Towards using Fluorescent Nanodiamonds to Study Chronological

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Towards using Fluorescent Nanodiamonds to Study Chronological Ageing in Saccharomyces Cerevisiae Kiran van der Laan, Julie Naulleau, Viraj G Damle, Alina Sigaeva, Nicolas Jamot, Felipe P Perona Martinez, Mayeul Chipaux, and Romana Schirhagl Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03431 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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Analytical Chemistry

Towards using Fluorescent Nanodiamonds to Study Chronological Ageing in Saccharomyces Cerevisiae Kiran J. van der Laan1, Julie Naulleau1, Viraj G. Damle1, Alina Sigaeva1, Nicolas Jamot1, Felipe P. Perona-Martinez1, Mayeul Chipaux1, Romana Schirhagl1* 1University

of Groningen, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AW Groningen

*Correspondence should be addressed to [email protected].

Abstract

One of the theories aiming to explain cellular ageing is the free radical theory of ageing, which describes the possible role of increased production and accumulation of free radicals. Fluorescent nanodiamonds (FNDs) are proposed to provide a tool to detect these radicals, as they function as magnetic sensors that change their optical properties depending on their magnetic surrounding. Therefore they could enable the study for ageing at a molecular level and unravel the exact role of free radicals in this process. In this study, important steps towards this goal are made. FNDs are introduced in chronologically ageing yeast cells. Furthermore, the behavior of FNDs in these ageing

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cells is studied, to demonstrate the potency of using FNDs in the search for causes of cellular ageing.

Keywords: nanodiamonds, free radicals, diamond magnetometry, ageing, yeast cells.

Understanding the mechanisms of cellular ageing is one of the emerging challenges in the current ageing population. The causes of ageing at a molecular level are still largely unknown, although some involved mechanisms have been appointed. Free radical formation and accumulation are believed to be one of the causes for cellular ageing, but their exact role is strongly debated in the past decades.1,2 We are aiming to use a new method called diamond magnetometry to monitor free radical activity in the future, by using fluorescent nanodiamonds (FNDs). FNDs can function as quantum sensors that change their optical properties depending on their magnetic surrounding, with single electron-spin sensitivity.3 Since unpaired electrons in free radicals are expected to influence coherence times, these FNDs could potentially enable the study on the role of free radicals in cellular ageing. The FNDs used in diamond magnetometry harbor a defect in the diamond structure, in which two carbon atoms in the lattice are substituted by a nitrogen atom (N) and an adjacent vacancy (V). Magnetic information from the environment can be read out optically because of these NV centers, causing the fluorescence signal of the nanodiamonds to change in the presence of electron spins. The stability of the NV centers and their fluorescence

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together with the excellent biocompatibility of FNDs,


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provide great promises to use its magneto-optical behavior as sensors in biomedical model systems.7–11 Until now FNDs have been used for several applications, such as labeling,12 monitoring temperature alterations

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and studying drug delivery.11,15–17 To provide

important prerequisites to study ageing, in this work the FNDs are introduced into yeast cells. Yeast is one of the most successful model organisms to study ageing for several reasons, including the conservation of ageing process among different organisms and its suitability for genetic manipulation.18 For proper functioning of the FNDs, the distance between the diamond probe and the target molecules needs to be as small as possible.19 Therefore it is necessary to introduce the nanodiamonds into the cells using a previously shown effective method.20 In this study, we have analyzed the internalization and behavior of nanodiamonds in ageing yeast cells. Furthermore, we have determined where exactly diamonds end up after internalization. All together, these results demonstrate the potency of using FNDs in yeast cells to study ageing at a molecular level.

Results An effective method to obtain fluorescent nanodiamond (FND) internalization in young yeast cells was earlier shown to be both biocompatible and highly effective.20 Here the cells were monitored after performing the uptake protocol in aged cells, in order to work towards the goal of using FNDs in the search for causes of cellular ageing.


