Resonance Raman Probes for Organelle-Specific Labeling in Live Cells

Abstract

Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging.

Resonance Raman Probes for Cells

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Andrey N. Kuzmin, Artem Pliss, Chang-Keun ,, Jeongyun Heo, Sehoon Kim,, Alexander , Bobo Gu,, Ken-TyeYong, ShangchunWen & Paras N. Prasad

dynamic changes in this organelle by Raman imaging.

Raman spectroscopic imaging is among the most powerful tools available for the analysis of molecular organization of cells and tissues. Raman spectroscopy relies on inelastic scattering of incident monochromatic light, wherein the energy of photons is being changed, either by a Stokes or anti-Stokes process, upon interaction with molecules of the sample.

Light scattering on dierent types of biomolecules generates corresponding bands in the Raman spectra, which enables to resolve certain amino acids, proteins, various classes of lipids and saccharides, as well as DNA and RNA. It is important to note that the intensity of Raman scattering is linearly dependent on the molecular concentrations at the site of spectral measurement, which uniquely allows for quantitative mapping of biomolecular distribution in situ1,2. This valuable feature of Raman spectroscopy has been realized using a Biomolecular Component Analysis (BCA), a powerful algorithm that identies concentrations of dierent molecular groups which collectively contribute to the Raman spectrum of the sample3. In this regard, Raman spectroscopy has a breakthrough potential for the development of innovatory omics technologies (e.g. proteinomics, metabolomics, and lipidomics) at a single-organelle level. An ultimate goal of these research disciplines is a comprehensive characterization and monitoring of biochemical composition in specic cellular organelles, to unravel mechanisms of cellular regulation4,5. Up to date, most of the data on the molecular composition of subcellular structures have been obtained by mass-spectroscopy. Using this technique, several thousand diverse molecular species have been identied in various cellular compartments58. However, molecular proling with mass-spectroscopy involves cell fractionation and extraction of biomolecules from various organelles, which inherently produces artifacts and is not compatible with live systems9. At the same time, conventional live cell imaging techniques, such as uorescence microscopy, can identify only a few molecular species at a time, which signicantly limits their efficiency for comprehensive molecular characterization of single cells and subcellular structures1013.

In comparison, Raman spectroscopy interrogates all molecules present in the sampling volume of the excitation beam, independently of any extrinsic labels, which is a major strength of this technique. Besides, the Raman signal intensity is not susceptible to photobleaching, thus enabling for long term monitoring of biological

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samples. Although Raman spectroscopy provides multiple benets for molecular analysis of a cell, its capabilities as a single-organelle tool are rather limited. A major obstacle of this technique for subcellular analysis is that the location of specic organelles should be resolved to target acquisition of Raman spectra and analyze the content of a single organelle. Meanwhile, subcellular compartments, with a partial exception of mitochondria1,14, do not exhibit organelle-specic vibrational bands and, therefore, cannot be recognized by the Raman imaging of intrinsic cellular components.

To circumvent this limitation, several research groups, including ours, have applied a bi-modal uorescence/ Raman approach, wherein conventional uorescence reporters are used to target Raman spectral acquisition to specic cellular compartments3,15,16. However, it became apparent that conventional uorescence reporters are not well suitable for Raman imaging, mainly due to a strong uorescence background, masking the Raman signal. Moreover, uorophores are quickly photobleached at high signal excitation power densities used in the Raman technique, thus making extended monitoring of organelles in live cells impossible17.

In parallel to experimentations with uorescence probes, a rst generation of designated Raman reporters has been developed on the basis of deuterium, nitrile or alkyl containing molecular groups, which produce distinctive vibrational bands in the biologically silent region of Raman spectrum17,18. This advancement, for the rst time, enabled detection of specic cellular structures in the Raman modality. However, the signal intensity of the aforementioned Raman tags does not exceed that of native cellular biomolecules, which implies severe limitations in the detection sensitivity. Since, exogenous molecular probes are typically applied at low (nanomolar to micromolar) concentrations to avoid cytotoxicity, the signal intensity from currently existing probes may not be sufficient for detection of labeled organelles in live cells.

Plasmonic enhancement of Raman scattering on metal surfaces, known as Surface Enhanced Raman Spectroscopy (SERS)19, has been used to increase signal from Raman probes and improve the detection sensitivity. However, advancing the SERS technology for mapping of intracellular biomolecules is extremely challenging20,21.

