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Super-resolution fluorescence imaging of biocompatible carbon dots Godefroy Leme´nager,†a Elisa De Luca,†a Ya-Ping Sunb and Pier Paolo Pompa*a
Received 11th April 2014 Accepted 27th May 2014 DOI: 10.1039/c4nr01970a www.rsc.org/nanoscale
Carbon Dots (CDs) are a new promising type of small (5 nm), biocompatible and multicolor luminescent nanoparticle. Here, we demonstrate super-resolution imaging of CDs at the nanoscale through STimulated Emission Depletion (STED) microscopy. In addition, we report the application of STED for detection of CD localization in both fixed and living cells, achieving a spatial resolution down to 30 nm, far below the diffraction limit, showing great promise for high resolution visualization of cellular dynamics.
recently developed nanomaterial, i.e. carbon dots (CDs), quantum-sized nanocrystals composed of carbon.6 CDs were shown to be biocompatible7 and highly photoluminescent, and offer multicolor emission.8,9 However, despite these important advantages for biological applications, no super-resolution imaging with CDs has been reported so far in the literature. We successfully employed STED microscopy to image CDs in cells, demonstrating a >6-fold improvement of the spatial resolution, compared to conventional confocal microscopy.
Introduction Far eld microscopy is one of the main techniques used for sample imaging in several elds, from condensed matter to life science. Because of the diffraction, however, the optical resolution is limited to about Dr z l/(2N.A.) z 200 nm (with l the wavelength of light and N.A. the numerical aperture of the lenses). In recent years, several breakthroughs, usually referred to as super-resolution techniques, appeared in the literature, overcoming the diffraction limit and signicantly improving the optical resolution.1–5 Available for different types of emitters, from organic dyes1 to quantum nanostructures,2 such techniques can be grouped in two major systems, based on uorescent inhibition properties or on stochastic methods.3,4 The rst group, including stimulated emission depletion (STED) microscopy, deactivates the uorophores selectively to enhance the resolution imaging in that area.5 The second group of techniques uses sequential activation and time-resolved localization of uorophores to create the super-resolved image.3,4 These methods were typically applied to life science investigations but are limited to compatible emitters, though the diversity of techniques now offers quite a long list of useful uorophores. In this work, we studied for the rst time a a
Istituto Italiano di Tecnologia, Center for Bio-Molecular Nanotechnologies@UniLe, Via Barsanti – 73010, Arnesano (Lecce), Italy. E-mail: [email protected]; Fax: +39-0832-1816230; Tel: +39-0832-1816214
b
Department of Chemistry and Laboratory for Emerging Materials and Technology, Clemson University, Clemson, South Carolina 29634, USA † These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2014
Results and discussion STED imaging of carbon dots Representative TEM and confocal microscopy images of CDs are shown in Fig. 1. From the TEM size distribution histogram, an average particle size of 5.5 0.7 nm was estimated. Excited at 405 nm, the emission spectrum showed a peak around 490 nm and a red tail up to 600 nm, allowing efficient STED at 592 nm. To demonstrate the potential of super-resolution microscopy for the imaging of CDs, we rst applied STED to CDs dispersed on a coverslip. The comparison of the confocal image with the STED counterpart (Fig. 2) shows a strong improvement of the lateral resolution. This is more evident in the closer view reported in Fig. 2(c). The ability of STED to break the diffraction limited resolution and to improve the quality of CD imaging is also shown by the analysis of the uorescence line proles. Fig. 2(d) and (e) show two typical row and Gaussian tted proles of CDs, characterized by a FWHM of 54 nm (d) or 61 nm (e) for STED nanoscopy. To calculate the mean values of the FWHM in confocal versus STED modes, 50 individual line proles were analyzed. The histogram of the size occurrences in Fig. 3 indicates that the distributions for the confocal and STED modes are well separated. The average FWHM value measured in confocal mode was 192 nm, a spot size close to the theoretical diffraction limit of confocal microscopy, while the mean FWHM measured in STED mode was 71 nm. It is noteworthy that the minimal lateral resolution obtained with the STED was as low as 30 nm, far
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Fig. 1 Representative (a) TEM and (c) confocal microscopy images of CDs. (b) CD size distribution by TEM. (d) Emission spectrum of CDs (lexc: 405 nm).
below the limit of the confocal resolution. This supports the potential of STED nanoscopy for the imaging of uorescent CDs. Likely, a further increase of resolution might be possible using, for example, ultra-bright uorescent probes, such as the recently described cross-linked CDs,10 because of the additivity of their uorescent properties leading to higher emission intensities.
