaneous absorption of two photons is less probable. The scattering of the light from cell or tissue is smaller for longer wavelength and higher for shorter wavelength. Therefore, 2PE fluorescence imaging is a useful technique to reduce the background noise, increase the optical sectioning, and suppress the photobleaching effect.46, 50 In this imaging technique, the focused laser beam is scanned in a raster pattern to generate images which increases the optical sectioning effect for the highquality image. The optical sectioning effect is higher in 2PE microscopes because the axial spread of the point spread function is significantly lower in comparison to single-photon excitation, which improves the resolution along the Z-dimension. The emission intensity of 2PE fluorescence is quadratically proportional to the excitation intensity, therefore the excitation light for 2PE is tightly focused in the small volume. The 2PE fluorescence imaging has been used for the study of different

fields of biology including physiology, neurobiology, embryology and tissue engineering.51-54 In this work, we have utilized 2PE fluorescence imaging approach to probe the location of DA in hDAT overexpressed human embryonic kidney (hDAT-HEK293) cell. We could expect higher probability of DA-hDAT interaction in a certain region of hDAT-HEK293 cell where DA is located. Therefore, we performed 2PE fluorescence imaging before all modeselective SERS experiment. Surface-enhanced Raman spectroscopy (SERS) is already used as a powerful experimental approach for the characterization of dopamine-human dopamine transporter (DA-hDAT) interactions in living cells.55 Natural Raman cross-section of many biomolecules are extremely small, therefore SERS technique is preferably used to enhance Raman cross-section of biomolecules.56, 57 In SERS technique, rough surface or nanoparticles of gold or silver are used as a substrate, which increase the Raman crosssection of analyte molecules by the factor of 105-106. 58, 59 The electromagnetic (EM) and charge transfer (CT) mechanisms are used to explain the SERS enhancement. The metal nanoparticles used in SERS are also useful to obtain fluorescence-free and high-quality Raman spectra. It is reported that metal nanostructures have a higher fluorescence quenching efficiency than protein or potassium iodide.60-62 Other forms of Raman spectroscopy are also successfully utilized to study the huge range of molecules.63-109 However, SERS technique is more established and powerful analytical approach for the label-free characterization and analysis of biomolecules like neurotransmitters, enzymes, membrane protein, and highly organized systems such as membrane preparation and photosynthetic bacteria.110-116 Although SERS has many important applications, it has certain limitations. Bare SERS substrates such as AgNPs denature the protein molecules during their interactions. The denaturation of proteins is reported due to bond formation between Ag+ ions or AgNPs with sulfhydral groups.117, 118 Therefore, the preventive measure for the direct contact between metallic SERS substrates and biomolecules is highly required and recommended to avoid denaturation of proteins. To avoid the direct interaction of metal with protein or other biomolecule, we have fabricated an ultra-thin layer (4-6 nm) of silica (SiO2) on the surface of silver nanoparticles (AgNPs), which effectively prevents the possible damage of biomolecules from Ag+ ions or AgNPs without reducing the electromagnetic field enhancement of Raman intensity (Figure S1).119, 120 Besides conventional SERS, we have coupled 2PE fluorescence imaging technique and modeselective Raman imaging technique to develop a background-free and label-free imaging approach and probed the atomic level interaction between dopamine (DA) and human dopamine transporters (hDAT) in living cells. The mode-selective Raman measurement requires some modification in the experimental set up of conventional Raman experiment. For example, the mode-selective Raman measurement requires the replace of long pass filter used


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in conventional SERS experiments by a digital minichrom monochromator ((DMC1-03) to select a desired wavenumber from scattered Raman light.

