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. 2023 Feb 3;15(2):mfad006. doi: 10.1093/mtomcs/mfad006

The use of synchrotron X-ray fluorescent imaging to study distribution and content of elements in chemically fixed single cells: a case study using mouse pancreatic beta-cells

Kira G Slepchenko 1,✉, Si Chen 2, Kathryn L Corbin 3, Robert A Colvin 4, Craig S Nunemaker 5
PMCID: PMC9933206  PMID: 36737500

Abstract

Synchrotron X-ray fluorescence microscopy (SXRF) presents a valuable opportunity to study the metallome of single cells because it simultaneously provides high-resolution subcellular distribution and quantitative cellular content of multiple elements. Different sample preparation techniques have been used to preserve cells for observations with SXRF, with a goal to maintain fidelity of the cellular metallome. In this case study, mouse pancreatic beta-cells have been preserved with optimized chemical fixation. We show that cell-to-cell variability is normal in the metallome of beta-cells due to heterogeneity and should be considered when interpreting SXRF data. In addition, we determined the impact of several immunofluorescence (IF) protocols on metal distribution and quantification in chemically fixed beta-cells and found that the metallome of beta-cells was not well preserved for quantitative analysis. However, zinc and iron qualitative analysis could be performed after IF with certain limitations. To help minimize metal loss using samples that require IF, we describe a novel IF protocol that can be used with chemically fixed cells after the completion of SXRF.

Keywords: metallome, SXRF, insulin, single cell imaging, beta-cells, zinc, calcium, iron

Graphical Abstract

Graphical Abstract.

Graphical Abstract

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In this work, we compared the metallome of chemically fixed pancreatic beta-cells detected with synchrotron X-ray fluorescence (SXRF) and exposed to optimized and standard immunofluorescent (IF) protocols. We found that zinc and iron distributions are well preserved after optimized IF, but calcium is not. Additionally, we describe a novel immunofluorescent protocol to be used after SXRF to determine cell type or proteins of interest.

Introduction

Synchrotron X-ray fluorescence microscopy (SXRF) provides unambiguous determination of metal identity and subcellular distribution. In addition, SXRF data can be quantified to show the total metal content in each cell. The elements examined with the SXRF technique are presented as 2D projections of the 3D cellular structure, resulting in maps of elemental distributions at the subcellular level. Multiple cell types have been investigated using SXRF, including cells from lung, ovaries, neurons, and many others, as reviewed by Pushie.1

There is no standardized method to preserve cells for SXRF, but two major methods have been employed: cryopreservation and chemical preservation. After cryopreservation cells can be scanned hydrated (under cryo-temperatures)2–7 or dehydrated.7–13 For chemical preparation different fixatives have been used: formaldehyde,14–16 paraformaldehyde6,7,17–24 and alcohol (methanol).25,26 The chemically preserved cells are scanned at room temperature. There exists few direct comparisons of the different preservation methods, but the existing comparisons suggest that cryopreservation is a superior method for the preservation of elements.2,3,6,7 However, the cryopreservation and scanning hydrated cells are not always readily available, which exacerbates the already limited availability of SXRF data. In addition, the major difficulty for our study was the poor preservation of beta-cells using standard cryopreservation methods,27,28 thus necessitating alternative preservation of cells for SXRF. We developed protocols aimed at preserving beta-cells with chemical fixation based on protocols described by Jin6 and further optimized for beta-cells.15

Immunofluorescence (IF) is a classic method used to visualize cellular distributions of organelles and proteins of interest and is based on the specificity of antibodies binding to organelles or protein targets. Combining metal distribution data obtained using SXRF with IF imaging can yield a better understanding of metal homeostasis on the cellular level; however, protocols combining these two techniques are lacking for single cells. In this study, we compared optimized and standard IF protocols to minimize metal loss before SXRF. The data suggest that IF protocols result in substantial metal loss and should not be performed for metal quantification; however, qualitative data can be obtained for zinc and iron, but not for calcium. Our study required identification of cell type (beta-cells) from a mixed cell population (pancreatic islets) and quantification of cellular metallome; thus, we describe a new IF protocol that can be performed after the metallome has been quantified with SXRF.