CDs are efficient probes for high resolution STED imaging in xed cells To evaluate the potential of CDs as new probes for high resolution STED imaging in xed cells, CDs were incubated in MCF7 cells. We rst assessed that CDs are not toxic to cells. In particular, MCF7 cells were incubated with increasing concentrations of CDs (up to concentration values as high as 170 nM) and the cellular viability was tested by a cell proliferation assay. As shown in Fig. 4(a), even the highest doses of CDs did not exert any detectable toxic effect, as the cell viability remains around 100% of the control also aer longer incubation times. To verify the cellular internalization of CDs and to probe their intracellular fate, we performed CD imaging in combination with LysoTracker probe, a specic dye for the acidic lysosomal compartments. As shown in Fig. 4(b), CDs are efficiently taken up by cells and mostly localize in lysosome compartments,11 as conrmed by the yellow colocalization signals (bottom right image). The strong colocalization is also conrmed by the white colocalization mask (the white signal represents overlapping regions of green and red signals), and by the scatter plot. The cells were then xed and the nanoscale arrangement of CDs was analyzed by comparing the confocal image (Fig. 5(a)) with its STED counterpart (Fig. 5(b)). The analysis of the two imaging modes demonstrates a clear visual improvement in the STED
8618 | Nanoscale, 2014, 6, 8617–8623
imaging. Two representative examples of line proles of confocal spots are shown in Fig. 5(c) and (d). Interestingly, unlike confocal microscopy showing only one non-resolved spot, STED microscopy reveals the presence of smaller aggregates of CDs. Fitting the proles by Gaussian curves, FWHM values of 140 and 66 nm (Fig. 5(c), peaks (i) and (ii), respectively) and 98, 54 and 30 nm (Fig. 5(d), peaks (i)–(iii), respectively) were extracted. Notably, despite the fact that the resolution may be inherently limited, due to the agglomeration of CDs during their internalization in the endocytic vesicles, it was however possible to detect CDs with a resolution down to 30 nm, as similarly observed in the analysis of the CDs dispersed on glass (Fig. 3). Our experimental results show that the good uorescent signal of our CDs (28% quantum yield) along with their strong resistance to photobleaching makes them valuable probes for high resolution imaging, opening the perspective of nanoscale investigations of cellular structures, through their conjugation with selective antibodies or other specic biomolecules. Thanks to their brightness and biocompatibility, CDs can be thus considered as promising nanomaterials for applications in cellular biology,12,13 such as immunolabeling of cells,14 as an alternative to the traditional uorescent dyes and proteins.
Non-perturbative STED imaging of CDs in living cells CDs are non-toxic, non-blinking and strongly resistant to photobleaching,15–19 all fundamental properties of uorescent probes suitable for live cell imaging. Therefore, we explored the efficiency of STED microscopy for high resolution cellular imaging of CDs in living cells. As shown in Fig. 6(a), CDs appeared very bright and clearly distinguishable both as larger aggregates (brighter green spots) and as few smaller ones (weaker green spots), due to lysosomal accumulation.
This journal is © The Royal Society of Chemistry 2014
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Typical raw images of CDs observed with (a) confocal and (b) STED microscopy. (c) Zoom-in view of the regions indicated in (a) and (b), respectively. (d and e) Graphs showing raw data (light color and symbols) and Gaussian fit (thicker line) of two representative intensity plots of fluorescent spots in confocal (red squares) and STED (green triangles) microscopy.
Fig. 2
Sequential images were captured in STED mode. No prominent bleaching effect or phototoxicity was observed over the entire acquisition. Fig. 6(b) shows two representative peaks with a FWHM of 67 and 76 nm obtained from a uorescent spot in the cytoplasm of the cell. In contrast, the same spot appeared as an unresolved one in confocal mode. This result demonstrates that CDs can be imaged at the nanoscale level in living cells under physiological conditions with a resolution down to 70 nm.