First, targeted molecules of interest greatly outnumber plasmonic nanoparticles that can be tolerated by cells without any adverse eect. Second, the size of most biomolecules is incomparably smaller than that of these nanoparticles. Therefore, any intracellular gradient in the distribution of biomolecules cannot be identied by SERS. Besides, bulky metal nanoparticles can disturb activities of biomolecules and produce mechanical damage and other artifacts. Finally, SERS is not applicable in xed cells, as plasmonic nanoparticles do not penetrate into the cell, even aer very extensive permeabilization of cellular membranes. All these factors limit the utility of the SERS technology for intracellular molecular probing.

An optimistic strategy to achieve enhancement of Raman scattering utilizes the Resonance Raman (RR) phenomenon22. In this approach, the energy of Raman excitation is adjusted to overlap with an electronic transition of the molecule of interest, such as a Raman reporter, which results in a signicant amplication of the light scattering process. This RR technique has been successively applied for biomedical tissue analysis and detection of cancer related abnormalities23,24. It has also been successfully applied for ultra-sensitive detection of nucleic acids and protein biomolecules. However RR enhancement of cellular biomolecules utilizes highly phototoxic UV light, which is a prohibitive limitation for most live cell studies. At the same time natural pigments, which absorb in visible light, have long been a target RR spectroscopic probing of live cells and tissues22. Besides, the advantages of RR and SERS can be combined into an approach known as Surface Enhanced Resonance Raman Spectroscopy (SERRS) when the RR enhancement for some analytes, or reporters, could be accomplished at the excitation wavelength which also excites the plasmonic particles and creates SERS. SERRS was reported to generate unsur-passed enhancement of Raman signal, and has been successfully used for cell-free assays25 . However as discussed above, the plasmonic particles have only a limited value for labeling of intracellular molecules. A new concept for Raman molecular probes, which produces unprecedentedly strong Raman signal through RR enhancement and provide capability for intracellular labeling, recently was developed by our group14. Our reporters utilized azobenzene (AZO) tags, which were modied to produce RR enhancement under excitation with visible light at 532nm, which is far less cytotoxic than UV light used in conventional RR spectroscopy. We further synthesized an AZO-RR probe for organelle-specic labeling in live cells and demonstrated its exceptionally high photostability, enabling long term monitoring of the same organelle14.

Current Raman microscopy involves fairly long acquisition time, up to several minutes per square micrometer of the sample as well as intense laser illumination (typically 10 mW or higher), and therefore the risk of photodamage to a living specimen has to be carefully considered. In this regards, it has been demonstrated that selecting the excitation wavelengths in the spectral region from the red to the infrared, signicantly reduces phototoxicity and enables repeatable spectra acquisition, without compromising functions of the organelle26.

Therefore, development of RR probes excitable by biologically safe wavelengths will be highly benecial for Raman microscopy of live samples.

In this paper, we introduce a novel RR molecular recognition probe, designed for identication of organelles or other cellular structures and demonstrate its application by Raman imaging using excitation in a biologically safe wavelength region. A RR reporter based on BlackBerry Quencher 650 (BBQ-650), was developed to produce RR enhancement under excitation in red spectral range, where cellular biomolecules practically do not absorb, thus minimizing phototoxicity. Amplication of Raman signal by the resonance mechanism drastically increases the detection threshold sensitivity as compared to that of conventional spontaneous Raman probes. Besides, this probe produces low uorescence background that oen limits the sensitivity of Raman technique. Using this novel RR reporter, we synthesized a probe for tracking lysosomes in live cells and demonstrate rst Raman detection and RR imaging of this type of cellular organelles. An inherent advantage of our approach is that RR imaging can be combined with the mapping of unlabeled cellular macromolecules by spontaneous Raman technique.

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Figure 1. Chemical structure of (a) BBQ650-NHS and (b) BBQ650-Lyso (lysosome targeting moiety is shown in red). (c) Absorption spectrum of 14M BBQ650-NHS and BBQ650-Lyso in DMF.

Figure 2. Dependence of the Raman signal of BBQ650-NHS on the excitation wavelength and concentration. (a) A correlation between the concentration of BBQ650-NHS solution in DMF and the intensity of Raman spectra under 633nm excitation. (b) Raman spectra of the BBQ-NHS solution in DMF obtained with dierent excitation sources, as labeled; the band at 10871133cm1 was used for BBQ-NHS detection. It shows that excitation with 633nm generates a signicant RR signal enhancement.

