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OPEN

Cathodoluminescence imaging
of cellular structures labeledwith luminescent iridiumor rhenium complexes at cryogenic
temperatures
Marie Vancová1,2*, Radim Skoupý3, Eva Ďurinová1,2, Tomáš Bílý1,2, Jana Nebesářová1,4,
Vladislav Krzyžánek3, Aleš Kolouch5 & Petr Horodyský5
We report for the first time the use of two live-cell imaging agents from the group of luminescenttransition metal complexes (IRAZOLVE-MITO and REZOLVE-ER) as cathodoluminescent probes.
This first experimental demonstration shows the application of both probes for the identification ofcellular structures at the nanoscale and near the native state directly in the cryo-scanning electron
microscope. This approach can potentially be applied to correlative and multimodal approaches andused to target specific regions within vitrified samples at low electron beam energies.
Abbreviations
CL	Cathodoluminescence
SEM	Scanning electron microscopy
Electron microscopy reveals cellular structures in incredible detail; however, the specific localization of nanoscale
structures in its native, cryo-fixed environment is still in its infancy. The current approaches for localizing thestructures at room temperature include immuno- and affinity-gold labeling, and less often, chemical ­labeling1.
Another possibility is incorporation of bioorthogonal labels, genetically expressed tags or fluorescent probes,
whose detections are correlated to the ultrastructure in the same cell (correlative light and electron microscopy)
with super-resolution microscopy and cryo-fluorescence m­ icroscopy2–5. These imaging technologies, whichhave advanced rapidly in recent years, provide significant progress in understanding how molecules functionin the structural context.
Here, we explore the use of IRAZOLVE-MITO and REZOLVE-ER probes for cathodoluminescence (CL)imaging. CL is the emission of photons from a material in response to excitation by accelerated electrons. Detection of CL emission from biological ­specimens6,7, organic ­fluorophores8–10, proteins, and semiconductor quantum­dots11 is extremely challenging due to low intensity optical signals that further bleach rapidly under the beam ofaccelerated electrons due to the structural damage of the biological material. In contrast, luminescent inorganic
nanocrystals (e.g., nanodiamonds or rare-earth element-doped nanocrystals) have gained increasing importanceas bright and stable CL probes with narrow emission s­ pectra12–18. Rare earth element-doped nanocrystals can bepotentially useful for correlative (multicolor) CL electron microscopy. Specifically, Y­ VO4:Bi3+, ­Eu3+, and ­Y2O3:Tb3
nanocrystals were simultaneously detected with CL and BSE at low electron acceleration voltages (≤ 2 kV) withinhuman vascular endothelial cells in epoxy resin blocks using Focused Ion Beam Scanning Electron Microscopy(FIB-SEM)19. FIB-SEM was used to detect ­LaF3:Tb3+ particles within endocytic compartments of human cells
embedded in epoxy ­resin12. The nanocrystals (e.g., ­Y2O3∶Tb3+, ­Y2O3∶Eu3+, ­LaF3:Tb3+), which have around 10 nm
in diameter, showed a remarkable resistance of the CL signal against electron beam exposure even at a highacceleration voltage (80 keV, 2 nA, 100 ms/pixel). They retained the CL intensity of more than 97% comparedto the initial intensity for 1 m­ in20.

