Sulforhodamine Nanothermometer for Multiparametric Fluorescence

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Sulforhodamine nanothermometer for multi-parametric FLIM imaging James Jenkins, Sergey M Borisov, Dmitri B. Papkovsky, and Ruslan I Dmitriev Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02675 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 6, 2016

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Analytical Chemistry

Sulforhodamine nanothermometer for multi-parametric FLIM imaging James Jenkins1$, Sergey M. Borisov2$, Dmitri B. Papkovsky1, Ruslan I. Dmitriev1* 1

School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Graz, Austria 2

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these authors contributed equally to this work.

*

Address correspondence to: University College Cork, Biosciences Institute, Western Road, Cork. E-mail: [email protected], phone: +353-21-4901339. Fax : +353-21-4901382

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Abstract Live cells function within narrow limits of physiological temperature (T), O2 and metabolite concentrations. We have designed a cell-permeable T-sensitive fluorescence lifetime-based nanoprobe based on lipophilic sulforhodamine, which stains 2D and 3D cell models, shows cytoplasmic localization and robust response to T (~0.037 ns/K). Subsequently, we evaluated the probe and FLIM technique for combined imaging of T and O2 gradients in metabolically active cells. We found that in adherent 2D culture of HCT116 cells intracellular T and O2 are close to ambient values. However in 3D spheroid structures having size >200 µm, T and O2 gradients become pronounced. These microgradients can be enhanced by treatment with mitochondrial uncouplers or dissipated by drug-induced disaggregation of the spheroids. Thus, we demonstrate the existence of local microgradients of T in 3D cell models and utility of combined imaging of O2 and T.


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Introduction Temperature (T) affects numerous physical, chemical and biological processes in the cell, including production of energy, diffusion of biomolecules and drugs, viscosity, enzymatic and acid-base reactions1-4. In higher multicellular organisms tissue T is usually kept within narrow physiological limits via a number of pathways, including thermogenesis in skeletal muscle and brown adipose tissue5-7. The success of diagnostics and therapy of multitude of diseases, including cancer, is also highly dependent on careful control and imaging of temperature8,9. Therefore, tissue T is regarded as important marker for medical imaging and cancer biology. The infrared thermal imaging systems 10 are accurate for medical imaging but have limited spatial resolution and 3D imaging capabilities. Fluorescence-based techniques and dedicated T-sensing probes can overcome these limitations and allow quantitative measurement and imaging of tissue T and gradients in real time, in 2D and 3D cellbased models3,11,12. A number of nanoparticle and small molecule fluorescent probes have been developed in recent years, not only for tissue T but also for intracellular compartments (the cytosol, endoplasmic reticulum, mitochondria)13-17, including those utilizing fluorescence lifetime measurements (FLIM)15,16,18. However, their performance is still compromised by aggregation, cross-sensitivity to viscosity, biomolecules and ions and instability of calibration. This often leads to misrepresentation of the T gradients, especially at subcellular level. The gradients of as high as 4 °C between cytoplasm, nucleus, ER and mitochondria measured with some T-sensitive probes can be questioned, based on the actual ability of the cell to generate such amount of heat and heat dissipation rates19-21. Therefore, it is necessary to carefully evaluate possible measurement artifacts and cross-interferences of the current and newly introduced T probes with different biological models to confirm the validity of FLIM data and probe T calibration in situ. On the other hand, a reliable T sensing probe can allow multiplexed FLIM imaging of O2, autofluorescence, pH, viscosity, Ca2+ and protein interactions22-29, while avoiding artifacts of intensity-based imaging. In particular, molecular oxygen (O2) can be imaged with dedicated cell-penetrating phosphorescent probes and PLIM method28,3032 . Transport and local gradients of O2 in respiring samples28 are profoundly influenced by T, and they play important roles in tissue function and tissue models such as 3D tumor spheroids33,34. Mimicking cell morphology, gradients of O2, nutrients and drugs in spheroid models allows better understanding of tumor progression and prediction of responses to drug treatment35,36. However, simultaneous high-resolution imaging of T and O2 in such tissue models has not been demonstrated so far. Here we report the design of a sulforhodamine-based nanoprobe for high-resolution imaging of temperature. We thoroughly evaluated the effects of encapsulation of sulforhodamine in different polymer nanoparticles and the effects of microenvironment (pH, serum, viscosity, intracellular location) on its T-sensitivity. We assessed its performance with adherent (2D) and tumor spheroid (3D) models, and combined with the O2-sensing probe to visualize the O2 and T gradients by FLIM-PLIM method. We demonstrate that in 2D models sub-cellular T and O2 gradients are negligible, but they become pronounced in spheroids of size larger than 200 µm and they correlate with spheroid size and drug treatments. The study also demonstrates the application potential of spheroid aggregates as physiological tumor tissue models.