Conclusions
Fig. 3 Statistical analysis of FWHM obtained with Gaussian fit of 50 individual emitting spots. Notice that the mean resolution measured in STED mode is about 70 nm and that the minimal FWHM detectable is 30 nm.
This journal is © The Royal Society of Chemistry 2014
In this work, we reported STED nanoscopy of uorescent CDs with a resolution down to 30 nm. Moreover, we demonstrate that it is possible to apply STED for the imaging of CDs in both xed and living cells, obtaining an improvement of the lateral resolution, >6-fold, compared to the diffraction limited confocal resolution. Taken together, these results show the possible
Nanoscale, 2014, 6, 8617–8623 | 8619
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Fig. 4 (a) Viability of MCF7 cells after the exposure to increasing doses of CDs, evaluated by the WST-8 assay. Viability of NP-treated cells is expressed relative to non-treated control cells (Ctrl), while positive control refers to cells treated with 5% DMSO (P Ctrl). Error bars indicate the standard deviation. (b) Representative confocal images of MCF7 cells incubated for 48 hours with CDs (170 nM). The upper panels display a large field of view with several cells, while the lower panels focus on a single cell (green: fluorescent CDs; red: lysosomes by LysoTracker Red staining, right panels: merged images). Note the high level of CD uptake, and the colocalization (yellow) of the CDs with lysosomes. (c) Colocalization mask (white) showing overlapping regions of green and red signals (left panel) and scatter plot of green intensities versus red intensities, with the pixels representing the degree of colocalization in the middle of the plot (right panel).
application of CDs for high resolution STED imaging, opening a broad variety of applications in life sciences. A rst advantage of using CDs as nanoparticles for super-resolution imaging of dynamic cellular processes is that they offer a broad possibility for surface functionalization.18 Hence, CDs could be applied for the labeling of cellular compartments (membranes and
8620 | Nanoscale, 2014, 6, 8617–8623
organelles) and for the sub-diffractive tracking of the diffusion dynamics of specic intracellular proteins over time. Another important advantage is that, unlike cadmium-based quantum dots,20,21 CDs are highly biocompatible,22 so they could be useful for the detailed visualization of cellular events over long periods of time or in in vivo experiments.
This journal is © The Royal Society of Chemistry 2014
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Fig. 5 Confocal (a) versus STED (b) imaging of CDs in fixed MCF7 cells (CD concentration: 170 nM, 48 h incubation). (c and d) Two representative line profiles extracted from signals of CDs in the cell. Raw data (light color with symbol) and Gaussian fits (thicker line) are shown in confocal (red color and square) and STED (green, triangle) mode. The FWHM values extracted from the Gaussian fit are (c) 140 nm (i), 66 nm (ii) and (d) 98 nm (i), 54 nm (ii) and 30 nm (iii).
Live cell imaging of CDs by STED microscopy. (a) STED imaging of CDs in MCF7 cells incubated with CDs for 48 hours. The white dotted line delineates the nuclear region. (b) Line profile (light green with triangle) and Gaussian fit (green) of STED CD signals indicated with the white line in panel (a). A FWHM resolution of 67 and 76 nm was obtained.
Fig. 6
Materials and methods Carbon dots The carbon nanoparticles were produced by laser ablation of a carbon target,6 with an average diameter of 5 nm. The CDs were
This journal is © The Royal Society of Chemistry 2014
surface-functionalized with diamine-terminated oligomeric (poly-ethylene glycol) H2NCH2(CH2CH2O)nCH2CH2CH2NH2 (average n z 35, PEG1500N). In order to test the STED technique on these NPs, we used two different approaches to prepare our samples. A water suspension of CDs was drop cast on a
Nanoscale, 2014, 6, 8617–8623 | 8621
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coverslip; aer evaporation, the samples were covered by Mowiol (81381 Aldrich) as embedding medium in order to avoid mismatch of refractive index. The second approach was to dilute directly the CDs in the Mowiol and then to drop cast the blend on a coverslip. The two conditions did not interfere or change the conclusions presented in this work.
Published on 03 June 2014. Downloaded by University of Regina on 26/10/2014 20:46:09.