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FND uptake in stationary phase yeast cells In the earlier mentioned study, the effectiveness of FND uptake was shown in yeast cells in their active growth phase (mid log phase). In order to study chronological ageing however, cells were grown until the stationary growth phase of yeast. Cells were restricted to chronological ageing by transferring them to water after they had reached the stationary growth phase. In this research, the effectiveness of the uptake method in stationary phase cells was checked, as well as the viability of aged cells after uptake of FNDs. First, the effectiveness of the uptake method in stationary phase cells was checked, as well as the viability of aged cells after uptake of FNDs. The number of internalized FNDs per cell was increased after performing the chemical transformation protocol, although the increase was not in all cases significant (figure 1a-b). Cells were additionally categorized based on the number of internalized objects or particles and the distribution of the percentage of cells per category is shown in figure 1c-d. An object is defined as connected FND positive pixels, which means it can also be an aggregate. Out of the total intensity of the objects, the number of single particles is calculated using the intensity of a single bright pixel. Showing both numbers gives an impression on the state of aggregation of particles. Compared to the control sample, there was a greater percentage of cells with a high amount of both objects and particles. The low number of particles and objects that are found in the control sample, could be attributed to large aggregates on the cell surface which give false positives. For the transformation protocol we find particles that are unambiguously inside the cells, stressing the need for


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the transformation protocol. Moreover, the viability of the cells was not changed as a result of performing the chemical transformation and adding the FNDs (figure 1e). The cell samples subjected to FNDs and the uptake protocol did not show altered viability compared to the untreated control sample.

Figure 1. Efficiency and viability of FND uptake in stationary phase yeast cells. Cells were grown until stationary phase of cell growth, before performing the chemical transformation and adding the FNDs. The particle uptake was quantitatively analyzed directly after the transformation protocol.


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(a-d) Quantitative analysis of FND internalization. Objects are composed of adjacent FND signal positive pixels and particles reflect the actual number of FNDs, derived from the total intensity of the objects. For each sample, 124-125 cells were analyzed (black n=125, green n=125, blue n=124). a and b show the average number of FNDs per cell. Error bars represent standard errors of the mean. Statistical differences were tested by comparing to the control (untreated cells with FNDs) with an unpaired, two-tailed ttest with Welch’s correction. c and d display a histogram of cells containing a range of FNDs. (e) Survival of stationary phase cells. Colony forming units (CFUs) are shown as a measure for the viability of stationary phase yeast cells after the chemical transformation procedure. Bars represent averages out of three independent experiments (n=3), each performed in triplicate. The grey bar indicates the area that differs 20% with the control sample, in which the viability is considered as normal. Significance is tested compared to the control situation by performing an unpaired, two-tailed t-test with Welch’s correction; no statistical differences were found. Error bars represent standard errors of the mean.

FND behavior during chronological ageing In order to investigate the effect of FND internalization on the ageing process, a chronological life span assay was performed. Cells were restricted to age chronologically by keeping them in a non-dividing state. After the addition of FNDs at day 1, the cells were kept in water and the survival of the cells was measured by assessing the proliferative ability every two days. This resulted in the chronological


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lifespan curve (figure 2a). The decline in viability we see in all the samples is the chronological ageing effect. There are several theories about the reasons and mechanisms involved in this decline, one of them is the above mentioned free radical theory1,2. In the first week, the functional decline of the cells seemed to happen at a similar rate, despite small differences. At later ageing time points though, the differences in the percentage of remaining viable cells became greater but not significant. Interestingly, from day 2 the viability decreased in parallel with the control sample (figure 2b). Similar decline rates were detected, but starting from a different percentage of cells at day 2 and thus ending up at lower number of cells at day 6-day10.

Figure 2. Chronological life span. The number of viable cells was analyzed in the days after chemical transformation was performed to achieve uptake. Cells were kept in water, to keep them in a non-dividing state. (a) Chronological survival of cells. After FND uptake, the survival was determined by testing the proliferative capacity every 2 days. Per condition, the value of a time point was compared to the number of viable cells with that condition at day 1, which was set as 100%. (b) Decline rate per measured


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time period. This part shows the absolute numbers of viable cells that are lost in between the time points that are measured. Given values of both (a) and (b) are averages out of three biologically independent experiments (n=3), each performed in three technical replicates. Error bars represent standard errors of the mean. Significance is tested compared to the control sample (untreated cells without FNDs), with an unpaired, two-tailed t-test with Welch’s correction (no significant differences).