Experimental section ACS Paragon Plus Environment

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Materials MitoImage-NanO2 and pH-XtraTM probes were from Luxcel Biosciences (Cork Ireland). LipidureTM 96-well plates were from Amsbio (UK). SII-0.2+ probe was prepared as described before37. Cholera Toxin, subunit B, Alexa Fluor 488 conjugate, MitoTracker Green, LysoTracker Red and TMRM were from Invitrogen (BioSciences, Dublin Ireland). CellTiter-Glo kit was from Promega (MyBio, Ireland). Ibidi µ-slide 8/12-well chambers were from Ibidi (Martinsried, Germany). Sulforhodamine B acid chloride (Lissamine™ Rhodamine B Sulfonyl Chloride), bisBenzimide Hoechst 33342, cantharidin, FCCP, oligomycin and all the other reagents were from Sigma-Aldrich (Dublin, Ireland). Synthesis of 2-(3-diethylamino-6-diethylazaniumylidene-xanthen-9-yl)-5-(Ndodecylsulfamoyl)benzenesulfonate In a flame-dried Schlenk tube, 60 µl (0.344 mmol) of N,N-diisopropylethylamine and 116 mg (0.625 mmol) of dodecylamine were dissolved in 8 ml anhydrous THF. Then 200 mg (0.345 mmol) of sulforhodamine B acid chloride dissolved in 8 ml anhydrous THF were added and the reaction mixture was stirred at room temperature overnight. After quantitative conversion 150 ml of deionized water were added. The aqueous layer was extracted with dichloromethane (3 x 20 ml) and washed with deionized water 4 times. The organic phase was dried over Na2SO4 and the solvent was removed under reduced pressure. The product was purified by column chromatography (silica gel, ethyl acetate:methanol (8:2 v/v). to give a red solid. Yield: 104 mg (41 %). MS (MALDI): m/z [M]+ 726.3611 calc., 726.3793 found. 1H NMR (300 MHz, CD2Cl2), ppm: 8.56 (s, 1H), 7.87 (d, 1H), 7.16 (dd, 3H), 6.77 (d, 2H), 6.66 (s, 2H), 4.80 (t, 1H), 3.50 (m, 8H), 2.96 (q, 2H), 1.21 (m, 32H), 0.79 (t, 3H). Preparation of nanoparticles 1 mg of the indicator and 200 mg of RL-100 were dissolved in 100 ml acetone, acetone:methanol (60:40 v/v) or acetone:THF:methanol (55:40:5) mixture (Fig. S9A). Then 300 ml of H2O were added within 2 s under vigorous stirring. The solvent was removed under reduced pressure and the dispersion of nanoparticles was concentrated to ~15 ml. Aggregates were removed by centrifugation. Analysis of size and characterisation of the nanoparticles in solution were performed as described previously37. Cell culture Wild-type and SCO2-/- human colon carcinoma HCT116 cells38 were handled as described previously37. Cells and spheroids were stained by adding 1 µg/ml of the T probe or 5 µg/ml of the O2 probe (SII-0.2+ or NanO2) to the growth medium and incubating for 16 h followed by two washing cycles. For SII-0.2+ probe ‘continuous staining’ procedure27 was used. Staining with LysoTracker Red (100 nM), MitoTracker Green (50 nM), Cholera toxin (2.5 ng/ml), Hoechst 33342 (1 µM) and TMRM (20 nM) was performed by incubating cells for 30 min and one washing step. Prior the imaging, cells were equilibrated in Phenol Red-free DMEM medium supplemented with 10 mM glucose, 1 mM pyruvate, 2 mM glutamine, 10mM HEPES, pH 7.2. Spheroids were allowed to attach (3-8 h) on collagen-poly-D-lysinepre-coated glassware.