RESULTS AND DISCUSSION Conventional and mode-selective Raman measurement. Our earlier work showed that Raman wavenumber at 807 cm-1, and 1076 cm-1 are the signature of the bound states of DA molecules in the human dopamine transporter (hDAT).114 Here, we have reproduced and validated our earlier findings. Briefly, Raman spectral analysis of DA, parent line human embryonic kidney (HEK293) cells, hDAT-HEK293 cells, DA-HEK293 cells, and DAhDAT-HEK293 cells shows that five new signature peaks at 807, 839, 1076, 1090, 1538, and 1665 cm-1 are related to DA-hDAT interaction.114 These new five peaks could be contributed from several changes that happen during DAhDAT interactions. The X-ray crystallographic studies show the bond formation by hydroxy and primary amine group of DA with protein residues of dopamine transporter (Figure 1). 36, 37 The direct measurement of these bond formation in living cell is particularly difficult for Raman experimental approach since it is faster dynamic processes. 120, 121 The electron densities on protein residues and DA are changed due to DA-hDAT interaction, which is associated to the change of certain Raman intensity and can be used to probe DA-hDAT interactions. The measurement and analysis of changes in electron density of DA is more convenient and effective to study the DA-hDAT interaction rather than the analysis of other protein residues since DA has comparatively smaller and simpler structure. Our DFT calculation shows that the electron density distribution of DA in its bound and unbound states are different. It also shows that HOMO of bound states of DA is stable than it unbound states by 0.466 eV energy (Figure S2A). This stabilization energy interprets that the DAhDAT interaction is an energetically feasible process. In addition, DFT calculation shows that Raman wavenumber of a certain bond of DA changes according to the change of its electrons density. The combined approach of Raman experiments and theoretical calculations shows that the two Raman peaks at 807 cm-1 and 1076 cm-1 are signature peaks for the bound states of DA, which could be utilized to probe the progress of DA-hDAT interactions in living cells. The coordinate of DA for DFT calculation was obtained from the crystal structure of dDATmfc (Figure 1). In this crystal structure, the amine group of dopamine interacts with the carboxylate group of Asp46 at 3 A°. The catechol group of dopamine is trapped in a cavity formed by Ala117, Val120, Asp121, Tyr124, Ser422, and Phe325 and interacts with the carboxylate group of Asp121 through a hydrogen bond. The meta-hydroxyl group of dopamine interacts with the side chain of Asp121 at 2.7 A° orienting itself towards Ser422 in TM8 at 3.8 A°. The para-hydroxyl group interacts with the carbonyl oxygen of Ala117 and the carboxylate of Asp121 at distances of 2.8 and 3.1 A° respec-

tively. The Raman wavenumber of bound states of DA is calculated without geometry optimization using density functional theory (DFT) method on a B3LYP level with a basis set of 6-31 G(d), and Gaussian 09 package. The geometry optimization is skipped for this state to conserve coordinates. However, the DFT Raman calculation of unbound state of DA is carried out in the same basis set with the geometry optimization. The analysis of DFT calculated Raman spectra of bound and unbound states of DA (Figure S2B) shows that two Raman modes at 807 and 1076 cm-1 are landmarks for the bound states of DA which is also observed in experimental result. We use these two Raman peaks to probe the DA-hDAT interactions in living cells. DFT Raman and experimental SERS spectra show a convenient way to probe the progress of protein-ligand interactions.

Figure 1. The crystal structure of dDATmfc (PDB ID: 4XP1) showing the binding site of dopamine. The crystal structure of dDATmfc mimics structure of human dopamine transporter (hDAT) where DA is located at the binding site surrounded by transmembrane helices (TMs). The amine group of dopamine interacts with carboxylate group of Asp46 at 3 A°. The catechol group of dopamine is trapped in a cavity formed by Ala117, Val120, Asp121, Tyr124, Ser422, and Phe325 which interacts with carboxylate group of ASP121 by the formation of hydrogen bond. The meta-hydroxyl group of dopamine interacts with the side chain of Asp121 at 2.7 A° orienting itself towards Ser422 in TM8 at 3.8 A°. The para-hydroxyl group interacts with both the carbonyl oxygen of Ala117 and the carboxylate of Asp121 at distances of 2.8 and 3.1 A° respectively. We used coordinates of DA molecule located in binding site of dDATmfc crystal to calculate HOMOLUMO energy calculation and DFT Raman spectrum of bound states of DA. DFT Raman calculation and conventional SERS study are useful to determine the characteristics peaks related to DA-hDAT interactions. Here, we have utilized the mode-selective Raman imaging approach along with combined approach of DFT calculation and conventional SERS experiment, which can visualize the high-quality