To record quantum measurements with the purpose of measuring free radical activity and its role in ageing, measurements will have to be taken at different ageing time points. Therefore, the persistence of FND internalization in aged cells was examined. In figure 3, the number of internalized FNDs in the cells is depicted as a function of time. Although there are still cells with internalized FNDs in the days after the initial uptake attempt at day 1, the number of FNDs inside cells got lower over time. This could be explained by either excretion of FNDs in some way, or by cell death of the cells with internalized FNDs. The latter explanation did not seem to be the case, since an additional experiment showed that the addition of diamonds did not cause decreased viability at the time-points were the number of internalized diamonds was diminished (figure S2).


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Figure 3. FND presence over time. The number of internalized FNDs was analyzed in the days after chemical transformation was performed to achieve uptake. After one day, the biggest drop in number of internalized objects (a) and particles (b) was observed. This effect was most obvious in the sample were the cells were additionally treated with a Triton wash, directly after the chemical transformation. Given values are relative numbers that are normalized against the control by subtraction of the values found in the control sample, in which diamond uptake was not stimulated. For all samples, per condition and time point, approximately 100 cells were analyzed: (green n=125 at day 1, n=103 at day 2, n=111 at day 3, n=99 at day 4; blue n=124 at day 1, n=104 at day 2, n=107 at day 3, n=82). Error bars represent standard errors of the mean.

Visual inspection of scanning electron microscopy (SEM) images revealed the subcellular localization of the nanodiamonds. Cells were embedded directly after performing the uptake protocol and adding the nanodiamonds, therefore images represent the location of FNDs shortly after uptake. Qualitative analysis of the images revealed that the nanodiamonds were predominantly located around the nucleus


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(figure 5a) and other membrane-enclosed, spherical structures (figure S1). Interestingly, budding yeast cells with internalized FNDs were captured (figure5a, figure S1), once more indicating that cells remained functional after uptake of FNDs. Using a focused ion beam SEM, a detailed 3D visualization of a cell with FNDs was obtained (figure 4), with the suspected nanodiamonds again localized around these spherical structures and the nucleus.

Figure 4. FIB-SEM 3D visualization of yeast cells with internalized FNDs. A 3D visualization was obtained using focused ion beam SEM. The scale bar represents 1.00 µm and applies to all three subfigures (a, b and c). The stack of 2D SEM images (a) was processed to a 3D reconstruction of a complete yeast cell (b and c). The bright spots that are proposed to be FNDs, are again found close to membrane-enclosed, spherical structures (b) and close to the nucleus (c). Based on their location, the membraneenclosed structures are in this case most likely representing mitochondria. The largest rectangle in (a) shows a suspected mitochondrion with three nanodiamonds attached to it. (purple = nucleus, red = membrane-enclosed structures, green = endoplasmatic reticulum, light blue and framed in yellow = fluorescent nanodiamonds)


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To confirm that the structures observed in SEM images are indeed nanodiamonds and not holes through the sections as an artifact resulting from the sample preparation, or other cellular structures - atomic force microscopy (AFM) analysis was conducted. As expected, the structures that were identified as diamonds were very stiff compared to the rest of the cell, three times as stiff, and did not deform at al. The size of the suspected diamonds was also in reasonable agreement with what was expected. Additionally, the characteristic sharp edge structure of diamonds was found. By measuring the height of the diamond particles, they could be differentiated from through holes in the section which would also have very high stiffness.