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Analysis of probe toxicity on cells The effects of nanoparticle staining on total cellular ATP and extracellular acidification rate (ECAR) were analyzed as described previously37, using CellTiterGloTM reagent (Promega) and pH-XtraTM probe (Luxcel Biosciences), respectively. Briefly, cells grown in microplates were exposed to various concentrations of the T (0-2 µg/ml) and SII-0.2+ (0-20 µg/ml) probes for 16 h, then washed and measured using Victor2 time-resolved fluorescence reader (Perkin Elmer). ATP and ECAR values were normalized for total protein content, determined by BCA assay. Microscopy O2 calibrations of SII-0.2+ at different temperatures were performed on a widefield fluorescence microscope Axiovert 200 (Carl Zeiss) equipped with and oil-immersion objectives 40×/1.3 EC Plan Neofluar and 100x/1.4 Plan Apochromat, 590 nm LED excitation module, time-gated CCD camera, emission filter (590/40 nm ex., 760 nm em.), ImSpector software (LaVision BioTec, Germany), and CO2/O2 control chamber (PeCon) 27. The two-site model fitting39 gave us the following analytical function: [O2, µM] = (0.63485/(0.63485-1+τ/38800)-1)/0.02486, where τ represents phosphorescence lifetime (µs). Analysis of cell staining, spheroid culture, T calibrations and FLIM-PLIM microscopy measurements were performed on an upright Axio Examiner Z1 microscope (Carl Zeiss) equipped with 20×/1.0 W-Plan-Apochromat and 63x/1.0 WApochromat objectives, temperature control (external incubator and heated stage, 25 o C-42 oC) with motorized Z-axis control, DCS-120 time-correlated single photon counting confocal scanner (Becker & Hickl GmbH), R10467U-40 and R10467U-50 photon counting detectors (Hamamatsu Photonics K.K.) and dedicated TCSPC hardware (Becker & Hickl GmbH)27,37. The probes were excited with a tunable picosecond supercontinuum laser SC400-4 (Fianium, UK) at 488 nm (Cholera toxin, MitoTracker Green), 540 nm (T probe, LysoTracker Red, TMRM) and 620 nm (SII0.2+) with emission collected at: 512-536 nm (for 488 nm), 565-605 nm (for 540 nm) and 760-810 nm (for 620 nm excitation). NanO2 and Hoechst 33342 were excited with a 405 nm picosecond laser BDL-SMC (Becker & Hickl GmbH) with emission collected at 635-675 nm and 438-458 nm, respectively. Fluorescence lifetimes for T probe were calculated in SPCImage software (Becker & Hickl GmbH) by monoexponential decay fitting (T1=50, T2=150, binning factor=4, threshold=10, shift=0, offset=0). Cells were pre-incubated for 10 min at desired T prior to the measurements. T calibration function produced was the following: T (oC) = 105.6-0.0277*τ, r2=0.99 where τ is in ps. Phosphorescence lifetimes of SII-0.2+ probe were determined from mono-exponential decay fits (T1=120, T2=225-240, binning factor=3, threshold=10, shift=0, offset=0). Calculated lifetimes were exported and further processed in Microsoft Excel, Origin 6.0, Fiji and Adobe Illustrator software. Data assessment and statistics Statistics were carried out using the results of at least 3 independent experiments. Experiments were evaluated for statistical differences using t-test with confidence levels of P20 µg/ml, in agreement with previous data37 and for the T probe at >5 µg/ml. Thus, working concentrations for cell staining were selected as 1-5 µg/ml for O2 and 1-2 µg/ml for T probes. Staining efficiency of multicellular spheroids was also assessed (Fig. S14). The O2 probe showed limited penetration in pre-formed spheroids and therefore it required continuous staining procedure27, whereas the T probe was seen to stain multiple cell layers, within 16 h incubation time. The T and O2 probes have very distinct spectral characteristics and luminescence lifetimes (2-3 ns and 10-40 µs, respectively), comparable brightness, and therefore are suitable for multiplexed imaging.