Results and Discussion

To develop a Raman marker producing resonance signal enhancement upon excitation in the biologically safe spectral region, we selected a commercial BBQ650 quencher. This molecule is composed of the electron accepting nitro group and the electron donating julolidine group coupled to a bis-azobenzene structure (Fig.1a), which induces a signicant red-shi of the electronic absorption spectrum to ~610 nm due to strong intramolecular charge transfer and extended -conjugation. Figure1b shows the structure of the RR reporter for tracking lysosomes (BBQ650-Lyso), prepared by tethering BBQ-650 and a lysosome targeting moiety designated here as Lyso. The absorption properties of the BBQ650 and BBQ650-Lyso probes indicate that RR enhancement can be obtained under the red light excitation in the 600700 nm range (Fig.1c). Notably, developed as a uorescence quencher, the BBQ650 probe produces very little uorescence background, which is an additional valuable benet of BBQ650 as a Raman probe.

To explore the efficiency of RR enhancement, we have measured the spectra of BBQ650-NHS dissolved in DMF, using the excitation wavelengths of 532, 633 and 785nm (Fig.2a). The obtained Raman spectra contained several distinctive bands in the region of 10001400cm1, with the most intense peak at 10871133cm1. Among the tested excitation wavelengths, the highest Raman signal intensity for BBQ650-NHS was obtained under 633 nm excitation (Fig.2a). This strong Raman signal is most likely resulting from a favorable combination of high extinction coefficient at 633nm but with a moderate reabsorption of the Raman signal.

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Figure 3. Cytotoxicity of BBQ650-Lyso, evaluated by the MTT colorimetric assay on HeLa cells. As indicated on the plot, cells were treated with 2.520M of BBQ650-Lyso for 3h or 24h. Control (Ctrl) corresponds to untreated cells.

At this excitation wavelength, we determined a detection threshold for BBQ650-NHS as 7 M with a 10 s of signal accumulation time (Fig.2b), which is two to three orders of magnitude lower than that of common biomolecules, under same experimental measurement conditions27. Hence, we concluded a high potential of BBQ650 as a RR probe for cellular bioimaging.

To introduce RR probes for detection of organelles in live cells, we designed a lysosome tracking RR reporter based on BBQ-650 N-hydroxysuccinimide ester (BBQ650-NHS), Fig.1(a). The lysosome probe (designated as BBQ650-Lyso) was obtained by simple amidation between the NHS ester and the amine modied targeting moiety. As shown in Fig.1(b), we utilized N,N-dimethyl ethylenediamine for lysosome targeting by permeation and accumulation of the monobasic amine28,29.

The biocompatibility of the obtained BBQ650-Lyso probe was evaluated by the MTT cytotoxicity assay. In these experiments, cells were incubated with dierent concentrations of BBQ650-Lyso, ranging from 2.5 M to 20M, for either 3h or 24h, and then processed under a standard MTT protocol. As shown in Fig.3, there was almost no toxic eect for cells following 3h incubation, for all tested concentrations of the probe. A only signi-cant decrease in the cellular viability was observed, when the probe was used at its highest concentration of 20M for 24h. Considering that organelle labeling in live cells, involves signicantly lower concentrations of the probe in the nanomolar to micromolar range, and a shorter incubation time which usually does not exceed one or two hours, we concluded that the BBQ650-Lyso probe is unlikely to cause any measurable cytotoxicity and is well applicable for live cell studies.

Next, we validated the organelle-targeting specicity of the newly synthesized BBQ650-Lyso probes. Here, live cells were incubated both with BBQ650-Lyso and the commercial uorescent lysotracker, and the uorescence and the Raman signal distribution were concurrently acquired (see Materials and Methods). As could be expected from lysotracker, both probes were accumulated in the lysosomes and also were diusely distributed in the cytoplasm. Our imaging data indicated a signicant degree of co-localization of both the uorescence and the Raman signals in lysosomes, (Fig.4) which indicates that the BBQ650-Lyso probe synthesized in our study, targets lysosomes with specicity similar to that of commercial probes.