STED and confocal microscopy Fluorescence imaging was performed with a SP8-STED (Leica Microsystems, GmbH, Germany). A continuous doughnut-shaped STED laser emitting at lSTED ¼ 592 nm was coupled to a diode laser at lex ¼ 405 nm for the excitation of the CDs. The laser beams were focused by a 100 oil immersion objective (HCX APO from Leica with a numerical aperture of 1.4) on the sample. With an emission peak centered around 490 nm, the spectral window to observe CDs was 450–550 nm. In order to compare confocal and STED imaging, the acquisitions were made in sequential mode: in the rst channel were applied both the 405 nm laser and the STED laser, while in the second channel was applied only the 405 nm laser for confocal imaging. For both acquisitions a pixel size of 15–20 nm was chosen with a pixel dwell time of 600 ns. A spectrometer (iHR 320, Horiba) was coupled to the microscope in order to measure the emission spectrum using a CCD (Synapse, Horiba). Cell cultures MCF7 cells (human mammary gland adenocarcinoma cell line ATCC HTB-22) were cultivated in DMEM with 50 mM glutamine, supplemented with 10% FBS, 100 U mL1 penicillin and 100 mg mL1 streptomycin. Cells were incubated in a humidied controlled atmosphere with a 95% to 5% ratio of air/CO2, at 37 C. WST-8 assay MCF7 cells were seeded in 96 well microplates at a density of 5000 cells per well at a nal volume of 50 mL and incubated for 24 h in a humidied atmosphere at 37 C and 5% CO2 to obtain a subconuent monolayer (60–70% of conuence). CDs were added to the single well obtaining nal concentrations of 170, 17, 1.7, and 0.17 nM in a nal volume of 100 mL for each well. The metabolic activity of all cultures was determined aer 24, 48 and 72 hours of exposure to CDs, using a standard WST-8 assay (Sigma). Assays were performed following the procedure previously described by Malvindi et al.23 Data were expressed as mean SD. Differences in cell proliferation (WST-8) between cells treated with CDs and the control were considered statistically signicant performing a Student's t-test with a p-value of
Published on 03 June 2014. Downloaded by University of Regina on 26/10/2014 20:46:09.
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Cite this: Nanoscale, 2014, 6, 8617
View Journal | View Issue
Super-resolution fluorescence imaging of biocompatible carbon dots Godefroy Leme´nager,†a Elisa De Luca,†a Ya-Ping Sunb and Pier Paolo Pompa*a
Received 11th April 2014 Accepted 27th May 2014 DOI: 10.1039/c4nr01970a www.rsc.org/nanoscale
Carbon Dots (CDs) are a new promising type of small (5 nm), biocompatible and multicolor luminescent nanoparticle. Here, we demonstrate super-resolution imaging of CDs at the nanoscale through STimulated Emission Depletion (STED) microscopy. In addition, we report the application of STED for detection of CD localization in both fixed and living cells, achieving a spatial resolution down to 30 nm, far below the diffraction limit, showing great promise for high resolution visualization of cellular dynamics.
recently developed nanomaterial, i.e. carbon dots (CDs), quantum-sized nanocrystals composed of carbon.6 CDs were shown to be biocompatible7 and highly photoluminescent, and offer multicolor emission.8,9 However, despite these important advantages for biological applications, no super-resolution imaging with CDs has been reported so far in the literature. We successfully employed STED microscopy to image CDs in cells, demonstrating a >6-fold improvement of the spatial resolution, compared to conventional confocal microscopy.