Materials and methods

Materials

Chemicals were purchased from Sigma-Aldrich. Windows for SXRF scanning were purchased from Norcada. Cell culture medium and supplies were purchased from Gibco or VWR.

Pancreatic islet isolation and dispersion into single cells

To generate a cell monolayer, mouse pancreatic islets were first isolated, as described previously.29,30 Briefly, islets are collagenase digested, separated by Histopaque gradient centrifugation. Islets were then observed under the dissecting microscope and picked out of the solution by pipetting and transferring into fresh media, while the acinar tissue was left behind. After 12–24 h of recovery from isolation, the islets are further processed into single cells by trypsin digestion, as described previously.15 After dispersion into single cells, cells were attached to a silicon nitride (SiN) window (Norcada #NX5150D) coated with poly-d-lysine (this coating has been routinely used to attach pancreatic cells to glass coverslips31). To coat the windows, they were placed in sterile 12-well plates (one window per well). About 50 μl of sterile 0.01% of poly-d-lysine solution in water was added to the center of each window, and the windows were incubated in a sterile hood at room temperature for 20–60 min. The windows were washed twice with sterile water (150 μl per window), and after aspiration of water, windows were left to dry in sterile hood until completely dry (takes about 2 h).

Chemical fixation and drying

After cellular attachment to windows (24 h after dispersion), cells were washed briefly with phosphate buffered saline (PBS) and fixed at room temperature with 4% formaldehyde (EM-grade, methanol-free) for 20 min. After fixation was completed, cells were rinsed by gently dipping the windows in PBS. The cells were then rinsed twice with freshly prepared Tris–glucose buffer (10 mM Tris base, 260 mM glucose, and 9 mM acetic acid), as described in Finney and Jin.22 The excess solution was removed by using absorbent wipes that were cut into small strips and by gently touching the strips to the frame of the windows. Next, cells were dehydrated in a vacuum-sealed desiccator overnight (12 h or longer). These cells can be stored and transported in a desiccated condition before scanning with SXRF.

Scanning cells with SXRF

A hard X-ray scanning nanoprobe (Bionanoprobe), located at sector 9-ID-B of the Advanced Photon Source was used, as described previously.15 Before scanning cells with SXRF, a light microscope equipped with a motorized stage was used to take pictures of cells and record the coordinates of the cells of interest using μProbeX software. To select beta-cells from the mixed population on a window, their size and shape were used. Beta-cells are 10–15 μm and are round to oval, while other cell types found in the islets are larger than 15 μm and not round. After the coordinates of cells of interest were recorded, the window was placed into SXRF beam, and the coordinates were used to find the cells on the windows.

To collect SXRF data for this work, a monochromatic (10.5 keV) X-ray beam was focused with zone plate optics to sub-100 nm and directed at the sample. Desired areas on the sample were raster scanned through the beam for measurement, firstly with a coarse spatial resolution to confirm the cell type, followed by fine scans with a 100 nm pixel size. At each scanning point, a full X-ray fluorescence (XRF) spectrum was collected using a silicon drift detector located at 90° with regard to the incident beam. A detailed explanation of the physics of the SXRF can be found in a comprehensive review by Pushie1 and is beyond the scope of this work.

Optimized immunofluorescent protocol

Free zinc was used as a proxy for metal retention. To visualize free zinc, the cells were plated on an 8-chamber glass slide coated with poly-d-lysine. Live cells were loaded with 5 μM Zinpyr-1 in PBS for 30 min in humidity incubator at 37°C. After washing twice with PBS, the cells were fixed with 4% formaldehyde, freshly diluted with PBS from 16% stock solution. The fixation was performed for 20 min at room temperature without agitation. The cells were rinsed three times with PBS. Cells were then blocked for 10 min with 10% goat serum in PBS with the following TritonX-100 (TX100) concentrations: 0%, 0.01%, 0.05%, and 0.1%. Primary antibodies against early endosomal marker protein EEA1 were diluted at 1:200 in 10% goat serum in PBS with different detergent concentrations, and the primary antibodies were incubated with cells for 30 min. Cells were washed in PBS three times, followed by 30 min incubation with 1:1000 fluorescent secondary antibody in 10% goat serum in PBS. After cells were washed three times with PBS, the chamber slide was disassembled, and mounting media (with 4′,6-diamidino-2-phenylindole (DAPI)) was added on top of the cells. The slide was sealed with a glass coverslip. Images were recorded with a Nikon Microphot SA fluorescent light microscope (Supplementary Fig. S1).