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Visualization of T and O2 gradients in 2D and 3D cell models Local O2 gradients in individual cells and 2D monolayer cultures can be insignificant, but they become profound in cell aggregates and spheroid structures of sizes > 50 µm 35,36,58 . We hypothesized that subcellular T gradients are also negligible in monolayer cultures, but they become visible in multicellular aggregates. To test this, we grown metabolically active HCT116 cells as 2D (monolayer) and 3D (spheroids) cultures and analyzed them with the T probe at resting conditions at 37 and 40 oC: Fig. 2 shows that in the 2D culture intracellular T values were found to be very close to environmental (37 oC, 18.6% O2 in the incubator). In contrast, in spheroids we detected prominent gradients of T, which were size-dependent and resulted in approximately 2-3 oC higher T in the core (Fig. 2). However, the spheroids of 200 and 300 µm in diameter showed similar maximal T values of ~ 41oC. When we exposed resting spheroids to different T in the incubator (Fig. 3), we also observed the effect of T on the oxygenation of spheroid core and periphery: at 40 oC spheroid core heated up, but became more oxygenated. To confirm that T and O2 gradients are caused by spheroid formation, we performed reverse experiment (Fig. 4): we treated HCT116 spheroids with cantharidin59, decreasing cell-cell contacts and leading to disaggregation of spheroids. As expected, upon disassembly, multicellular structures increased their O2 and T approaching ambient steady-state levels. Marker of mitochondrial membrane potential TMRM also showed brighter staining (i.e. high viability and metabolic activity) in spheroids before disaggregation, while treatment with cantharidin decreased them. To see what happens to observed T and O2 gradients in spheroids upon metabolic stimulation and drug treatment, we produced spheroids from wild-type and deficient in oxidative phosphorylation SCO2-/- HCT116 cell lines38, stained these spheroids with T and O2 probes and treated them with drugs FCCP (uncoupler, activates cell respiration) and oligomycin (inhibitor of ATP synthase) to achieve maximal mitochondrial uncoupling60. We found (Fig. 5) that in the wild-type spheroids such treatment significantly increased deoxygenation (from 50 to 10 µM) due to increased O2 consumption61, and concomitantly increased spheroid core temperature (by ~ 2-3 o C, for 200-250 µm spheroid, seen by color FLIM image and on bar charts for selected ROIs). In contrast, the non-respiring SCO2-/- cell-based spheroids showed no changes in T and O2 upon such treatment. Compared to wild-type samples, SCO2-/spheroids also showed higher oxygenation levels and nearly ambient T in the core (Fig. 5 B). Thus, in tumor spheroid model we observed dynamic behavior of T and O2 gradients, which were dependent on the size of the aggregates and their metabolic activity. Conclusion The rational design and characterization of the novel formulation of sulforhodamine dye in cationic RL100 nanoparticles produced a fluorescent probe highly useful for quantitative T imaging in cell and tissue models by FLIM technique. The encapsulation in nanoparticles made the probe largely unaffected by environmental factors such as ionic strength, pH and serum proteins while retaining or improving its T sensitivity in fluorescence lifetime (Fig. 1, S1-S11). Upon addition to growth medium, the T probe provided efficient staining of HCT116 cells with distribution in the cytoplasm, in both 2D (monolayer cells) and 3D cell culture (spheroids). The spectral and decay time characteristics of the T probe allowed its combined use with the phosphorescent intracellular O2-sensitive nanoparticles for quantitative T and O2


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imaging. Chemical structure of sulforhodamine is similar to SR10162, indicating that T probe must be also compatible with two-photon FLIM. Using the 2D cell cultures in conjunction with the T probe and FLIM, we observed nearly homogeneous distribution of T across cell cytoplasm, with no visible effects of micro-heterogeneity of pH and viscosity within the cell. On the other hand, combined T and O2 imaging in spheroid cultures revealed pronounced T and O2 gradients, which depended on spheroid size and metabolic activity. O2 gradients were previously described for spheroid models27,61,63, while T gradients were demonstrated here for the first time. T gradients were studied in HeLa cells spheroids14 but were undetectable, most likely due to their small dimensions and low metabolic activity of highly glycolytic HeLa cells. Our results highlight the interdependence of T and O2 gradients for tumor tissue models; both parameters are influenced by their metabolic state and microenvironment. We found that the temperature of spheroids never exceeded ~ 41 o C (Fig. 2), which we explain by adaptation of the cells to the temperature produced by their metabolic activity and decreasing their heat production and activating heatshock pathway. Cancer cells should avoid generation of excess of heat, to prevent the heat shock and following death. The increase of cell oxygenation in the spheroid core exposed at 40 oC (Fig. 3) can be due to reduced consumption and faster diffusion of O2 and altered balance of energy production pathways (OxPhos to glycolysis)60. On the other hand, this can be explained by the decrease of cell viability occurred due to the critical temperature values. Such ‘increased oxygenation’ effect was also in contrast with the temperature dependence of O2 solubility in aqueous phase64 and thus should be accounted for cell metabolic activity and viability. The data also demonstrate that in terms of T and O2 regulation, 3D tissue models such as spheroids are more physiological than conventional 2D cultures. This is particularly important for understanding of tumor biology and development of anticancer drugs and therapies and interpretation of effects on metabolism. Altogether, imaging of cell and tissue T, and particularly combined T and O2 FLIM-PLIM imaging, have high application potential with various tissue models, in regenerative medicine and cancer biology. Supporting information available Supplementary table S1 and figures S1-S14 are combined in Supplementary data PDF file. This material is available free of charge via the Internet at http://pubs.acs.org . Abbreviations 2D – two-dimensional; 3D – three-dimensional; FCCP – carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone; FLIM -fluorescence lifetime imaging microscopy; OXPHOS - oxidative phosphorylation; PLIM - phosphorescence lifetime imaging microscopy; PMMA-AA – poly(methyl methacrylate –co-acrylic acid); RL100 – Eudragit RL100 polymer; TCSPC – time-correlated single photon counting; TMRM - tetramethylrhodamine methyl ester;


