The dynamics of molecules in living cells hampers precise imaging of molecular patterns by functional and super-resolution microscopy. We developed a method that circumvents lethal chemical fixation and allows on-stage cryo-arrest for consecutive imaging of molecular patterns within the same living, but arrested, cells. The reversibility of consecutive cryo-arrests was demonstrated by the high survival rate of different cell lines and by intact growth factor signaling that was not perturbed by stress response. Reversible cryo-arrest was applied to study the evolution of ligand-induced receptor tyrosine kinase activation at different scales. The nanoscale clustering of epidermal growth factor receptor (EGFR) in the plasma membrane was assessed by single-molecule localization microscopy, and endosomal microscale activity patterns of ephrin receptor A2 (EphA2) were assessed by fluorescence lifetime imaging microscopy. Reversible cryo-arrest allows the precise determination of molecular patterns while conserving the dynamic capabilities of living cells.
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The authors would like to thank M. Reichl, J. Luig and P. Glitz for excellent technical assistance and A. Krämer for help in writing the manuscript. This study was funded by the Fraunhofer Society and the Max Planck Society for the Promotion of Science (CryoSystems grant to G.R.F. and P.I.H.B.) and the European Research Council (ERC AdG 322637 to P.I.H.B.).
The authors declare no competing financial interests.
Integrated supplementary information
Shown are different representations of the assembly of the cryo-stage. (a) Computer-aided design (CAD) model of separated core parts of the cryo-stage. (b) CAD model of assembled stage without mounting viewed from below. (c) Photographic representation of the stage mounted on a microscope. (1) silver block with temperature control; (2) aluminum block with medium inlet and outlet (3) and 100 μm thick channel with a cover slide (4); (5) insulating polyvinyl chloride plate adapted for commercial microscopy stages; (6) nitrogen inlet connected to liquid nitrogen reservoir; (7) nitrogen outlet connected to nitrogen pump; (8) medium inlet connected to automated syringe; (9) medium outlet; (10) connection for electronic temperature measurement and control.
HeLa cells transfected with EGFR-mCitrine were incubated with QDots bound to EGF for 5 min and cryo-arrested. (n=5 cells) Single QDots were tracked over 2 min. (a) Mean squared displacement (MSD) of the confined fraction of tracks. Dashed line represents the MSD value related to the localization precision. (b) Fractions of detected molecule tracks that are classified as mobile and confined.
FRET-FLIM in living cryo-arrested HeLa cells co-expressing EGFR-mCitrine and PTB-mCherry. Cells were cryo-arrested in three consecutive cycles. (a) Average fluorescence lifetime (τ), of EGFR-mCitrine in four different cells cryo-arrested three times without stimulation compared to average fluorescence lifetime (τ) of EGFR-mCitrine at 37°C (4 cells). (b) Difference in average fluorescence lifetime (△τ) of EGFR-mCtrine in cryo-arrested HeLa cells before and 5 min after stimulation with 200 ng/mL EGF plotted against mean PTB-mCherry fluorescence intensity per cell.
Supplementary Figure 4 Localization of ERK-KTR upon stimulation with EGF and in untreated cells before and after cryo-arrest.
Representative images of HeLa cells at 37°C expressing ERK-KTR upon stimulation with 200 ng/mL EGF (upper row) or before and after a cryo-arrest cycle (lower row). Scale Bar: 10 μm
HeLa cells were transfected with a SNAP-EGFR construct, which was labeled with Alexa647 and the liquid-ordered domains in the plasma membrane were marked with the fluorescent dye DiOC18. (a) Representative confocal images of the basal membrane of untreated HeLa cells as well as scatterplots showing fluorescence intensity of Alexa-647 labeled EGFR versus DiOC18 fluorescence before (37°C, upper row) and during cryo-arrest (-45°C, lower row). (b) Representative images of HeLa cells, treated with 10 mM methyl-β-cyclodextrin for 60 min to extract cholesterol, before (37°C, upper row) and during cryo-arrest (lower row). (c) Quantification of co-localization of SNAP-EGFR with DiOC18 in HeLa cells with (n=12) and without cholesterol depletion (n=6) at 37°C and ‑45°C by image correlation quotient (ICQ); data is represented as mean ± s.d.; ns: p > 0.05 using student’s t-test. Scale bars: 10 μm.