Figure 5. Subcellular localization of FNDs. (a) SEM visualization of yeast cells with internalized FNDs shows the location of FNDs within the cells. A representative image is shown wherein FNDs are located around in close proximity of the nucleus. Furthermore, FNDs were found in budding yeast cells. The scale bar represents 1.00 µm and applies to all three subfigures (a, b and c). (b) Confirmation of the localization in AFM stiffness mode. In stiffness mode, diamonds show a significantly higher stiffness (3: 37 GPa) than the surrounding cell material (2: 17 GPa). The outside of the cell (1) has


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an even lower stiffness of 13 GPa. The circled line shows a vesicle structure, visible in the height image that is constructed by taking the height at every point in AFM. (c) Confirmation of localization in AFM deformation mode. This shows the same AFM image, but in deformation mode. Here, the sharp edge of the diamond is clearly visible.

FND uptake in aged yeast cells Since the chronological ageing was shown to be slightly affected (figure 2) and the internalization of FNDs was shown to be transient (figure 3), it was attempted to obtain FND internalization in aged cells. Cells were chronologically aged until an age of 4 or 8 days old, before adding the nanodiamonds. The FND uptake was attempted by performing the established chemical transformation protocol. A significant number of internalized FNDs could be obtained, both in 4-day and 8-day old cells (figure 6a-b). After FND uptake, the viability of these cells was determined as well. There was no difference in viability of the aged cells after the uptake of FNDs, compared to the control aged cells (figure 6c-d). Additionally, an optically detected magnetic resonance (ODMR) spectrum was recorded at these ageing time points as well (figure 6e). An ODMR signal typical for NV center containing nanodiamonds could be recorded at all tested ageing time points using the homebuilt magnetometer.


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Figure 6. Internalization of FNDs in aged cells. In order to have FNDs in older cells, 4day and 8-day old cells were subjected to FNDs and the chemical transformation. (a-b) The uptake of objects and particles after performing the uptake protocol in 4-day old or 8-day old cells. The number of internalized diamonds was significantly higher after


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performing the chemical transformation, compared to the control sample in which diamonds were added without further treatment of the cells. Two times approximately 100 cells were analyzed (day 4: black n=127, green n=261, blue n=268, day 8: black n=123, green n=219, blue n=182). Given values represent the average number of FNDs per cell with the standard errors of the mean. Statistical differences were tested compared to the control with an unpaired, two-tailed t-test with Welch’s correction (*** p≤0.001, **** p≤0.0001). (c-d) Survival of cells to the chemical transformation procedure at the age of 4 days old and 8 days old. Bars represent averages of replicates out of three independent experiments (n=3). The grey bar indicates the area that differs 20% with the control sample (cells without FNDs and chemical transformation), in which the viability is considered as normal. Significance is tested compared to the control situation by performing an unpaired, two-tailed t-test with Welch’s correction; no statistical differences were found. Error bars represent standard deviations. (e) ODMR spectra were taken from diamonds in both 1-day, 4-day and 8-day old cells. All samples showed the typical ODMR signal, one representative spectrum is shown here.

Discussion Yeast as a model for ageing Taken together, we have shown the effective uptake of nanodiamonds in both stationary phase and aged yeast cells using the chemical transformation protocol. This


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is a necessity for using FNDs to take intracellular quantum measurements in ageing cells in the future, with the goal of studying the process of ageing. It should be noted that the uptake observed into yeast cells is lower than what has been observed for some mammalian cells. The reason is that these mammalian cells naturally tend to ingest particles, while for yeast cells this is an entirely artificial process. Additionally, the disappearance of FNDs after their cellular uptake has not (yet) been observed in mammalian cells, although it might also happen in cells that have not been studied for longer time periods so far. These fundamental differences between cell types underline the necessity to study these cells in detail instead of assuming similar behavior from what is known in the literature. For replicative ageing in yeast cells, it is known that the cell wall thickness increases over time because a chitin ring that is involved in the budding process remains in the mother cell wall.21 This means the cell wall thickness is increased in the stationary phase cells and will also increase during replicative ageing. In chronological ageing however, cells are not dividing and thus the cell wall is not getting thicker by chitin remainders. This could explain why we observed that the uptake was even more efficient in aged cells (both 4 and 8-day old cells), as a weaker cell wall is likely to be more easily disrupted. Although the cell wall is an obstacle that had to be overcome by performing a chemical transformation, there are numerous advantages of using yeast as a model for ageing research. One of them is the conservation of cellular ageing pathways, that are retained from the unicellular yeast cells to complex vertebrate organisms. Moreover,