1

Institute of Parasitology, Biology Centre CAS, České Budějovice 37005, Czech Republic. 2Faculty of Science,
University of South Bohemia, České Budějovice 37005, Czech Republic. 3Institute of Scientific Instruments
CAS, Brno 612 000, Czech Republic. 4Faculty of Science, Charles University in Prague, Prague 128 00, Czech
Republic. 5CRY​TUR​, Spol. S R.O., Turnov 511 01, Czech Republic. *email: vancova@paru.cas.cz
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The IRAZOLVE-MITO and REZOLVE-ER probes were designed for time-lapse ­imaging21,22. REZOLVE-ER,
chemically fac-[Re(CO)3(1,10-phenanthroline)(4-pyridyltetrazolate)], specifically localizes to the nuclear membrane and endoplasmic reticulum and also allows the detection of exocytotic events at the plasma ­membrane21.
IRAZOLVE-MITO (iridium complexed with cyclometalated 2-phenylpyridine and the 5-(5-(4-cyanophen-1-yl)
pyrid-2-yl)tetrazolate ligand) has a high specificity for mitochondria in live cells, and its emission spectrum
is in the range of 505–625 ­nm22. Both probes rapidly penetrate the cell membrane, have low cytotoxicity, areresistant to photobleaching, and in contrast to organic fluorophores, have long excited state lifetimes ranging
between hundreds of nanoseconds to ­milliseconds23. The extended excited state lifetimes stem from the structuralarrangement of the probes and excited triplet multiplicity nature of Ir and R­ e21,22. Transition metal complexes
enable access to new electron states due to the presence of d-block metal centres. This gives rise to their uniquephotophysical and photochemical properties (high resistance to photobleaching, extended excited state lifetime,and low cytotoxicity over long exposure time), making these probes valuable for long-term live-cell ­imaging23–25.

Results and discussion

We employed at first holographic microscopy in combination with fluorescence to visualize in vivo REZOLVEER and IRAZOLVE-MITO staining patterns in Vero cells. The dyes were used at concentrations 50 µM and20 µM, respectively, and incubated for either 30 min or overnight (see Supporting Information). REZOLVE-ER
staining was observed on the reticular network around the nucleus, whereas IRAZOLVE-MITO accumulated
within vesicle-like structures, probably lysosomes. However, in cells that were pre-fixed before IRAZOLVE-MITO
application, the staining pattern changed and the granular and filamentous structures extending throughout
the cells were labeled (Fig. 1). A similar observation was seen in Vero cells stained with a mitochondrial stain
MITOBLUE (fluorescent bisbenzamidine derivative)26.
CL imaging was performed on stained, high-pressure frozen cells that were freeze-fractured before cryoscanning electron microscopy (cryo-SEM) observation. We found that REZOLVE-ER (Fig. 2, left panel) and
IRAZOLVE-MITO (Fig. 3) exhibit strong CL signal that closely coincided with the fluorescence (Fig. 1).
In the cryo-SEM, CL enabled quick mapping of stained organelles over large areas and allowed to distinguishfrom intact cells embedded within the vitrified medium, which facilitated navigation to the regions of interest.
At low magnification, CL clearly revealed the shape of cells and nuclei position. The consecutive secondary electron images provided topographic data of the sample, which allowed for identification of additional organelles(Fig. 2, left panel; Fig. 3D,E). Since the acquisition parameters did not match for the topographic (1 kV) and CL(4 kV) measurements, the registration was performed manually by using defined landmarks. Unstained cells
were used as negative controls to confirm that the settings used for visualization of both IRAZOLVE-MITO and