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Acknowledgment This work was supported by Science Foundation Ireland, grants 13/SIRG/2144 and 12/RC/2276. The authors thank Andreas Steinegger, Anna Walcher and Matthias Schwar (Graz University of Technology) for their help in the preparation and characterization of the T probe. References (1) Jarmuszkiewicz, W.; Woyda-Ploszczyca, A.; Koziel, A.; Majerczak, J.; Zoladz, J. A. Free Radical Biol. Med. 2015, 83, 12-20. (2) Mahmoudi, M.; Abdelmonem, A. M.; Behzadi, S.; Clement, J. H.; Dutz, S.; Ejtehadi, M. R.; Hartmann, R.; Kantner, K.; Linne, U.; Maffre, P. ACS Nano 2013, 7, 6555-6562. (3) Zhou, H.; Sharma, M.; Berezin, O.; Zuckerman, D.; Berezin, M. Y. ChemPhysChem 2016, 17, 27-36. (4) Vysniauskas, A.; Qurashi, M.; Gallop, N.; Balaz, M.; Anderson, H. L.; Kuimova, M. K. Chem. Sci. 2015, 6, 5773-5778. (5) Echtay, K. S. Free Radical Biol. Med. 2007, 43, 1351-1371. (6) Mahmmoud, Y. A.; Gaster, M. Br. J. Pharmacol. 2012, 166, 2060-2069. (7) Whittle, A.; Relat-Pardo, J.; Vidal-Puig, A. Trends Pharmacol. Sci. 2013, 34, 347-355. (8) Repasky, E. A.; Evans, S. S.; Dewhirst, M. W. Cancer Immunol. Res. 2013, 1, 210-216. (9) Chakraborty, M.; Mukhopadhyay, S.; Dasgupta, A.; Banerjee, S.; Patsa, S.; Ray, J.; Chaudhuri, K. In SPIE Medical Imaging; International Society for Optics and Photonics, 2016, pp 97853I-97853I-97857. (10) Ring, E.; Ammer, K. Physiol. Measurement 2012, 33, R33. (11) Wang, X.-d.; Wolfbeis, O. S.; Meier, R. J. Chem. Soc. Rev. 2013, 42, 7834-7869. (12) Fischer, L. H.; Harms, G. S.; Wolfbeis, O. S. Angew. Chem., Int. Ed. 2011, 50, 45464551. (13) Arai, S.; Lee, S.-C.; Zhai, D.; Suzuki, M.; Chang, Y. T. Sci. Rep. 2014, 4. (14) Arai, S.; Suzuki, M.; Park, S.-J.; Yoo, J. S.; Wang, L.; Kang, N.-Y.; Ha, H.-H.; Chang, Y.-T. Chem. Comm. 2015, 51, 8044-8047. (15) Hayashi, T.; Fukuda, N.; Uchiyama, S.; Inada, N. PloS One 2015, 10, e0117677. (16) Itoh, H.; Arai, S.; Sudhaharan, T.; Lee, S.-C.; Chang, Y.-T.; Ishiwata, S. i.; Suzuki, M.; Lane, B. Chem. Comm. 2016. (17) Tanimoto, R.; Hiraiwa, T.; Nakai, Y.; Shindo, Y.; Oka, K.; Hiroi, N.; Funahashi, A. Sci. Rep. 2016, 6. (18) Paviolo, C.; Clayton, A.; McArthur, S.; Stoddart, P. J. Microsc. 2013, 250, 179-188. (19) Baffou, G.; Rigneault, H.; Marguet, D.; Jullien, L. Nat. Methods. 2014, 11, 899-901. (20) Suzuki, M.; Zeeb, V.; Arai, S.; Oyama, K.; Ishiwata, S. i. Nat. Methods. 2015, 12, 802803. (21) Okabe, K.; Inada, N.; Gota, C.; Harada, Y.; Funatsu, T.; Uchiyama, S. Nat. Commun. 2012, 3, 705. (22) Aigner, D.; Dmitriev, R. I.; Borisov, S. M.; Papkovsky, D. B.; Klimant, I. J. Mater. Chem. B 2014, 2, 6792-6801. (23) Betolngar, D.-B.; Erard, M.; Pasquier, H.; Bousmah, Y.; Diop-Sy, A.; Guiot, E.; Vincent, P.; Mérola, F. Anal. Bioanal. Chem. 2015, 407, 4183-4193. (24) Poëa-Guyon, S.; Pasquier, H.; Mérola, F.; Morel, N.; Erard, M. Anal. Bioanal. Chem. 2013, 405, 3983-3987. (25) Kuchibhotla, K. V.; Lattarulo, C. R.; Hyman, B. T.; Bacskai, B. J. Science 2009, 323, 1211-1215. (26) Becker, W.; Shcheslavskiy, V.; Studier, H. In Advanced Time-Correlated Single Photon Counting Applications, Becker, W., Ed.; Springer International Publishing: Cham, 2015, pp 65-117. (27) Dmitriev, R. I.; Zhdanov, A. V.; Nolan, Y. M.; Papkovsky, D. B. Biomaterials 2013, 34, 9307-9317.