Standard immunofluorescent protocol

The cells were fixed with 4% formaldehyde for 20 min at room temperature, rinsed three times with PBS, and blocked with 10% goat serum in PBS containing 0.1% TX100 for 1 h. Primary antibody was incubated for 1 h at room temperature (1:200 EEA1 diluted in 10% goat serum in PBS/0.1%TX100). Subsequently, cells were washed three times with PBS. Secondary antibody was incubated for 1 h (1:1000 diluted in 10% goat serum in PBS/0.1%TX100). Cells were washed in PBS (three times) and dried as described above in ‘Chemical fixation and drying’.

Immunofluorescent protocol after SXRF scanning

To identify different cell types in the pancreatic islet, immunofluorescent protocols were performed after scanning with SXRF: To identify beta-cells, insulin-specific antibodies were used, and to identify alpha-cells, glucagon-specific antibodies were used. After SXRF was performed, the windows were shipped back to our lab in dry conditions overnight and stored in a vacuum desiccator (up to a month). Before immunofluorescent experiments, the cells were rehydrated by graded ethanol rehydration, 5-min incubations in each grade: 100% ethanol, 90% ethanol, 70% ethanol, and water. After rehydration, cells were blocked with 10% goat serum in PBS for 1 h at room temperature.

Primary antibodies were diluted 1:100 with 10% goat serum in PBS. To label beta-cells, an insulin-specific antibody was used, and a glucagon-specific antibody was used to label alpha-cells. The antibodies were added as a mixture and incubated with cells simultaneously. The cells were incubated with the primary antibodies for 1 h at room temperature. Cells were washed by gently dipping the windows in PBS. The washing was repeated three times, using fresh PBS. Secondary antibodies conjugated to fluorescent probes were used at 1:1000 dilution in 10% goat serum in PBS. After incubation for 1 h at room temperature, the windows were rinsed three times by dipping in PBS.

The windows were mounted on the glass slide, and prior to adding the mounting medium, small coverslip shards were added to the glass slide to surround the window (at least four shards at each corner of the window). Mounting medium with DAPI (to label the nucleus) was added on top of the window, and a glass coverslip was gently placed on top of the window on the glass slide. The coverslip with the window was sealed with nail polish. The fluorescent images of insulin, glucagon, and DAPI were recorded with a Nikon Microphot SA fluorescent microscope.

Data analysis: quantification of SXRF data to determine the total content of elements in single beta-cells

Spectrum fitting and elemental quantification per pixel were done with MAPS.32 An AXO (AXO DRESDEN GmbH) standard thin film was measured with the same beamline configuration and used for elemental concentration calibration. Whole cell content analysis was also performed with MAPS.32 The region of interest (ROI) tool was used when all elements were open and displayed in the MAPS window. To include the whole cell in the analysis, the differential phase contrast image of the cell was used to draw the ROI around the cell. To quantify the background metal content, three ROIs were drawn in the area outside the cell. The mean background fluorescence was subtracted from the cell fluorescence to quantify the cellular content of the elements.

Statistical analysis

The statistical analyses were performed using GraphPad Prizm 8 or R-Studio. To identify outliers, all cells were subjected to robust regression followed by outlier identification method—ROUT.33 The normality of the data was tested by a Shapiro–Wilks test and Q–Q plots. The variance of the data was tested with Levene's test for homogeneity. According to the results of the normality and equal variance tests, the appropriate statistical tests for data analysis were performed. The alpha (α) = 0.05 and P-value < 0.05 were considered significant. The graphs were generated with GraphPad Prism 8. The language to describe statistical evidence has been adapted from Muff.34 We summarize the data in the graphs using descriptive statistics (mean and standard deviation) as recommended by Barde.35

Results and discussion

Comparison of standard and optimized immunofluorescent protocols performed prior to SXRF

We have optimized the IF protocol to prevent metal loss and improve metallome preservation by finding minimal necessary detergent concentration (0.01% TritonX-100) and minimizing the incubation times during the IF procedure (Supplementary Fig. S1). To test if the optimized IF protocol resulted in better metallome preservation, we compared chemically fixed cells without IF to cells that underwent the standard or optimized IF approaches (Fig. 1).