REZOLVE-ER did not detect endogenous CL (data not shown).
We also aimed to find how the individual steps of the conventional SEM preparation protocol at room temperature affect the CL signal. In contrast to frozen-hydrated cells (Fig. S1A), aldehyde-fixed cells dehydratedusing organic solvents (ethanol, acetone) at room temperature did not display CL signal. The CL signal waspartially preserved after air-drying (or rotary-pump drying); however, it bleached almost immediately underthe electron beam excitation (Fig. S1B,C).
The CL was taken independently using two distinct CL detectors: the Crytur CL detector (Figs. 2, 3, Figs. S1,
S3, S5) or the MonoCL4 + CL detector from Gatan (only Fig. S2) mounted to the SEM Magellan 400L, where
we also measured the CL emission spectra at 5 kV, 0.1 nA, and − 140 °C. The CL spectrum of REZOLVE-ER
staining showed a dominant initial emission band centered around 550 nm (Fig. 4A), which correspondedwith an emission profile for REZOLVE-ER between 500 and 650 ­nm27. The dominating CL emission band of
IRAZOLVE-MITO was in the 520 nm region (Fig. 4B). However, the absorbed electron dose, which linearly
increased during serial CL spectral acquisition, could bleach the iridium complexes and thus impair the recordingof spectra at longer wavelengths. The IRAZOLVE-MITO excited at 403 nm had an emission interval from 505
to 625 n­ m24. Both CL emission maxima were obtained at -140 °C and are presumably blue-shifted compared toluminescence emission maxima obtained at room temperature. The blue shift of luminescence emission maxima
of organometallic complexes obtained at − 196 °C versus room temperatures was observed earlier and explainedwith the rigidochromic e­ ffect27.
The lowest accelerating voltage generating the CL emission of both probes was 2 kV; however, the optimal
signal-to-noise ratio was obtained at 4–5 kV (Fig. S3A) and 30–300 pA probe currents (Fig. S3B,C).
At these parameters, we estimated the spatial and lateral resolution of the CL signal based on the Monte
Carlo simulation of electron scattering. The simulations allow to quantitatively estimate the CL generation in abulk sample (here amorphous ice as the predominant part of a biological sample) within the electron interaction volume (Fig. S4A). The highest CL intensity around the actual beam position decreases significantly withincreasing radius but still interferes into hundreds of nm with significant background (Fig. S4B). In the z-axis,
we estimated that most of the CL signal is generated at depths around 250 nm under the surface. It is given bysignificantly higher irradiated volume even if less than 20% of initial beam intensity reaches this depth. In xyplane, most of the signal (more than 60%) is generated from a radius < 8 nm (simulated beam diameter is 4 nm).
These theoretical values are strongly affected by sample inhomogeneities, CL emitting particles/compounds
distribution or light refraction/absorption in real samples. The resolution can be enhanced only with decreasing primary beam energy or using an electron transparent sample where the characteristic electron interaction
pear-shape is not being fully developed. Next limiting factors are CL detector sensitivity, working distance, andstability of specimens under the electron beam.
Under selected parameters (4 kV, 15 µs, 300 pA, − 140 °C), we compared the decay of the CL signal from
intensity profiles measured on the IRAZOLVE-MITO stained structures without and with gold sputter coating (~ 2 nm) (Fig. 5). From measurements of CL intensities over 25 uncoated CL areas from the first and theScientific Reports |Vol:.(1234567890)(2022) 12:13432 |https://doi.org/10.1038/s41598-022-17723-w