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and background-free Raman images of DA-hDAT interactions in live HEK293 cells. The optical requirements for mode-selective Raman imaging approach are met by the combination of 488 nm continuous wave (CW) argon ion laser, inverted confocal microscope (Axiovert 135) equipped with a XY piezo-controlled scanning stage (physic instrumente), oil immersion objective (Zeiss FLUAR, 100×; 1.3 NA), digital mini-chrom monochromator (DMC1-03), and single photon avalanche photodiode (APD) (PerkinElmer SPCMAQR-14). The schematic representation of experimental set up is shown in Figure 2. The photons from a Raman scattering are selected using digital mini-chrom monochromator and focused on APD to obtain mode-selective Raman images. In addition to the mode-selective Raman images, we have collected the mode-selective Raman spectra focusing similar photons to spectrophotometer (Triax 550, Jobin Yvon). The consistency between the occurrence of mode-selective Raman images and mode-selective SERS spectra is required to test the validity of mode-selective Raman image. The occurrence of mode-selective Raman images and spectra in our experiments are highly correlated (Figure 3). For mode-selective Raman measurement, we allowed the photons corresponding to Raman wavenumber 807 cm-1 and 1076 cm-1 (~508 nm and ~515 nm) to pass through digital mini-chrom monochromator. The mode-selective Raman images related to wavenumber 1076 cm-1 and 807 cm-1 are shown in Figure 4E and 3F respectively. The corresponding Raman peaks of these wavenumber are shown in Figure 5E and 4F respectively.

Figure 3. Summary of the observations from modeselective Raman measurements on 50 hDAT expressed HEK293 cells after the addition of DA. The black and green bars represent the appearance of mode-selective Raman images and mode-selective Raman spectra respectively. The observations are based on cells which were cultured in seven separate petri dishes; 6-8 DA rich cells were taken from each petri dish for the experiments. Mode-selective Raman images and spectra were collected form the regions where the presence of dopamine was confirmed by 2PE fluorescence imaging. Experiments show the appearance of Raman wavenumber 1287 cm-1 in 50 and 1463 cm-1 in 43 DA rich cells. The Raman wavenumber 1076, and 807 cm-1 appeared in 38 DA-hDATHEK293 cells. The Raman wavenumber 1287 cm-1 and 1463 cm-1 are signature for DA and hDAT protein respectively. The Raman frequencies 1076 cm-1 and 807 cm-1 are signature for DA-hDAT interactions, and they are originated from the change of electron density of DA due to interactions.

Figure 2. Schematic representation of the optical setup for the fluorescence, and Raman imaging (not to scale). For twophoton excited (2PE) fluorescence imaging, frequency doubled 532 nm femtosecond pulsed laser (Chameleon Discovery, coherent, ~ 100 fs fwhm) was used. For the Raman experiment, 488 nm continuous wave argon ion laser was used. Mirrors (M) and digital mini-chrom monochromator (MC) are foldable and do not block the next optical alignments


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Figure 4. Two-photon excited (2PE) fluorescence and mode-selective Raman images of hDAT-HEK293 cells. 2PE fluorescence image of (A) autofluorescence background from hDAT-HEK293 cells before addition of DA. (B) the 2PE fluorescence signal intensity produced from DA in hDAT-HEK293 cells. The fluorescence signal intensity of DA in hDAT-HEK 293 cells was 42 ± 5 % intense than autofluorescence background of the cells. (C)The modeselective Raman images of hDAT-HEK293 cells before DA addition which are corresponding to 1463 cm-1 and are signature for the hDAT protein; (D) The mode-selective Raman images of hDAT-HEK293 cells after the DA addition, which are corresponding to 1287 cm-1 and are signature for DA. (E) and (F) are the mode-selective Raman images of hDAT-HEK293 cells after the DA addition which are corresponding to 1076 cm-1 and 807 cm-1 respectively and are signature for DA-hDAT interactions.

Figure 5. The consistence between conventional and mode-selective SERS spectra. (A) and (B) are the Raman spectrum from hDAT-HEK293 cells before and after the addition of DA. (C) The mode-selective Raman spectrum corresponding to Raman wavenumber 1463 cm-1 which is signature for the hDAT protein. (E) and (F) are the modeselective Raman spectrum corresponding to 1076 cm-1 and 807 cm-1 respectively which are signature for DA-hDAT interactions. The dotted lines show the consistency between signature peaks observed in the mode-selective Raman spectrum, and conventional Raman spectrum.