Fig. 1.

Fig. 1

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Comparison of metal distribution in chemically fixed beta-cells exposed to standard and optimized IF protocols. White dashed line highlights presumed cell borderers and white dotted line highlights presumed nuclear region. (A–C) Cellular metal distributions in chemically fixed beta-cells. Scale bars are 10 μm. Pseudo-color represents metal density. (A) Chemically fixed cells without IF: Zn (max = 0.2 μg/cm2), Ca (max = 0.5 μg/cm2), Fe (max = 0.25 μg/cm2). (B) Standard IF protocol: Zn (max = 0.02 μg/cm2), Ca (max = 0.1 μg/cm2), Fe (max = 0.05 μg/cm2). (C) Optimized IF protocol: Zn (max = 0.05 μg/cm2), Ca (max = 0.1 μg/cm2), Fe (max = 0.5 μg/cm2).

Qualitative SXRF data, showed zinc was localized at higher density in perinuclear region of beta-cells after all the tested procedures (Fig. 1), suggesting that qualitatively overall subcellular zinc distribution was not affected by IF optimization. On the other hand, calcium distributions were different between the groups, with high calcium in perinuclear region of chemically fixed cells without IF (Fig. 1A) and amorphous calcium distribution after standard IF (Fig. 1B), while after optimized IF, some calcium was observed in nuclear and perinuclear regions (Fig. 1C). These observations suggest that calcium distribution is distorted after IF protocols, and in general, qualitative data collected from cells after IF cannot be used to study calcium distribution in beta-cells. Iron distribution after the standard IF protocol was comparable to cells that did not undergo IF (Fig. 1A and B) and was observed in highly dense circular iron puncta as we reported previously.15

The cellular distribution of the elements collected with SXRF does not identify which organelle the elements are localized in, necessitating additional labeling of cells with organelle-specific antibodies using IF protocol. Although metal loss during the washing steps of the IF protocol is inevitable, we show that optimized detergent concentration and shortening times of incubation (optimized IF) preserve the qualitative distributions of zinc and iron and can be used with SXRF to identify organelles that accumulate zinc and iron; however, calcium localization should not be attempted. Standard IF protocol also sufficiently preserves qualitative distribution of zinc and iron, but not calcium.

We have previously published quantification of the metallome of chemically fixed beta-cells,15 and when compared to cells that underwent IF, zinc concentration had almost a 5-fold decrease after IF. When quantification of total zinc content was compared between standard and optimized IF, there was no evidence of a significant difference with 30.4 ± 33.4 fg total zinc after standard IF, compared to 12.9 ± 4.13 fg (t(5) = 1.36, P = 0.23). Similar observations were found for calcium and iron, with total calcium of 38.5 ± 34.2 fg after standard IF in comparison to 57.3 ± 47.9 fg after optimized protocol (t(5) = 0.49, P = 0.64). When compared to chemically fixed cells that did not undergo IF protocol, there is 4- to-6-fold decrease in total calcium. There was no evidence that total iron content was affected by the method of IF used, where cells that underwent standard IF had total iron of 23.4 ± 17.7 fg and 13.3 ± 7.23 fg after standard and optimized IF, respectively (t(5) = 1.19, P = 0.29), however this was about 2-fold decrease when compared to chemically fixed cells that were not processed with IF.15

Quantitative SXRF data suggests that both IF protocols resulted in significant loss of all three observed metals when compared to cells scanned without IF protocol. However, there was no evidence that the total content of zinc, calcium, or iron was affected by the optimization of IF procedures. This was a surprising observation because we expected improved preservation of total metal content after optimized IF procedure. Despite the small number of cells scanned, these data suggest that detergent concentrations and time of incubation do not have significant effects on the total content of zinc, calcium, and iron in chemically fixed beta-cells as detected by SXRF.