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Figure 1.  REZOLVE-ER and IRAZOLVE-MITO probes were used for imaging of endoplasmic reticulum andmitochondria of Vero cells, respectively. Holographic microscope 3D Cell Explorer (Nanolive). Bar 20 µm. RI(the refractive index).

following three SEM images, we calculated the mean pixel intensity for each image (area 6 was excluded, seeFig. 5A; Fig. S5).
We found that the CL signal decreased between the acquisition of the first and the second SEM image inuncoated samples by ~ 7.4%, then ~ 3.4% and ~ 5.5% to the value of ~ 83.8% (the fourth SEM image) (Fig. 5A,left). In the coated samples, CL decreased by ~ 5.4%, then ~ 3.2%, and ~ 4.8% to the value of ~ 86.7% (Fig. 5B,
right). The gray-scale intensities of one selected area are shown in Fig. 5B. In comparison, organic fluorophores
bleached under the electron beam (2 kV, 0.8 nA, equivalent to a dose of 0.04 nC/μm2) almost ­immediately19.
Quantum dots (CdSe/CdS) exhibited highly reduced CL emission (by 30%) within the first 10 s (equivalent to

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Figure 2.  SEM of freeze fractured Vero cells stained in vivo with REZOLVE-ER. Corresponding images weretaken using Crytur cathodoluminescence detector (CL) and the Everhart–Thornley detector (SE) at − 140 °C
and 5 keV. The CL image was colourised and merged with the SE image to illustrate the exact structural
overlay. The CL signal helped find positions of stained cells in the fractured area and identify labeled cellularcompartments. The arrow marks the intact cell. SEM JEOL 7401F. Bars: 10 µm.
a dose of 0.08 nC/μm2), whereas the drop in emission intensity of rare-earth element-doped nanocrystals was
10–20% within the first 10 s (equivalent to a dose of 0.04 nC/μm2)19.
Here, we demonstrated that IRAZOLVE-MITO and REZOLVE-ER d­ yes21,22 produce CL signal, which canbe used for correlative studies combining live and CL-SEM imaging at cryogenic temperatures. The advantageof this approach is that one can perform localization studies and structural imaging at cryogenic temperatures
simply in one SEM instrument without multiple sample transfers typical for non-integrated cryo-fluorescenceSEM ­workflows4,5. Cryo CL-SEM imaging approach may unleash the potential of the CL to localize moleculesin the cellular volume using the cryo-FIB-SEM, cryo-block-face imaging techniques, and it can be an alternative
to integrated cryo-correlative fluorescence electron microscopy workflow (e.g., “iCorr” or Photon Ion Electron
microscope)28,29.
In contrast to rare-earth element doped nanocrystals and nanodiamonds mainly used to specifically label
components in the endocytic pathway, IRAZOLVE-MITO and REZOLVE-ER have been designed for bioimaging as alternative organelle-specific dyes. Widespread application of CL in bio-imaging is currently limitedby the availability of high-intensity CL probes targeting structures of interest, their insufficient stability underthe electron beam, and lack of probes with distinct emission spectra that would allow multicolor CL-SEM. CL
properties also need to be tested for IRAZOLVE-ALKYNE and REZOLVE-ALKYNE tags as well as other novel
photoluminescent iridium or rhenium complexes, now well-established probes used in life science r­ esearch23,30–32.
For example, rhenium(i) tricarbonyl complexes incorporating 1,10-phenanthroline or the 3-chloromethylpyridyl
bipyridine that accumulate in m
­ itochondria33,34, a rhenium complex linked to 4-cyanophenyltetrazolate (commercially available as REZOLVE-L1), which has an affinity to polar lipids (specifically to phosphatidylethanolamine, sphingomyelin, sphingosine), and finally a rhenium 3-pyridyltetrazolate complex, which localizes withinthe ­lysosomes34. Another new probe, a cyclometalated iridium complex, was designed to visualize microtubulesby light and electron m­ icroscopy35. Phosphorescent organometallic iridium complexes have also been recently
used to detect diatoms ­viability36. Other luminescent probes include iridium (III) polypyridine ­complexes37,
which exhibit photocytotoxic activity upon prolonged laser i­ rradiation38.
In conclusion, this study shows that luminescence probes from the group of iridium and rhenium complexes,
IRAZOLVE-MITO and REZOLVE-ER, generate detectable CL signals in vitrified samples at low electron beamenergies. We carried out CL and topography imaging on freeze fractured cells and found that the CL intensity of
IRAZOLVE-MITO dye was reduced by 16% in uncoated samples and 13% in coated samples between the firstand fourth CL image acquisition of the same region taken at − 140 °C, 4 kV, 300 pA, and 15 µs. As CL imaging
can be easily integrated into simultaneous multimodal imaging offered by the modern SEM, we believe in great
potential of using organelle-specific probes such as IRAZOLVE-MITO and REZOLVE-ER in high-resolution
biological imaging. The dyes can be used to select target regions before cryo-FIB milling or perform correlative
CL-SEM as demonstrated in this study.