Next, we explored the feasibility of simultaneous imaging of BBQ650-based RR probes as well as cellular macromolecules by the spontaneous Raman spectroscopy technique. In these experiments, cells were incubated with the BBQ650-Lyso probe, followed by acquisition of two sequential Raman imaging scans. First, cells were scanned using a 8mW laser at 633nm, to produce selective enhancement of the BBQ650-Lyso signal, through the RR mechanism. With a signal accumulation time of 0.2sec/pixel, an image acquisition of 2424m2 area corresponding to an average-size cell took about 40 seconds, which satises most of live cell imaging tasks. Next, the same cells were scanned with a 532 nm laser at 10 mW excitation power to image the distribution of cellular biomolecules. Despite the higher excitation power, the accumulation time had also to be increased tenfold up to 2sec/pixel, to obtain a sufficient signal from biomolecules in the absence of resonance enhancement (Fig.5). The obtained hyper-spectral data were analyzed with the Thermo Scientic OMNICxi imaging soware

using embedded image-rendering algorithms. The peak intensities assigned in earlier studies to proteins (2906 3015cm1)3,30,31, were integrated to generate Raman images (Fig.5b). Similarly, the signal at 10871133cm1 was processed, for detection of the BBQ650-Lyso probe (Fig.5c,d) in accordance with the Raman spectrum of this compound (Fig.3).

We found that owing to RR enhancement, the signal from the BBQ650-Lyso probe at 10871133 cm1 was very intense under 633 nm excitation, which enabled for condent imaging of lysosomes in the cell. Raman spectrum of BBQ650-Lyso in lysosomes has the same major peaks at 1080, 1160, 1240 and 1370cm1, as that of BBQ650-NHS solution. At the same time, in comparison with BBQ650-NHS, where Raman peak centered at 1080 cm1 demonstrated the highest intensity, BBQ650-Lyso does not show this feature. As shown in the

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Figure 4. Co-localization of the uorescence signal from the commercial lysotracker and the Resonance Raman signal from the BBQ650-Lyso probe. The cell was simultaneously stained with the BBQ650-Lyso probe and the commercial lysotracker. Both types of lysosome probes are accumulated in the cytoplasm, and co-localize in the lysosomes, while the nucleus was not stained. Signals from (a) commercial uorescent lysotracker and (b) BBQ650-Lyso probe. (c) The yellow color on the right panel represents the overlap of the two signals. White bar in (c) corresponds to 10m. The scanning rate is 4s/pixel (full image time scanning ~4s).

Figure 5. Raman images of HeLa cell incubated with the BBQ-NHS lysosome marker. (a) Transmittedlight image snap. (bd) Raman images, generated from the spectral peaks assigned to dierent molecular constituents. (b) Spontaneous Raman image under 532nm excitation corresponding to the proteins 2906 3015cm1 band. The signal depicts distribution of intracellular proteins in the cell nucleus and cytoplasm.

The highest proteins concentration corresponds to the nucleoli (dark domains in (a)). (c) Resonance Raman image of the BBQ650-Lyso probe (10871133cm1 band) under 633nm excitation. The BBQ650-Lyso probe accumulated into bright foci in the cytoplasm, consistent with the lysotrackers labeling pattern. (d) Spontaneous Raman image for the 10871133cm1 band obtained under 532nm excitation. The signal is present both inthe cell nucleus and the cytoplasm, and does not resemble staining from the lysosome probes. The White barin (a) corresponds to 10m. The scanning rates are 2s/pixel (full image time scanning ~10min) for (b,d) and 0.2s/pixel (full image time scanning ~1min) for (c). (e) Averaged Raman spectra for the entire cell excited by 532nm (black curve, spontaneous mode, excitation power 10mW) and BBQ650-Lyso (red curve, resonance mode, excitation power 8mW). A comparison of the signal intensity at 750100cm1 and 1750400cm1 demonstrates that under 633nm excitation by, the signal from cellular macromolecules was at the background level, due to short accumulation time.

integrated Raman spectra the non-resonant Raman signal from the cellular macromolecules was only at the background level due to the short signal accumulation time (Fig.5e). Contrarily, with the 532 nm excitation, the signal from the BBQ650-Lyso probe was barely detected, despite the prolonged accumulation time. Indeed, mapping the signal of the 10871133cm1 band, does not reveal a similarity between the images obtained under 532 and 633 nm excitations. Most likely, this mismatch can be explained by accumulation of the Raman signal from biomolecular vibrations attributed to C-N and C-C bonds, the tryptophan ring in cellular proteins, and the O-P-O backbone in DNA/RNA32,33, rather than that of the RR from BBQ650-Lyso, due to the low intracellular concentration of the probe and insignicant levels of RR enhancement with 532nm excitation.