Introduction Far eld microscopy is one of the main techniques used for sample imaging in several elds, from condensed matter to life science. Because of the diffraction, however, the optical resolution is limited to about Dr z l/(2N.A.) z 200 nm (with l the wavelength of light and N.A. the numerical aperture of the lenses). In recent years, several breakthroughs, usually referred to as super-resolution techniques, appeared in the literature, overcoming the diffraction limit and signicantly improving the optical resolution.1–5 Available for different types of emitters, from organic dyes1 to quantum nanostructures,2 such techniques can be grouped in two major systems, based on uorescent inhibition properties or on stochastic methods.3,4 The rst group, including stimulated emission depletion (STED) microscopy, deactivates the uorophores selectively to enhance the resolution imaging in that area.5 The second group of techniques uses sequential activation and time-resolved localization of uorophores to create the super-resolved image.3,4 These methods were typically applied to life science investigations but are limited to compatible emitters, though the diversity of techniques now offers quite a long list of useful uorophores. In this work, we studied for the rst time a a
Istituto Italiano di Tecnologia, Center for Bio-Molecular Nanotechnologies@UniLe, Via Barsanti – 73010, Arnesano (Lecce), Italy. E-mail: [email protected]; Fax: +39-0832-1816230; Tel: +39-0832-1816214
b
Department of Chemistry and Laboratory for Emerging Materials and Technology, Clemson University, Clemson, South Carolina 29634, USA † These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2014
Results and discussion STED imaging of carbon dots Representative TEM and confocal microscopy images of CDs are shown in Fig. 1. From the TEM size distribution histogram, an average particle size of 5.5 0.7 nm was estimated. Excited at 405 nm, the emission spectrum showed a peak around 490 nm and a red tail up to 600 nm, allowing efficient STED at 592 nm. To demonstrate the potential of super-resolution microscopy for the imaging of CDs, we rst applied STED to CDs dispersed on a coverslip. The comparison of the confocal image with the STED counterpart (Fig. 2) shows a strong improvement of the lateral resolution. This is more evident in the closer view reported in Fig. 2(c). The ability of STED to break the diffraction limited resolution and to improve the quality of CD imaging is also shown by the analysis of the uorescence line proles. Fig. 2(d) and (e) show two typical row and Gaussian tted proles of CDs, characterized by a FWHM of 54 nm (d) or 61 nm (e) for STED nanoscopy. To calculate the mean values of the FWHM in confocal versus STED modes, 50 individual line proles were analyzed. The histogram of the size occurrences in Fig. 3 indicates that the distributions for the confocal and STED modes are well separated. The average FWHM value measured in confocal mode was 192 nm, a spot size close to the theoretical diffraction limit of confocal microscopy, while the mean FWHM measured in STED mode was 71 nm. It is noteworthy that the minimal lateral resolution obtained with the STED was as low as 30 nm, far
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Fig. 1 Representative (a) TEM and (c) confocal microscopy images of CDs. (b) CD size distribution by TEM. (d) Emission spectrum of CDs (lexc: 405 nm).
below the limit of the confocal resolution. This supports the potential of STED nanoscopy for the imaging of uorescent CDs. Likely, a further increase of resolution might be possible using, for example, ultra-bright uorescent probes, such as the recently described cross-linked CDs,10 because of the additivity of their uorescent properties leading to higher emission intensities.
CDs are efficient probes for high resolution STED imaging in xed cells To evaluate the potential of CDs as new probes for high resolution STED imaging in xed cells, CDs were incubated in MCF7 cells. We rst assessed that CDs are not toxic to cells. In particular, MCF7 cells were incubated with increasing concentrations of CDs (up to concentration values as high as 170 nM) and the cellular viability was tested by a cell proliferation assay. As shown in Fig. 4(a), even the highest doses of CDs did not exert any detectable toxic effect, as the cell viability remains around 100% of the control also aer longer incubation times. To verify the cellular internalization of CDs and to probe their intracellular fate, we performed CD imaging in combination with LysoTracker probe, a specic dye for the acidic lysosomal compartments. As shown in Fig. 4(b), CDs are efficiently taken up by cells and mostly localize in lysosome compartments,11 as conrmed by the yellow colocalization signals (bottom right image). The strong colocalization is also conrmed by the white colocalization mask (the white signal represents overlapping regions of green and red signals), and by the scatter plot. The cells were then xed and the nanoscale arrangement of CDs was analyzed by comparing the confocal image (Fig. 5(a)) with its STED counterpart (Fig. 5(b)). The analysis of the two imaging modes demonstrates a clear visual improvement in the STED
8618 | Nanoscale, 2014, 6, 8617–8623
imaging. Two representative examples of line proles of confocal spots are shown in Fig. 5(c) and (d). Interestingly, unlike confocal microscopy showing only one non-resolved spot, STED microscopy reveals the presence of smaller aggregates of CDs. Fitting the proles by Gaussian curves, FWHM values of 140 and 66 nm (Fig. 5(c), peaks (i) and (ii), respectively) and 98, 54 and 30 nm (Fig. 5(d), peaks (i)–(iii), respectively) were extracted. Notably, despite the fact that the resolution may be inherently limited, due to the agglomeration of CDs during their internalization in the endocytic vesicles, it was however possible to detect CDs with a resolution down to 30 nm, as similarly observed in the analysis of the CDs dispersed on glass (Fig. 3). Our experimental results show that the good uorescent signal of our CDs (28% quantum yield) along with their strong resistance to photobleaching makes them valuable probes for high resolution imaging, opening the perspective of nanoscale investigations of cellular structures, through their conjugation with selective antibodies or other specic biomolecules. Thanks to their brightness and biocompatibility, CDs can be thus considered as promising nanomaterials for applications in cellular biology,12,13 such as immunolabeling of cells,14 as an alternative to the traditional uorescent dyes and proteins.