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single celled organisms like yeast have pro- and anti-apoptotic protein pathways that might be useful in multicellular organisms e.g. to block tumor growth or to promote survival in degenerative diseases.22,23 Next to this, the feasibility of handling and culturing the cells is convenient especially for use in ageing experiments. The lifespan of yeast is about one month and cells are easily separated into young and old cells based on cell size.21 The genetic modification possibilities are also favorable, as they will allow to study the effect of specific mutations on the levels of free radicals. Using the nanodiamonds in genetically modified yeast strains, provides the chance to monitor the role of free radicals in relation to specific mutations.24 The effects on cellular characteristics should still be extensively studied, to not misinterpret any FND-related effects for age-related effects. For example, whether or not there is an oxidative stress response to the FNDs or internalization process should be investigated. Transmission of FNDs during yeast cell budding In this study, cells were aged chronologically to mimic ageing of cells in a nondividing state such as neurons.24 However, the yeast cells are also able to age by replication. Replicative ageing is defined by a fixed number of cell divisions that can be performed, before undergoing cell death. Introducing the nanodiamonds in replicating cells would similarly answer interesting questions, for example the transmission of nanodiamonds during division and how the nanodiamonds would be (re)distributed over the mother and daughter cells. The SEM visualizations in this study, rise the suggestion that nanodiamonds are not passed on to the next generation of daughter cells while budding. There was no


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transfer of FNDs from mother to daughter cells observed. The cells that were captured while budding, do not show nanodiamonds in the smaller daughter cells. A similar observation was made by Sen et al., who studied the transmission of gold nanoparticles in budding yeast cells. They showed transmission electron microscopy (TEM) images indicating that accumulated gold nanoparticles in the mother cell are not transmitted to the new daughter cell. This indicates that the accumulation is not transmittable and that the nanoparticles are not redistributed over the cells.25 The behavior of nanodiamonds during cell division was also studied in other cell types. Liu et al. used FNDs to specifically label and track cell division in cancer and stem cells. Nanodiamond particles were found to be equally separated onto two daughter cells of cell division.26 The differences can possibly be explained by the different modes of action between the mammalian cell division and the yeast cell budding process. The asymmetric redistribution of nanodiamonds during yeast cell budding could be interesting for a method to distinguish mother and daughter cells, based on a cell’s diamond fluorescence signal. Subcellular localization of FNDs Visual inspection of electron microscopy images revealed the accumulation of FNDs in the cytoplasm, but specifically closely located around the nucleus, the mitochondria or other membrane-enclosed structures, also called microbodies. Microbodies are organelles containing enzymes and other proteins, surrounded by a single phospholipid bilayer membrane. Examples are peroxisomes, involved in enzymatic reactions with the creation of hydrogen peroxide as a byproduct.27 The presence of


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intact peroxisomes is as well important in chronological ageing, since cells with mutated peroxisome matrix proteins show a reduced chronological lifespan.28 The subcellular localization of FNDs was as well studied in other cell types. Correspondingly, FNDs were found to accumulate in the cytoplasm in human kidney cells,29 human lung epithelial cells and human fibroblasts.30 However, they did not report any specific organization of the accumulated FNDs in the cytoplasm.

Conclusion To conclude, these results demonstrate the excellent opportunities for using nanodiamonds in the research of cellular ageing in yeast cells. Chemical transformation was shown to be effective in both stationary phase and aged yeast cells, paving the way for future quantum measurements from inside the cells during ageing and thus to study ageing at a molecular level. Additionally, diamonds were found to be close to membrane-enclosed structures after the uptake of cells. Although the chemical transformation proved to be effective for obtaining uptake of FNDs in older cells, the uptake was less efficient compared to uptake in mid log phase cells in terms of the number of internalized FNDs. However, a low number of internalized FNDs is not necessarily a problem. For quantum sensing it would be preferred to measure the signal of one diamond, to be able to properly interpret the signal. For other purposes, like labeling, it would be desirable to have a higher number of diamonds inside the cells.