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Figure 6. The two-photon excited (2PE) fluorescence images from parent line HEK293 cells. The two-photon excited (2PE) fluorescence images of (A) cleaned empty cover glass (B) spin coated 30 µl solution of 100 nM DA. (C) autofluorescence background from HEK293 cells before addition of DA. (D) The 2PE fluorescence signal from DA in HEK293 cells. The 2PE fluorescence signal intensity from DA in HEK293 cells was 41 ± 6 % intense than autofluorescence background from the HEK293 cells. (E) and (F) are mode-selective Raman im-1 -1 ages of HEK293 cells corresponding to 1414 cm and 1145 cm respectively. For 2PE fluorescence imaging approach, frequency doubled femtosecond 532 nm pulsed laser (Chameleon Discovery, coherent, ~ 100 fs fwhm) of average power 15±2 µW is used.

Figure 7. The conventional and mode-selective Raman spectrum from DA and parent line HEK293 cells. The Raman spectrum from (A) cleaned cover glass and (B) DA coated cover glass. The correspondence of figure 6A with 7A and 6B with 7B shows that the increase of fluorescence signal in the DA coated cover glass is contributed by DA. (C) The mode-selective Raman spectrum from DA coated cover glass corresponding to Raman wavenumber 1287 cm-1. Raman spectrum corresponding to HEK293 cells (D) before and (E) after addition of DA. The mode-selective Raman spectrum related to (F) 1414 cm-1 and (G) 1145 cm-1. The dotted red and black line show the consistency of Raman peak at 1145 cm-1 and 1414 cm-1 between modeselective Raman spectrum and full range Raman spectrum before and after the addition of DA. The dotted blue line shows the consistency of Raman peak at 1287 cm-1 in full range Raman spectrum of DA, DA added parent line HEK293 cells, and mode-selective Raman spectrum of DA. We tested our experimental results with many controlled experiments. For that, we collected the modeselective Raman images from HEK293 cells, DA-HEK293 cells, and hDAT-HEK293 cells. The mode-selective Raman image of HEK293 cells was collected using photons of


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Raman frequencies 1414 cm-1 (Figure 6E) and 1145 cm-1 (Figure 6F). The mode-selective Raman image was confirmed as we were also able to collect mode-selective Raman spectra corresponding to 1414 cm-1 (Figure 7F) and 1145 cm-1 (Figure 7G) from same spot. Furthermore, we obtained the mode-selective Raman image (Figure 4C) and spectrum (Figure 5C) from hDAT-HEK293 cells associated to the Raman wavenumber 1463 cm-1. According to our experimental analysis, this Raman wavenumber is the signature for the hDAT protein. On the basis of literature, this Raman wavenumber is originated from the individual or combined contribution of C-asym rocking of Ala, Cδbending of Lys, and Cγ1-asym rocking and Cδ-asym bending of Ile.121 The assignments of important Raman peaks from DA and cells are provided in supporting information (Table TS1 and TS2). In separate experiment, we have obtained the mode-selective Raman image of dopamine (Figure 4D) in DA-HEK293 and DA-hDAT-HEK293 cells utilizing its signature Raman wavenumber of 1287 cm-1. According to our DFT calculation and literature, the signature Raman mode 1287 cm-1 is related to in-plane –CH2 bending vibration arising from DA cationic form. We also obtained the mode-selective Raman spectra of this wavenumber (Figure 5D). The concentration of dopamine, we used in our experiment is ~4µM, which is a way smaller than the concentration of neuronal dopamine in the synaptic vesicles (~100s of mM).122 This experimental results demonstrate that our approach is capable for the direct imaging of dopamine present in brain cells. Two-photon excited (2PE) fluorescence imaging approach. Before the mode-selective SERS experiments, we used the two-photon excited (2PE) fluorescence imaging approach to visualize the quantitative changes of the fluorescence signal due to DA in parent line HEK293 cells and hDAT-HEK293 cells. For that, we analyzed the 2PE fluorescence images of HEK293 cells and hDAT-HEK293 cells in the absence and presence of DA. The red spots on Figure 4D and 5D represent the 2PE fluorescence signal intensity of DA in hDAT-HEK293 cells and HEK293 cells respectively. We observed the signature SERS peaks for DA, hDAT, other membrane proteins, and their interactions by a SERS spectrum originating from the bright red spot. The bright red spots obtained from 2PE experiments basically indicate the presence of DA in that area. However, this bright region does not necessarily represent the interaction of DA with hDAT or other membrane protein. The benefit of 2PE fluorescence is that: it works as a guide to find DA-rich region in cells where we could expect a higher probability for DA-hDAT interactions. This is how the combined approach of mode-selective Raman measurements and 2PE fluorescence imaging was used to probe and confirm the interaction of DA, hDAT and other membrane protein.