Methods

Cultures and staining and in vivo imaging. Human airway epithelial cells A549 were cultivated in
DMEM Low glucose medium with 10% bovine fetal serum, 1% ATB (Penicillin, Streptomycin), and 1% Glutamine Stable at 37 °C and 5% C
­ O2. Cell cultures of Vero E6 were cultivated in FLUOROBRITE DMEM highglucose medium with ATB at 37 °C and 5% C
­ O2. The cells were seeded to 25 c­ m2 tissue culture flasks or µ-Dish
35 mm Imaging Chamber with a glass-bottom (Ibidi) to achieve 80–100% confluence. REZOLVE-ER and IRAZOLVE-MITO (both ReZolve Scientific, see acknowledgments for further details) were dissolved in DMSO to
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Figure 3.  Correlative cryo-cathodoluminescence (CL) scanning electron microscopy of Vero cells stained with
IRAZOLVE-MITO. (A–E) In vivo stained cells were frozen, fractured, and imaged at − 135 °C using Crytur
CL detector (4 keV) and the Everhart–Thornley detector (ETD, 1 keV). (F–H) Chemically pre-fixed cells were
stained, frozen at high pressure, and corresponding images were taken at 4 keV using CL (F) and ETD (G). Bars:
1 µm (A–E), 10 µm (F–H). SEM JEOL 7401F. CL and topographic images were merged manually (A,H).
10 mM (stock solutions). Cells were stained in the presence of culture media by adding either the REZOLVEER stock solution (to final concentrations of either 33 or 50 µM) or IRAZOLVE-MITO (20 µM, 40 µM). Cells
were imaged in vivo by a holographic microscope 3D Cell Explorer (Nanolive) either after 30 min or overnightincubation. In the case of IRAZOLVE-MITO staining, the cells were stained and imaged after fixation in 4%
formaldehyde in 0.1 M HEPES for 1 h at room temperature, followed by a 30 min wash step.

Cryo‑scanning electron microscopy and CL imaging. Pelleted cells were high pressure frozen (EMICE, Leica Microsystems) in the presence of 20% bovine serum albumin and transferred into the scanningelectron microscope (SEM) JEOL 7401F (JEOL Ltd.) equipped with a cryo-attachment CryoALTO 2500 (Gatan,Inc.) precooled to − 140 °C. Cells were freeze- fractured, optionally freeze-etched at temperatures between− 98 °C and − 95 °C for 10 s, and gold-coated for 10 s with gold (~ 2 nm). Images were taken using the Everhart–
Thornley detector of secondary electrons and the CL detector designed by Crytur comp. The CL detector detects
panchromatic CL signals with a high signal-to-noise ratio in the spectral range 330–600 nm. This retractable CL
detector of the parabolic mirror type consists of a quartz light guide and the photomultiplier. The contrast andbrightness settings of the CL detector during CL intensity measurements were constant.
Independent observation was performed using the SEM Magellan 400L (ThermoFisher Scient.) equipped with
MonoCL4 + CL detector (Gatan, Inc.) at a range of temperatures from − 125 °C to − 150 °C. Freeze fracturing
and Au/Pt coating (appr. ~ 2.7 nm) was performed using the sputter coater Leica ACE 600 (Leica Microsystems).
CL images and spectra were taken at a beam energy of 7 keV and probe current of 0.1 nA.

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Figure 4.  The CL emission spectra of REZOLVE-ER (A) and IRAZOLVE-MITO (B) probes are shown inturquoise. (A) For comparison, the absorbance/emission spectrum of REZOLVE-ER is 350–405 nm/500–
650 nm (green) according to the datasheet. The broad emission band of IRAZOLVE-MITO in C
­ H2Cl2 iscentered at ~ 605 n­ m24. CL emission spectra were measured by the MonoCL4 detector at − 135 °C. The
dominating were bands at approx. 550 nm for REZOLVE-ER (A) and 525 nm for IRAZOLVE-MITO dye (B).

Preparation of cells for observation by SEM at room temperature. Cells stained in vivo by IRAZOLVE-MITO were fixed in 4% formaldehyde and 0.1% glutaraldehyde in 0.1 M HEPES for either 15 min orovernight. After washing, cells were, dehydrated with graded acetone series followed by two washes in 100%
acetone. After critical point drying, the glass cover slides were mounted onto stubs and coated for 10 s using
gold (Baltec SCD 050). Stained and fixed cells were optionally air-dried or dried with a rotary pump without
dehydration.
Image analysis of intensity and decay of CL.