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Figure 6. (a) Time sequence of the Raman spectra of lysosome, together with the Raman spectra of BBQ (grey curve) and of cellular lipids (magenta curve). (b) The residual spectra aer subtracting of 10, 20 and 30min sequential Raman spectra of lysosome from that of 0min (starting point of measurement). The spectra of BBQ and lipids were subtracted with corresponding weight coefficient as well. Abbreviations: Alaalanine, strstretch, irrirregular, narunspecied non-aromatic side-chain, defdeformation. The Grey box shows measurement standard error frame.

Finally, we utilized the RR signal from the BBQ650-Lyso probe to enable time-sequenced acquisition of Raman spectra from a single labeled organelle, and thus validate the utility of this RR probe for selective targeting of subcellular structures. In these experiments, lysosomes in live cells were labeled with the RR probe. The nal concentration of the RR probe was ~1M, which is within the recommended concentration range for lysotrackers. Labeled organelles were then localized by Raman spectroscopic imaging of cytoplasm and rendering the signal from RR probe. Then, a series of Raman spectra were acquired from single labeled organelles using 633nm excitation and a longer accumulation time of 120s for condent characterization of local molecular contents. An intense RR signal from BBQ650 enabled for monitoring of the lysosome position for each spectral measurement. All spectra in the sequence contained the characteristic 10871133 cm1 band assigned to BBQ650 (Fig.6a). As an example of the BCA technique for quantitative monitoring of specic types of molecular vibrations in an organelle, the spectra were processed as follows. First, the spectrum obtained at the rst time point was subtracted from the other spectra in the time series. This step enabled to identify changes in the spectra of lysosomes occur-ring in time. The residual spectra contained prominent contributions of BBQ650 and membrane lipid Raman components. Second, BBQ650 and lipid components were further subtracted from the analyzed spectra. The nal residual spectra, obtained aer the second subtraction step, are shown in Fig.6b. We found that during the monitoring time, the intensity of the Raman band centered at 1099cm1, a common marker of proteins assigned to the alanine C-C-stretch33, gradually increased, while another group of protein bands centered at 1248cm1 and 1316 cm1, which are correspondingly assigned to irregular conformations and deformations of non-aromatic side chains, were decreasing. Such changes reveal that the pool of proteins in lysosome is dynamically changing in time. Thus, the presented here technique can enable studies of biological signicance of such transformations in the molecular makeup of a specic organelle.

These data indicate that application of the RR probes may enable rapid location of the organelle of interest in live cells, making it available for monitoring with Raman microspectroscopy. This is a signicant step forward in comparison with current bioorthogonal spontaneous Raman markers that cannot be detected at biologically safe concentrations.

Summary

In this communication, we introduced a next generation of Raman probe for labelling organelles in live cells that utilizes a BBQ650-NHS structure. The proposed probe provides ultrasensitive molecular detection through the mechanism of resonance enhancement of Raman signal. A valuable feature of this probe is that the resonance excitation is achieved using biologically safe light wavelengths in the red region enabling non-invasive live cell imaging. The presented Raman reporter combines all the benecial features of small molecule probes, such as easy access to dense intracellular structures, absence of unwanted interference with cellular environment, while possessing high detection sensitivity comparable to that of plasmonically enhanced nanoprobes. Aer specic functionalization, this novel marker can serve as a molecular-selective probe, targeting distinct subcellular domains and making them available for Raman spectroscopic analysis. We have validated this concept for design of RR probes using a BBQ650-Lyso marker for targeting lysosomes in live cultured cells. Using this concept the possibility of dual-mode, resonant and non-resonant, Raman imaging was demonstrated. With this novel technique, the cellular organelles are condently identied by the signal from the external RR probe using physiologically safe concentration, comparable to that of uorescence probes. Simultaneously, the conventional Raman imaging of native cellular macromolecules at the site of interest is implemented. Photostability of the RR probe was sufficient for long term tracking of marked lysosomes to perform time-sequential local Raman probing

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and monitoring the changes of the biomolecular composition in a single organelle of a live cell. This approach will bridge together the advantages of highly versatile molecular recognition probes, currently available only in uorescence imaging techniques, and a label-free Raman imaging for quantitative biochemical characterization of subcellular structures.