We found that 2PE fluorescence imaging approach is important to probe the potential region of DA-hDAT interaction in HEK293 cells. The requirements for 2PE fluorescence imaging is achieved by the combination of frequency doubled femtosecond 532 nm pulsed laser (Chameleon Discovery, coherent, ~100 fs fwhm), inverted confocal microscope (Axiovert 135) equipped with a piezocontrolled scanning stage (physic instrumente), oil immersion objective (Zeiss FLUAR, 100×; 1.3 NA), and APD (PerkinElmer SPCMAQR-14). The bandpass filter (FES0500) is positioned before the entrance slit of APD to collect the fluorescence emission originated from twophoton excitations. The percentage transmittance of the dichroic mirror (chroma, ZT532rdc) and bandpass filter (FES0500) show that they are suitable optics to meet the criteria of 2PE (Figure S3A and S3B respectively). The analysis of emission and excitation of dopamine supports the phenomenon of 2PE fluorescence (Figure S4 and S5). The 2PE fluorescence images of HEK293 cells (Figure 6C) are originated from their intracellular contents, the characteristic SERS spectrum of HEK293 cells is shown in Figure 7D. When HEK293 cells were treated with 4 µM DA solution for ≥ 30 minutes, the 2PE fluorescence signal of DA were detected in cells as a function of dopamine concentration. According to our quantitative analysis, it is found that the 2PE fluorescence signal intensity of DA in HEK293 is 41 ± 6 % more intense after addition of DA (Figure 6D). Figure 7C represents the mode-selective Raman spectra from HEK293 cells after addition of dopamine. HEK293 cells were washed three times with warm PBS buffer before 2PE fluorescence imaging experiments which is required to avoid the interference from extracellular DA fluorescence. We have also measured the function of DA concentration in hDAT-HEK293 cells to produce dopaminergic fluorescence intensity. For that, we analyzed the intensity of 2PE fluorescence image obtained from hDAT-HEK293 cells in the absence (Figure 4A) and 2PE fluorescence signal intensity of DA in hDAT-HEK293 cells (Figure 4B). To test the sensitivity of the dopamine in hDAT-HEK293 cells, the 4µM concentration of DA solution was treated with cells sample and waited for ≥30 minutes for the incubation. This time allows DA to bind with hDAT protein and enter hDAT-HEK293 cells. The 2PE fluorescence signal intensity of DA in hDAT-HEK293 cells was (Figure 4C) was 42 ± 5 % more intense than 2PE auto fluorescence obtained from intracellular proteins in hDAT-HEK293 cells (Figure 4B). The 2PE fluorescence images of DA in hDAT-HEK293 cells are useful to probe the location of DA and possible region for the DA-hDAT. The correlation between the appearance of characteristic SERS peak of DA and appearance of dopaminergic 2PE fluorescence signal intensity validates the 2PE fluorescence contributed from DA. Mode-selective Raman images (Figure 4D) and SERS spectra (Figure 5D) corresponding to Raman wavenumber 1287 cm-1 are appeared after DA treatments which is signature for the presence of DA compound. In other control experiment, we performed