At first, we took four consecutive images at the same
parameters (4 keV, 15 µs, 300 pA) of Vero cells stained in vivo with IRAZOLVE-MITO dye without coating orgold-coated (~ 2 nm) using the Crytur CL detector mounted to SEM JEOL 7401F at − 140 °C. Intensities anddecay of CL were analyzed from images using the Matlab tool as shown in Fig. S5. The centers with the highest CL intensities from 25 areas from the uncoated specimen and 9 areas from the gold-coated specimen weredetected by FAST-PEAK-FIND39 (Fig. S5A), with a threshold parameter equal to 145 as determined manuallyfrom the maximum intensity of cells in the background. For further analysis, we cut each area with the highest
CL intensities from the image out to a square area in size of 21 × 21 px (Fig. S5B). For each area, we determinedthe diameter of the circle defining the CL region (marked red in Fig. S5B) as the maximum of the second
derivative (marked red in Fig. S5D) obtained from the average intensities calculated in concentric circles with anincreasing diameter with the centre located in the middle of this square area (Fig. S5B, C). Total CL intensity wascalculated and statistically analyzed from this internal CL region (Fig. S5E) whereas the outer areas represented
the background (Fig. S5F). CL intensities were plotted over time (four consecutive photos), where the first value
represents 100% (Fig. 5).
The CL resolution simulated for amorphous ice and beam energy of 4 keV in a bulk sample was computedin Casino 2.540.

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Figure 5.  Decay of cathodoluminescence intensities of IRAZOLVE-MITO dye (without and with sputteredgold nanolayer) measured on four consecutive images taken at the same parameters of the CL detector andthe SEM (4 keV, 15 µs, 300 pA, 10,367 px/nm). (A) The graph shows changes in CL intensities measured at thefirst and consecutive three scans. (B) The CL intensities were measured over the representative uncoated area
(area 25 from Fig. S5 A is shown) from first to fourth images (from left to right). The blue plane represents thelevel of background mean value.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary
information files.
Received: 9 March 2022; Accepted: 29 July 2022

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Acknowledgements

We thank Crytur company for the possibility to use their CL detector, Jiří Vaněček (BC CAS) and Marek Dolejší(University of South Bohemia, USB, České Budějovice) for expert technical assistance with the SEM, Eva Výletová (USB) for providing us human airway epithelial cells, gift from R. Randall, University of St. Andrews, UK.
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www.nature.com/scientificreports/
IRAZOLVE-MITO and REZOLVE-ER staining probes, previously commercialized by ReZolve Scientific, werekindly provided by Prof. Sally Plush (University of South Australia; sally.plush@unisa.edu.au) and Prof. Max
Massi (from Curtin University, Perth, Australia, and ReZolve comp.; m.massi@curtin.edu.au); to enquire about
the availability of the probes and potential collaboration, please contact Sally Plush and Max Massi using theemail addresses provided.

Author contributions

M.V. designed the experiment, wrote the manuscript, prepared the samples, and performed the cathodoluminescence-SEM imaging. R.S. took part in the cathodoluminescence-SEM imaging and performed a simulation of
CL resolution. E.D. conducted cultivation and in-vivo imaging. T.B. performed image analysis. The manuscript
was written through the contributions of all authors. All authors have given approval to the final version of themanuscript.

Funding

This work was supported by the Technology Agency of the Czech Rep. (TN0100008), The Ministry of Education,
Youth and Sports of the Czech Rep. (Czech BioImaging LM2018129), and European Regional Development
Fund-Project “Mechanisms and dynamics of macromolecular complexes: from single molecules to cells” (No.
CZ.02.1.01/0.0/0.0/15_003/0000441).

Competing interests

The authors declare no competing interests.

Additional information

Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​022-​17723-w.
Correspondence and requests for materials should be addressed to M.V.
Reprints and permissions information is available at www.nature.com/reprints.
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Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons licence and your intended use is not
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