Taken together, these data suggest that quantification of metals should not be performed when using either standard or optimized IF protocol. However, reasonable qualitative preservation of zinc and iron distributions after IF protocol is useful to study organelle distributions paired with zinc and iron. The use of IF protocol will be less effective with calcium because calcium distributions are not conserved after IF procedures.

Confirmation of beta-cell identity (immunofluorescent protocol after scanning with SXRF)

The source of single cells in this study was a mixed population from pancreatic islets; however, we were interested in beta-cells specifically. We have shown previously that the dispersion protocol used in this study results in beta-cell populations at 77–78%.31 To confirm that the cells in the data set were beta-cells, insulin and glucagon antibodies were used after scanning cells with SXRF (insulin staining identifies beta-cells and glucagon staining identifies alpha-cells). The IF of the pancreatic islet cells on windows confirmed previous observations because most of the cells on the windows were insulin-positive (data not shown) and the cells that were selected to be beta-cells were confirmed with insulin-positive staining (Fig. 2A). These data suggest that cells retain an ability to bind antibodies after scanning with SXRF. There was concern that radiation damage after scanning would prevent antibodies binding to antigens (insulin and glucagon in this case); however, the antigenicity was retained after scanning (Fig. 2A and B). Interestingly, cells scanned with SXRF did not stain with DAPI nuclear staining (Fig. 2A). In contrast, the adjacent cells that were not scanned were successfully stained with DAPI (Fig. 2B). This result may indicate that DNA was damaged by the radiation, and the DAPI dye could not bind to the damaged DNA. These data suggest that for the study of specific cell type from mixed cell populations, it is possible to distinguish the cell identity using immunofluorescent protocol after SXRF scanning. Of note, the IF must be performed after the SXRF scanning to avoid metal loss, as described above.

Fig. 2.

Fig. 2

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Confirmation of beta-cell identity using IF after cells were scanned with SXRF. Merged image: insulin (green), glucagon (red), and DAPI (blue). Nuclear boundary of each cell is outlined with a white dotted line. Scale bar is 10 μm (right lower corner). (A) An example of an individual beta-cell scanned with SXRF. (B) Image of a cluster of islet cells that was not scanned with SXRF but was adjacent to cell that was scanned (shown in A). BF, brightfield; ins, insulin (green); gluc, glucagon (red).

Variation in total metal content within biological samples in chemically fixed beta-cells

To show reproducibility of SXRF data collected from chemically fixed beta-cells, we compared two windows from the same biological sample, in other words we compared technical replicates. The total metal content quantification per cell is presented in Fig. 3A–C. Total zinc content was found to be 153.9 ± 52.5 fg and 170.7 ± 73.2 fg in both technical replicates (t-test, t(19) = 0.59, P = 0.56) (Fig. 3A). Iron was found to be similar in both replicates with 30.6 ± 13.8 fg and 30.0 ± 7.78 fg (t-test, t(19) = 0.092, P = 0.923) (Fig. 3B). We found no evidence that total calcium content was different between the replicates with 221.9 ± 59.9 fg and 201.6 ± 87.9 fg in both technical replicates (t-test, t(19) = 0.61, P = 0.55) (Fig. 3C). These data suggest the reproducibility of chemically fixed samples for zinc, calcium, and iron. In addition, we quantified variability between the replicates, by calculating the coefficient of variability (CV) using the following formula (% CV = standard deviation of window means divided by the mean of window means). The CV for total zinc content was found to be 7.32%, for calcium 6.78%, and 1.28% for iron content. Note that a CV of 15% is considered acceptable variability for biological samples, and the data shows an acceptable margin of variability, providing another line of evidence that the SXRF data is reproducible in chemically fixed beta-cells.

Fig. 3.

Fig. 3

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Comparison of total metal content in biological and technical replicates of chemically fixed beta-cells. (A–C) Comparison of total metal content of two windows (W) (technical replicates). The columns represent means ± SD, each dot represents the value of an individual beta-cell. N(W1) = 15 cells and N(W2) = 6 cells. (D–F) Individual variability (biological replicates). Pooled = three different mice combined, individual = a single mouse. The columns represent means ± SD, each dot represents the value of an individual beta-cell. N(pooled) = 21 cells and N(individual) = 8 cells.