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2PE fluorescence imaging experiment and Raman measurement on blank and dopamine coated cover glass where fluorescence signal from blank cover glass (Figure 6A) was found negligible with comparison of dopamine coated cover glass (Figure 6B). Moreover, the Raman spectrum from a blank cover glass was just like a background line (Figure 7A) while characteristic SERS spectrum of dopamine was observed from the dopamine coated cover glass (Figure 7B). Correlation between mode-selective Raman image and spectra. We performed the mode-selective Raman measurements in fifty dopamine treated hDATHEK293 cells. For these experiments, cells were cultured in seven separate petri dishes. From each petri dish, 6-8 dopamine-rich hDAT-HEK293 cells were selected for the 2PE fluorescence imaging and mode-selective Raman measurements. The dopamine-rich cells were selected and confirmed by 2PE fluorescence imaging. In our experiment, we observed mode-selective Raman image and spectra associated to Raman wavenumber 1287 cm-1 from all 50 dopamine-rich cells. However, mode-selective Raman image and spectra associated with the Raman wavenumber 1463 cm-1 were seen only from 43 cells (Figure 3). These results indicate that hDAT protein was not expressed equally in all 50 HEK293 cells. The Raman frequencies: 1076 cm-1 and 807 cm-1 were observed only from 38 dopamine-rich cells. These results show that signature Raman wavenumbers related to DA-hDAT interactions were not found for all hDAT expressed dopamine-rich cells. We found that 5 out of the 43 cells which had both hDAT and DA were present; but, failed to produce DAhDAT signature peak. As we mentioned before, the Raman wavenumber 1287 cm-1 and 1463 cm-1 are signatures for DA and hDAT protein respectively, and 807 cm-1 and 1076 cm-1 are signatures for DA-hDAT interactions. These experimental results related to occurrence of modeselective Raman image and spectra indicate that DAhDAT interaction is not possible all the time even though DA and hDAT are found together. The results from the mode-selective Raman image (black bar) have good agreements with the occurrence of mode-selective Raman spectra (green bar) which are summarized in Figure 3. However, we found one case where a mode-selective Raman image associated to 1076 cm-1 was obtained but we did not find the corresponding mode-selective Raman spectra. This discrepancy could open another scope of research which is not focus of our current study. In summary, our experimental results show that dopamine, hDAT proteins, and their interactions can be effectively and sensitively probed and confirmed in living cells using combined approach of mode-selective Raman imaging and 2PE fluorescence imaging techniques.

CONCLUSION We have recorded SERS spectra of dopamine, HEK293 cells, and hDAT expressed HEK293 cells in the absence or presence of dopamine. The analysis of experi-

mental and DFT Raman spectra shows that Raman wavenumber of 807 cm-1 and 1076 cm-1 are benchmark for the DA-hDAT interactions. As a matter of fact, these Raman wavenumbers are generated from the bound states of DA. Utilizing these signature mode, we generated the modeselective Raman images and visualized the interaction between DA and hDAT proteins in living HEK293 cells. Apart from Raman measurements, we utilized twophoton excited (2PE) fluorescence imaging approach to probe the dopamine molecules in living cells which guides to find the potential region of the DA-hDAT interactions in living cells. Our technique could be developed as a simpler and effective technique to probe the location of DA-hDAT interactions in living cells, which could be further extended to probe the location and interactions of other proteins and signaling molecules including drugs and neurotransmitters in brain cells. The combined approach of surface-enhanced Raman scattering and twophoton excited (2PE) fluorescence imaging could be developed as the powerful tool to visualize the several signaling molecules, signaling proteins, and their interactions in living systems.

METHODS Synthesis of silica coated silver nano particles. Silver nanoparticles (AgNPs) are synthesized by a standard sodium citrate reduction method,123 followed by the addition of active sodium silicate to generate ultra-thin layer of silica over AgNPs.124 Silver nitrate (AgNO3), sodium citrate, and sodium silicate required for the synthesis of nanoparticles were purchased from Sigma Aldrich, and used without further purification. Then, the synthesized nanoparticles were characterized by transmission electron microscopy (TEM) and varian UV-Vis spectrophotometer (EL07013173). According to our TEM measurement, the average sizes of silica-coated silver nano particles were found 58 ± 8 nm with SiO2 shell thickness 5 ± 1 nm (Figure S1). The UV-Vis spectroscopy shows that the maxima (λmax) of the plasmon resonance band position related to silver nanoparticles is found at 420 nm, which was shifted to 407 nm after generating the ultra-thin layer of silica on its surface.114 HEK293 cells culture. The vial of HEK293 cells were thawed in a warm water bath (37oC) and decontaminated by spraying 70% ethanol. Then cells were transferred into a T-75 flask with 15 ml of complete medium. The complete medium contains Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich D5796), 10% fetal bovine serum (Sigma-Aldrich, F2442), and 1% penicillinstreptomycin (ATCC, 30-2300). Then, the T-75 flask containing cells was stored in the incubator at 37oC with 5% supply of CO2 atmosphere. After 24 hours, all the complete medium from the T-75 flask was aspirated out and replaced by fresh complete medium. When confluence of the cells reached to 70-80% on the surface of the T-75 flask, cells were harvested using trypsin EDTA and sub


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cultured on a 25-mm circular cover glass in 35 mm petri dish.

ly, the filter was washed twice with 25 μL of elution buffer to collect a total 50 µl plasmid solution.