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Figures

Figure 1. Chemical structure and characterization of the T probe. A: Structure of the synthesized sulforhodamine dye. Lipophilic tail is indicated in yellow. B: Normalized fluorescence spectra of the probe in solution (25 oC, exc.max at 560 nm, em.max at 578 nm). C: Confocal microscopy images of HCT116 cells stained with T probe (1 µg/ml, dye concentration 0.004 µg/ml, 16 h, shown in red) and counter-stained (green color) with Cholera toxin (endosomes), MitoTracker Green (mitochondria) and NanO2 O2 probe. D: FLIM images of resting cells stained with T probe and measured at different temperatures. Scale bar is in µm. Insert (right) shows examples of fitting decays for 25 and 40 oC. E: Fluorescence lifetime distribution histograms for the T probe and the free dye in cells at temperatures ranging 25-42 oC. F: T calibrations of sulforhodamine dye loaded in cells in free form (different concentrations) and as nanoparticle formulation.


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Figure 2. Evaluation of temperature gradients in adherent (2D) and spheroid (3D) cell cultures with T probe. FLIM images of intracellular T in HCT116 cells exposed to 37 and 40 o C (regions of interest are indicated in pink). Bottom panel: average calculated temperatures of the cells and spheroids for selected regions of interest. Scale bar is in µm. N=3.

Figure 3. Analysis of T and O2 gradients in resting HCT116 cell spheroids. Spheroids were stained with the O2 and T probes, exposed to different temperatures, and measured by FLIMPLIM microscopy at 0 and 30 µm depths (‘periphery’ and ‘core’ regions, respectively). A: Examples of FLIM images (left) and average T values for selected ROIs (right). B: Examples of PLIM images (left) and average O2 values for selected ROIs (right). Scale bar is in µm. N=3. Asterisks indicate significant differences, P = 0.05 (*), P = 0.001(***).


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Figure 4. Drug-induced disaggregation of spheroids dissipates local T and O2 gradients. HCT116 cell spheroids treated with cantharidin (CAN, 5 µM, 3 h) were analyzed. DMSO was used as control. A: Transmission light microscopy. B: Staining with mitochondrial membrane potential probe TMRM (20 nM). C: O2 Representative PLIM images (top) and calculated mean O2 values (bottom). D: Representative FLIM images (top) and average T values (bottom). Scale bar is in µm. N=3.


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Figure 5. Changes in T and O2 gradients in spheroids upon metabolic stimulation. Spheroids from respiring (wild-type, A) and non-respiring (SCO2-/-, B) HCT116 cells were stained with T and O2 probes and measured by FLIM-PLIM microscopy before and 10 min after stimulation with 4 µM FCCP/ 10 µM oligomycin. FLIM and PLIM images of spheroids (left) and average T and O2 values for selected ROIs (right). The cross-sections of spheroids were at 15 µm depth. Scale bar is in µm. N=3. Asterisks indicate significant differences, P = 0.05 (*), P = 0.001(***).


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For TOC only


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Sulforhodamine Nanothermometer for Multiparametric Fluorescence

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