There are recommendations to combine multiple animals to avoid individual variability, e.g. for gene expression studies, it has been recommended to pool pancreatic islets from at least three individual animals;36 using this rationale, we compared SXRF for the total content of zinc, calcium, and iron from two biological samples, one was pooled (three mice) versus an individual mouse (Fig. 3D–F). There was no evidence of any differences between total zinc content in pooled versus individual mice, where total zinc was 158.7 ± 57.9 fg for pooled samples and 130.4 ± 86.4 fg in an individual mouse (t-test, t(27) = 1.03, P = 0.31) (Fig. 3D). For the total calcium content, there was very strong evidence of a difference between pooled and individual samples: 216.1 ± 67.4 fg for pooled samples and 96.9 ± 44.7 fg in an individual mouse (t-test, t(27) = 4.61, P < 0.0001) (Fig. 3E). Total iron content was found to be 30.4 ± 12.2 fg for pooled samples and 16.5 ± 12.6 fg in an individual mouse (t-test, t(27) = 2.73, P = 0.01) (Fig. 3F), providing strong evidence that iron content is different in samples from pooled mice when compared to an individual mouse. These data are consistent with previous studies showing significant individual variation in pancreatic islet's gene expression36 and calcium dynamics,37 suggesting that individual variability should be taken into consideration when interpreting the SXRF data. Combining multiple individual animals for studies with SXRF should yield greater biological relevance.

Conclusions

Many metals play essential roles in cellular function and evidence is mounting that changes to metal homeostasis results in multiple human diseases, necessitating discoveries of novel tools to study metals in single cells and improvements of existing methods. In this work, we describe a combination of SXRF protocol to study metal distributions and IF protocols that can be used for the identification of organelles or proteins. In chemically preserved beta-cells, we found that IF protocols result in unreliable quantitative data; however, we describe optimized IF protocol that results in preservation of qualitative data for zinc and iron, suggesting that for zinc and iron, IF protocols can be used before SXRF if metal co-localization with organelles or proteins of interest is desired without the quantification of the metals. We described a novel IF protocol that can be performed after SXRF quantification of the metallome. Additionally, we showed that individual variability (cells from an individual mouse) is higher when compared to a sample of pooled animals (samples combining three different mice). Overall, each research group must determine empirically optimized sample preparation methods based on the cell type and metal of interest, and we propose that it may be beneficial to combine at least three individual animals to combat individual variability, thereby collecting data with greater biological relevance.

Supplementary Material

mfad006_Supplemental_File
Click here for additional data file. (420.1KB, docx)

Acknowledgements

We would like to thank Dr Ramiro Malgor for the use of his fluorescent microscope at Ohio University Microscopy Core. We would like to thank Dr Barry Lai for technical support with SXRF and for helpful discussions. We would like to thank Dr Qiaoling Jin for assistance with live-cell preparations at Argonne, scanning cells hydrated, and data interpretation. We would like to thank Evan Maxey for supporting the SXRF experiments.

Contributor Information

Kira G Slepchenko, D epartment of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA.

Si Chen, Advanced Photon Source, Argonne National Laboratory, Lemont, IL, USA.

Kathryn L Corbin, D epartment of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA.

Robert A Colvin, Department of Biological Sciences, Ohio University, Athens, OH, USA.

Craig S Nunemaker, D epartment of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA.

Funding

This work was funded by the Ohio University Heritage College of the Osteopathic Medicine and Osteopathic Heritage Foundation based on work originally funded by the NIH (R01 DK089182). Additional funding was provided by R15 DK121247 and the Ohio University Student Enhancement Award (Kira Slepchenko). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science user facility, operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Extraordinary facility operations were supported in part by the DOE Office of Science through the National Virtual Biotechnology Laboratory, a consortium of DOE national laboratories focused on the response to COVID-19, with funding provided by the Coronavirus CARES Act.

Conflict of interest

There are no conflicts of interest to declare.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.

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