Preparation of hDAT inducible HEK293 cells. After 24 hours of sub culture, we followed the standard protocol of Invitrogen for the preparation of hDAT inducible HEK293 cells. In brief, all the complete medium from the T-75 flask was aspirated out and replaced by fresh complete medium containing 1×10-4 gm/ml zeocin (invivoGen, ant-zn-1), and 1×10-3 gm/ml blasticidin (Millipore/Calbiochem, 203350). When the cells reached 7080% confluence on the surface of the T-75 flask, hygromycin B (Sigma-Aldrich, H3274) was added to obtain its final concentration 25 μg/μl, which kills all the cells within 2 days except some hygromycin resistant cells. Then these cells were isolated and cultured in complete media; when cells were attached (approx. 24 hrs) on the surface of the T-75 flask, doxycycline (Tocris Bioscience, 4090) was added to make the final concentration 0.5 µg/µl and waited for ≥ 48 hrs to obtain hDAT inducible HEK293 cells.

Transfection of pcDNA3.1-hDAT in HEK293 cells. For the efficient gene transfection, we followed a protocol of lipid-mediated transfection with Lipofectamine LTX and Plus reagent (Invitrogen, 11668-019). After the subculture of hDAT inducible HEK293, we normally waited for 1-2 days to allow them to attach onto the cover glass and cover 50% of its area. This was the condition when cells were ready for the hDAT expression. One day before the transfection, the growth media having antibiotics (Penicillin streptomycin) was replaced with the growth media without having antibiotics. For the transfection, 5 μL pcDNA3.1-hDAT plasmids was first added to 250 µl optiMEM solution. Then 5 µl of Plus reagent was added into this resulting solution. The 50 µl solution from this diluted plasmid was then mixed with the previously prepared solution of 50 µl opti-MEM and 4 µl of lipofectamine LTX (Invitrogen). This complex DNA reagent was incubated for 20 min at room temperature. Finally, a 50 µl solution of complex DNA reagent was added into the Petri dish containing cells and waited for 2-3 days for the expression of hDAT in HEK293 cells. The successful expression of hDAT was confirmed by a green fluorescence (Figure S6). Before the experiment, cells were washed with PBS solution to remove culture medium and used fresh PBS solution to avoid drying of cells.

Amplification of pcDNA3.1-hDAT plasmid. The green fluorescence protein (GFP) tagged pcDNA3.1-hDAT (32810) was purchased from Addgene and amplified with a standard method. Amplified plasmid was treated with hDAT inducible HEK293 cells for the expression of the human dopamine transporter (hDAT). Here the extraction of pcDNA3.1-hDAT from bacterial cells and transfection of plasmid in HEK293 has been explained briefly. Firstly, the pcDNA3.1-hDAT plasmid of the human dopamine receptor received as the bacteria in agar stab was streaked on the surface of LB agar plate using a sterile wire loop. All LB plates were kept inside the small incubator for 16 h at a temperature of 37°C. When bacteria were grown on LB plates as colonies with a copy of pcDNA3.1hDAT plasmid, one spot from a bacterial colony was taken out and transferred into a falcon round-bottom tube containing 6 mL of LB solution with 100 µg/ml ampicillin. This round bottom tube was placed in a rotating incubator to grow bacteria for 24 h at 37°C. In this solution, the bacteria having pcDNA3.1-hDAT plasmid were grown, which were extracted from LB solution in a 2 ml centrifuge tube by a centrifuging process. The liquid part was thrown out after each centrifuging process and the solid residue was finally deposited on the bottom of the centrifuge tube. This residue was dissolved with 250 ml of resuspension reagents (Thermo Scientific, Gene JET Plasmid Miniprep kit, cat. no. K0502), 250 ml of lysis solution, and 350 ml of neutralization solution to separate plasmid from other parts of bacterial cell. The resulting solution was centrifuged for 15 min at 1200 rpm, and the supernatant solution was taken out from the centrifuge tube and filtered through the miniprep gene jet spin column to remove filtrate. The content adsorbed on a filter was washed two times with 500 ml wash buffer and centrifuged to remove any remaining alcohol in plasmid. Final-