Materials and Methods

5 L of N,N-dimethylethylenendiamine was added to 1 mL of 1.4mM BBQ-650 N-hydroxysuccinimide ester (BBQ650-NHS, Berry & Associate) in dimethylformamide (DMF) at ambient temperature. The reaction mixture was stirred overnight at ambient temperature. The reaction was monitored by TLC (Ethylacetate:Hexane= 1:1). Aer disappearance of the BBQ650-NHS spot on TLC, the mixture was used for toxicity measurements and cellular imaging experiments without further purications.

Cell culture and MTT Assay and labeling of lysosomes. HeLa cells were grown in DMEM supplemented with a 2.5% fetal bovine serum (Gibco), 1% glutamax, 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) at 37C in a humidied atmosphere, containing 5% CO2. For the MTT Assay, cells were placed in 96-well plates and incubated with the experimental RR lysotracker at concentration ranging from 2.5 to 20 M for either 3 h or 24 h at 37 C. Next, cells were washed with PBS and incubated with 0.5 mg/mL of the MTT solution for 3h, followed by the addition of dimethylsulfoxide. Plates were placed in the microplate spectrophotometer system (Opsys MR, Dynex technologies) and absorbance was recorded at 490nm. Results were analyzed with the Revelation Quicklink soware and are presented as the percentage of the control values.

For staining of lysosomes, HeLa cells were incubated with 1M BBQ650-NHS probe and/or 500nM uores-cent lysotracker (ThermoFisher) for 30min.

Raman Single Spectra Measurement and Raman Imaging. Raman spectroscopic measurements were performed by using a confocal Raman microspectrometer based on the SpectraPro 2500i (Acton Research, Trenton, NJ) monochromator equipped with the Spec 10100B CCD camera (Princeton Instruments, Trenton, NJ) for Raman signal detection .The three laser sources used for excitation are: 532 nm Verdi V-6, 633 nm He-Ne laser (both Coherent, Santa Clara, CA) and 780 nm OPO Levante (APE, Germany) pumped by a High-Q Laser (High Q Laser GmbH, Austria). This conguration enables the measurements within the range of Raman shi of 6003000 cm1. The spectral resolution for a xed diraction grating position (wave number interval of 1210 cm1) was ~1.5 cm1. The Raman spectra of BBQ650 solutions were measured using the following excitation powers: 633nm25mW, 532nm25mW, 785nm200mW. Raman imaging was performed with the DXRxi Raman Imaging Microscope (Thermo Fisher Scientic, Madison, WI). For cell imaging, we used a 60x water immersion objective lens (Olympus, Japan).

The laser power at the sample plane during Raman imaging was 8mW for 633nm and 10mW for 532nm.

Live cells where lysosomes were labeled with both BBQ650-Lyso and commercial uorescent lysotracker, were imaged by the multimodal imaging system described in our earlier publications3,15. For imaging using uorescent lysotracker, 10 W excitation at 532 nm was used in combination with a 60 nm band-pass optical lter centered at 580 nm (FF01-580/60, Semrock, USA). The RR signal of BBQ650-Lyso, generated by 30 mW of 633 nm excitation, was used for imaging, employing a 30nm band-pass optical lter centered at 687nm (FF01-687/30, Semrock, USA).

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This work at Bualo (P. N. Prasad) was supported by the Air Force Office of Scientic Research grant FA9550-12-1-0226. The work at Changsha (S. Wen) was supported by the National Natural Science Foundation of China (Grant No. 11574079).

Author Contributions

BBQ650-Lyso probe was designed and synthetized by C.-K.L. with assistance of S.K. Cell growth, incubating and uptake were performed by A.P. and B.G. MTT cytotoxicity assay was performed by J.H. Raman imaging was performed by A.K.N., A.R. and A.P. Raman microspectrometry experiments were performed by A.N.K. and B.G. The processing and analysis of collected data was done by A.N.K., K.-T.Y., S.W. and P.N.P. The manuscript was written by A.K.N., A.P., P.N.P. and C.-K.L. All authors reviewed the manuscript.

Additional Information

Competing nancial interests: The authors declare no competing nancial interests.

How to cite this article: Kuzmin, A. N. et al. Resonance Raman Probes for Organelle-Specic Labeling in Live Cells. Sci. Rep. 6, 28483; doi: 10.1038/srep28483 (2016).

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