Localization precision histogram for EGFR-mEos2 molecules in HeLa cells that were chemically fixed by 4% formaldehyde (left) or cryo-arrested (right).
(a) Density-based spatial cluster analysis using a neighborhood radius of 40 nm of the EGFR-mEos2 distribution in the basal membrane of HeLa cells before (red) and 5 min after (blue) stimulation with EGF (left column) and cryo-arrested twice with an interval of 5 min without stimulation (middle column) as well as cells chemically fixed without EGF stimulation (red) and 5 min after (blue) stimulation (right column). In the upper two rows, clusters were grouped by the number of molecules they contain. The fraction of molecules per group are shown individually (first row) or as differential plot (second row). In the lower two rows, clusters were grouped by their diameter. The fraction of clusters belonging to each group is shown individually (third row) or as differential plot (fourth row). (b) Average node degree (representing the number of neighbors per cluster) histogram for the inter-cluster arrangement. Data represented as mean ± s.d.; n=8 cells for each condition.
Supplementary Figure 8 Comparative cluster analysis of EGFR between the central and peripheral area of the basal membrane.
(a) Representative example showing the localization of transiently expressed EGFR-mEos2 to the inner (orange) and outer (green) regions of the same cryo-arrested HeLa cell before and 5 minutes after EGF stimulation. Clusters are grouped according to their diameter. The ratio between outer and inner regions of the number of molecules and molecular density per cluster is shown in (b) and (c), respectively. Data represented as mean ± s.d; n=10 cells.
(a) Confocal FLIM measurements of LIFEA2 activation in Cos7 cells upon stimulation with pre-clustered ephrinA1-Fc (2 µg mL-1) for the indicated time (min) at 37°C. Upper row: representative images of LIFEA2 average fluorescence lifetime (t) at 37°C, lower row: representative images of LIFEA2 average fluorescence lifetime (t) at ‑45°C. Scale bars: 20 µm (b) Example of a Cos7 cell cryo-arrested 5’ after stimulation with ephrinA1-Fc (2 µg ml-1). Left: Spatial binning into 4 concentric radial bins of equivalent area (blue: bin 1 represents the area closest to the plasma membrane; dark red: bin 4 represents the innermost area around the nucleus). Right: mCitrine fluorescence intensity image of LIFEA2. (c, left) Representative average lifetime maps (t) of LIFEA2 on endosomes in Cos7 cells cryo-arrested 5 min and 20 min after stimulation. Endosomes were identified from Fourier transformation of mCitrine fluorescence intensity images (see Online Methods). (c, right) Corresponding α-maps. Scale bars: 20 µm (d) Deconvolved confocal FLIM z-scan through a representative HeLa cell cryo-arrested 5 min after stimulation with pre-clustered ephrinA1-Fc. Upper row: LIFEA2 donor (mCitrine) photon count, lower row: corresponding. α-maps of representative slices out of a total of 36. Total acquisition time 37’. Scale bars: 10 µm.
(a) Cover slides sticking to double-sided sticky tape with release liner remaining (black arrow heads) are placed into a 6-well dish and covered with cell culture medium containing cells. The cells are cultured in this arrangement for at least 24 h. (b) Anodized aluminum flow-through chamber from the bottom without (left) and with the cover slide (right) glued to it. The channel as well as the medium in- and outlets (white arrows) are not covered by the double-sided sticky tape (release liner removed; white arrow head).
Supplementary Figures 1–10 (PDF 1575 kb)
Representative region of the basal membrane of a HeLa cell expressing SNAP-EGFR labeled with Cy3. The video shows the diffusion of the receptor at 37 °C. Exposure time of each frame is 31.6 ms. (MOV 2554 kb)
Representative region of the basal membrane of a HeLa cell expressing SNAP-EGFR labeled with Cy3. The video shows the diffusion of the receptor at -45 °C. Exposure time of each frame is 31.6 ms. (MOV 1886 kb)
The video shows a 3D projection of a confocal FLIM z-scan through a representative HeLa cell cryo-arrested 5' after stimulation with pre-clustered ephrinA1-Fc (2 μg ml-1). (MOV 479 kb)
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Masip, M., Huebinger, J., Christmann, J. et al. Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution. Nat Methods 13, 665–672 (2016). https://doi.org/10.1038/nmeth.3921
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