Two photons excited (2PE) fluorescence imaging. The frequency doubled femtosecond 532 nm pulsed laser (Chameleon Discovery, coherent, ~100 fs fwhm) was used for the excitation of samples. The excitation laser was shone on samples through 532 nm dichroic mirror (Chroma, Zt532rdc) and oil immersed objective of the inverted microscope. The diffraction limited (300 nm) epi fluorescent light beam returns to the 532-nm dichroic mirror and was collected by single photon avalanche photodiode (APD) (PerkinElmer SPCMAQR-14). The bandpass filter (FES0500) was positioned in front of APD to prevent entry of any excitation laser and single photon excited (1PE) fluorescence. Surface enhanced Raman measurements. SERS spectra were collected using the home-modified confocal Raman microscope125 (Figure 2) using 488-nm continuous-wave (CW) argon ion laser of approximately 13±2 µw power. We used 30 second integration time for the recording of a SERS spectrum. Mercury lamp and cyclohexane were used to calibrate the setup before Raman measurements with spectral resolution 2 cm-1. For the analysis of conventional SERS spectrum, we selected the range of 700–1700 cm-1. We maintained the same experimental parameters throughout the experiment to avoid their effect. For the collection of conventional SERS spectra, the long-pass filter (HHQ495LP) was positioned in front of the entrance slit of monochromator (Triax 550, JobinYvon). The Raman spectra were collected by a liquid ni-



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trogen cooled CCD (LN2-CCD; Princeton instruments), which was cooled at about -100°C. Mode-selective Raman measurement. The modeselective Raman experiment requires some modification in the experimental setup of conventional Raman measurement. For that we replaced the long-pass filter (HHQ495LP) with the digital mini-chrom monochromator (DMC1-03). Similarly, mode-selective Raman images were collected using 488 nm CW argon ion laser as an excitation source, FES0500 band pass filter, dichroic mirror (Chroma, Zt488 drc), and avalanche photodiode (PerkinElmer SPCMAQR-14). Density functional theory calculations. Geometry optimization and Raman frequency calculations were performed using the density functional theory (DFT) method on B3LYP level with a basis set of 6-31G (d) and Gaussian 09 package to compare free state of DA with its bound state in hDAT. According to a comprehensive evaluation of Scott and Radom126, obtained Raman wavenumbers were scaled by a factor of 0.9614. Molecular orbitals were calculated with the same basis set and visualized with Avogadro software (Avogadro: an open-source molecular builder and visualization tool). All calculations were carried out on a vector processor (Ohio Supercomputer Center, Columbus, Ohio).

ASSOCIATED CONTENT Supporting Information: Raman spectroscopic measurement details, computational calculation of the Raman spectra, two-photon spectroscopy characterizations, and two-photon excitation spectroscopic imaging of living cells The supporting information is available free of charge on the ACS Publication website at DOI: AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected] ORCID H peter Lu https://orcid.org/0000-0003-2027-428X Author contributions: H.P.L. designed research; A.S. performed research; H.P.L. contributed new reagents/analytic tools; A.S. and H.P.L. analyzed data; and A.S. and H.P.L. wrote the paper. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

H.P.L. acknowledges the support from the Ohio Eminent Scholar endowment fund and the Ohio Attorney Gen-

eral’s Center for the Future of Forensic Science Research Fund, Ohio. ABBREVIATIONS DA, dopamine; HEK, human embryonic kidney; hDAT, human dopamine transporter; dDAT, drosophila dopamine transporter; mfc, minimal functional construct; 2PE, two photons excited; SERS, surface enhanced Raman spectroscopy; Ile, isoleucine; Lys, lysine REFERENCES

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ACS Chemical Neuroscience

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ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

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Mode-Selective Raman Imaging of Dopamine ... - ACS Publications

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