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Methods Yeast strain and cell preparation A BY4741 S. cerevisiae strain expressing Hxt6-GFP was used in this study, from the yeast GFP Clone Collection from ThermoScientific.31 The fusion of a glucose transporter in the cellular membrane (HXT6) with green fluorescent protein (GFP) allows visualization of the cellular membrane. Yeast cells were cultured in synthetic dextrose (SD) complete medium (2% glucose, 6.9g/L yeast nitrogen base without amino acids, 0.79g/L dropout complete mix) and plated on yeast peptone dextrose (YPD) plates (1% yeast extract, 2% peptone, 2% D-glucose, 2% agar). Nanodiamonds Fluorescent nanodiamonds with an average hydrodynamic diameter of 70 nm were used in this study (FND70 in aqueous solution, Adamas Nano). They are produced by the manufacturer by grinding HPHT diamonds followed by irradiation to increase the number of NV centers. As a result, each diamond contains about 300 centers. Since they are acid treated by the manufacturer, their surface chemistry is oxygen-terminated. The average size of the FNDs in the yeast medium was 136.03 nm ± 8.27 nm (average ± standard deviation, measured in triplicates). Internalization of FNDs was achieved by performing chemical permeabilization of the cell wall using a transformation mix with 66.6% (w/v) PEG4000 and 1M lithium acetate (protocol as described by Hemelaar et al. 201720). Uptake analysis


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Confocal laser scanning microscopy was used to monitor FNDs (ex/em 561nm/650nm) and GFP (ex/em 488nm/525nm), in order to analyze nanodiamond uptake. Directly after finishing the transformation protocol (figure 1a-d and 6a-b), cells were fixed in 3.7% paraformaldehyde for 15 minutes prior to imaging. Confocal images were taken at a Zeiss LSM 780 (Zeiss, Germany) and analyzed using Fiji software to quantify FND uptake (https://fiji.sc/). The home written Fiji script used for FND uptake analysis (as described in detail by Hemelaar et al. 2017 20), firstly determines the cell’s region in all three dimensions. Secondly, a volume is created holding only the cell of interest and therefore excluding the cell membrane from analysis. Finally, the actual particle counting is done by assuming pixels to be part of a nanodiamond particle when its intensity is greater than a preset threshold. This results in a number of objects and a number of particles. One object is composed of adjacent FND positive pixels and can actually be formed by a multiple nanodiamonds. Out of this, the number of particles is calculated from the total intensity of the objects to get a value for the actual number of single nanodiamonds. To account for a change in GFP signal in the membrane over time, values in figure 3 were normalized against the control. There was no correlation between the size of the cells, as estimated by the script based on the GFP signal, and the number of diamonds (data not shown). Chronological ageing assay by water-transfer Cells were restricted to ageing chronologically by replacing the medium with water, as described by Cohen et al. (2016).32 An overnight culture was diluted 1:200 in 100 mL SD complete medium and cells were grown at 30°C on a shaking platform (200 rpm).