Non-perturbative STED imaging of CDs in living cells CDs are non-toxic, non-blinking and strongly resistant to photobleaching,15–19 all fundamental properties of uorescent probes suitable for live cell imaging. Therefore, we explored the efficiency of STED microscopy for high resolution cellular imaging of CDs in living cells. As shown in Fig. 6(a), CDs appeared very bright and clearly distinguishable both as larger aggregates (brighter green spots) and as few smaller ones (weaker green spots), due to lysosomal accumulation.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 03 June 2014. Downloaded by University of Regina on 26/10/2014 20:46:09.
Communication
Nanoscale
Typical raw images of CDs observed with (a) confocal and (b) STED microscopy. (c) Zoom-in view of the regions indicated in (a) and (b), respectively. (d and e) Graphs showing raw data (light color and symbols) and Gaussian fit (thicker line) of two representative intensity plots of fluorescent spots in confocal (red squares) and STED (green triangles) microscopy.
Fig. 2
Sequential images were captured in STED mode. No prominent bleaching effect or phototoxicity was observed over the entire acquisition. Fig. 6(b) shows two representative peaks with a FWHM of 67 and 76 nm obtained from a uorescent spot in the cytoplasm of the cell. In contrast, the same spot appeared as an unresolved one in confocal mode. This result demonstrates that CDs can be imaged at the nanoscale level in living cells under physiological conditions with a resolution down to 70 nm.
Conclusions
Fig. 3 Statistical analysis of FWHM obtained with Gaussian fit of 50 individual emitting spots. Notice that the mean resolution measured in STED mode is about 70 nm and that the minimal FWHM detectable is 30 nm.
This journal is © The Royal Society of Chemistry 2014
In this work, we reported STED nanoscopy of uorescent CDs with a resolution down to 30 nm. Moreover, we demonstrate that it is possible to apply STED for the imaging of CDs in both xed and living cells, obtaining an improvement of the lateral resolution, >6-fold, compared to the diffraction limited confocal resolution. Taken together, these results show the possible
Nanoscale, 2014, 6, 8617–8623 | 8619
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Published on 03 June 2014. Downloaded by University of Regina on 26/10/2014 20:46:09.
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Fig. 4 (a) Viability of MCF7 cells after the exposure to increasing doses of CDs, evaluated by the WST-8 assay. Viability of NP-treated cells is expressed relative to non-treated control cells (Ctrl), while positive control refers to cells treated with 5% DMSO (P Ctrl). Error bars indicate the standard deviation. (b) Representative confocal images of MCF7 cells incubated for 48 hours with CDs (170 nM). The upper panels display a large field of view with several cells, while the lower panels focus on a single cell (green: fluorescent CDs; red: lysosomes by LysoTracker Red staining, right panels: merged images). Note the high level of CD uptake, and the colocalization (yellow) of the CDs with lysosomes. (c) Colocalization mask (white) showing overlapping regions of green and red signals (left panel) and scatter plot of green intensities versus red intensities, with the pixels representing the degree of colocalization in the middle of the plot (right panel).
application of CDs for high resolution STED imaging, opening a broad variety of applications in life sciences. A rst advantage of using CDs as nanoparticles for super-resolution imaging of dynamic cellular processes is that they offer a broad possibility for surface functionalization.18 Hence, CDs could be applied for the labeling of cellular compartments (membranes and
8620 | Nanoscale, 2014, 6, 8617–8623
organelles) and for the sub-diffractive tracking of the diffusion dynamics of specic intracellular proteins over time. Another important advantage is that, unlike cadmium-based quantum dots,20,21 CDs are highly biocompatible,22 so they could be useful for the detailed visualization of cellular events over long periods of time or in in vivo experiments.