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Water-transfer was done at the end of logarithmic growth (0.6-1.0 A600), usually after 1220 hours of growth. Cellular concentration at the start of chronological ageing assays was determined by measuring the optical density (OD) at 600 nm and cells in stationary phase were diluted to a final concentration of 1*108 cells/mL for 20 mL total volume. Cells were spun down by centrifugation (2400xG, 6 and 12 minutes), washed two times and resuspended in 20 mL sterile H2O. The water cultures were kept at 30°C with shaking. Every other day, dilutions of the cell samples were plated onto YPD plates (in triplicates) and incubated for 2 days at 30°C prior to counting the colony-forming units (CFUs) to get an estimation of the viability in the sample. Cells from the water cultures were washed every 2-4 days, to remove nutrients released from dead cells.24 For the chronological lifespan, 1mL antibiotic mix of 0.1%(w/v) penicillin and 0.05%(w/v) gentamicin was added to the 20 mL cell samples in order to prevent contamination with bacteria during the time cells were chronologically aged in water. Viability Yeast viability was quantified based on its proliferative ability. Conventional CFU counting was performed to assess the ability of a cell to proliferate and form a colony.33 Dilution series of cell samples were prepared, to avoid overcrowding, on yeast extract peptone dextrose (YPD) plates. After applying the cell dilutions on technical triplicates, the plates were incubated at 30°C for 2 days prior to CFU counting. Subcellular localization To track down the preferred subcellular localization of nanodiamonds in yeast cells, cells were imaged using scanning electron microscopy (SEM). Fixation of yeast cells was


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done in 1% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylic acid and embedding was further performed by the protocol described by Griffith et al. (2008).34 Areas containing the cell pellet appeared dark in the Epon and this darker area was sawn from the Epon block. Subsequently, 50 nm sections were cut with an ultramicrotome (Leica EM UC7) using a diamond knife and put on a silicon waver piece for imaging. Samples were imaged using Field Emission (FE-)SEM (figure 5, Hitachi SU5000) at 2.0kEV at 4.7 mm working distance and in secondary electron detection mode. 3D reconstruction was done using Focused Ion Beam (FIB-)SEM (figure 4, FEI Helios NanoLab 600i) with a gallium ion beam and was processed using the Fijij Volume Viewer plugin.35 To confirm the results of SEM and FIB-SEM, nanomechanical measurements were conducted using Atomic Force Microscope (Bruker Catalyst) to measure z-height map, specimen deformation and stiffness modulus of the cavities found in the cell samples. For these measurements, TESPA-V2 tips of spring constant (~42 N/m) were used with a peak force set-point and amplitude of 200 nN and 20 nm respectively. Furthermore, tips were calibrated using plain glass slide before every experiment. To determine section thickness to compare it against the depth of cavities in the sample, sections were imaged using AFM (Bruker Dimension 4) with DNP-10 non-conductive Silicon Nitride tip (k: 0.06 N/m). Section thickness was then calculated by image processing using Gwyddion and Nanoscope Analysis. Data analysis


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All data were analyzed using GraphPad Prism 6; both visualizations and statistical tests were performed using this software. Statistical tests were performed at a significance level of 0.05 (unless stated otherwise) and by comparing to the control. The control samples that are depicted in black throughout the whole paper, either represent cells that are not treated with diamonds and chemical transformation (in case of survival measurements; figure 1e, figure 2 and figure 6c-d) or cells that are only subjected to diamonds, without stimulating uptake (figure 1a-d, figure 6a-b).

Supporting Information Available Figure S1. SEM visualization of subcellular localization of FNDs. Figure S2. Viability of cells after treatment with chemical transformation.

Author information Corresponding Author Correspondence should be addressed to [email protected]. Author contributions K. van der Laan conceived and designed the experiments. K. van der Laan, J. Naulleau and N. Jamot performed the experiments. R. Schirhagl and V. Damle performed Scanning Electron Microscopy analysis. V. Damle performed AFM scans and their analysis. A. Sigaeva analyzed the FIB-SEM results and F. Perona-Matinez designed


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the FIJI particle uptake script. R. Schirhagl lead the research group. All authors reviewed the manuscript.

Acknowledgements This work is supported by a FOM projectruimte grant FOM-G-36. Part of the work was performed at UMCG Imaging and Microscopy Center (UMIC), which is sponsored by NWO grant 175-010-2009-023 (Zeiss LSM 780). RS is thankful for support via the ERC starting grant ERC-2016-StG – 714289. We would like to thank Annegreet Bittermann and ETH Zurich’s SCOPEM facility for their support with electron microscopy and FIB-SEM. Furthermore we would like to thank Michael Chang and Daniele Novarina for fruitful discussions on yeast biology and Fulvio Reggiori for taking a look at our EM images with experienced eyes.


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