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 03 June 2014. Downloaded by University of Regina on 26/10/2014 20:46:09.
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Nanoscale
Fig. 5 Confocal (a) versus STED (b) imaging of CDs in fixed MCF7 cells (CD concentration: 170 nM, 48 h incubation). (c and d) Two representative line profiles extracted from signals of CDs in the cell. Raw data (light color with symbol) and Gaussian fits (thicker line) are shown in confocal (red color and square) and STED (green, triangle) mode. The FWHM values extracted from the Gaussian fit are (c) 140 nm (i), 66 nm (ii) and (d) 98 nm (i), 54 nm (ii) and 30 nm (iii).
Live cell imaging of CDs by STED microscopy. (a) STED imaging of CDs in MCF7 cells incubated with CDs for 48 hours. The white dotted line delineates the nuclear region. (b) Line profile (light green with triangle) and Gaussian fit (green) of STED CD signals indicated with the white line in panel (a). A FWHM resolution of 67 and 76 nm was obtained.
Fig. 6
Materials and methods Carbon dots The carbon nanoparticles were produced by laser ablation of a carbon target,6 with an average diameter of 5 nm. The CDs were
This journal is © The Royal Society of Chemistry 2014
surface-functionalized with diamine-terminated oligomeric (poly-ethylene glycol) H2NCH2(CH2CH2O)nCH2CH2CH2NH2 (average n z 35, PEG1500N). In order to test the STED technique on these NPs, we used two different approaches to prepare our samples. A water suspension of CDs was drop cast on a
Nanoscale, 2014, 6, 8617–8623 | 8621
View Article Online
Nanoscale
coverslip; aer evaporation, the samples were covered by Mowiol (81381 Aldrich) as embedding medium in order to avoid mismatch of refractive index. The second approach was to dilute directly the CDs in the Mowiol and then to drop cast the blend on a coverslip. The two conditions did not interfere or change the conclusions presented in this work.
Published on 03 June 2014. Downloaded by University of Regina on 26/10/2014 20:46:09.
STED and confocal microscopy Fluorescence imaging was performed with a SP8-STED (Leica Microsystems, GmbH, Germany). A continuous doughnut-shaped STED laser emitting at lSTED ¼ 592 nm was coupled to a diode laser at lex ¼ 405 nm for the excitation of the CDs. The laser beams were focused by a 100 oil immersion objective (HCX APO from Leica with a numerical aperture of 1.4) on the sample. With an emission peak centered around 490 nm, the spectral window to observe CDs was 450–550 nm. In order to compare confocal and STED imaging, the acquisitions were made in sequential mode: in the rst channel were applied both the 405 nm laser and the STED laser, while in the second channel was applied only the 405 nm laser for confocal imaging. For both acquisitions a pixel size of 15–20 nm was chosen with a pixel dwell time of 600 ns. A spectrometer (iHR 320, Horiba) was coupled to the microscope in order to measure the emission spectrum using a CCD (Synapse, Horiba). Cell cultures MCF7 cells (human mammary gland adenocarcinoma cell line ATCC HTB-22) were cultivated in DMEM with 50 mM glutamine, supplemented with 10% FBS, 100 U mL1 penicillin and 100 mg mL1 streptomycin. Cells were incubated in a humidied controlled atmosphere with a 95% to 5% ratio of air/CO2, at 37 C. WST-8 assay MCF7 cells were seeded in 96 well microplates at a density of 5000 cells per well at a nal volume of 50 mL and incubated for 24 h in a humidied atmosphere at 37 C and 5% CO2 to obtain a subconuent monolayer (60–70% of conuence). CDs were added to the single well obtaining nal concentrations of 170, 17, 1.7, and 0.17 nM in a nal volume of 100 mL for each well. The metabolic activity of all cultures was determined aer 24, 48 and 72 hours of exposure to CDs, using a standard WST-8 assay (Sigma). Assays were performed following the procedure previously described by Malvindi et al.23 Data were expressed as mean SD. Differences in cell proliferation (WST-8) between cells treated with CDs and the control were considered statistically signicant performing a Student's t-test with a p-value of
