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Reversible Cryo-arrests of Living Cells to Pause Molecular Movements for High-resolution Imaging
采用活细胞可逆性冷冻停滞法暂停分子运动以进行高分辨率成像   

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Abstract

Fluorescence live-cell imaging by single molecule localization microscopy (SMLM) or fluorescence lifetime imaging microscopy (FLIM) in principle allows for the spatio-temporal observation of molecular patterns in individual, living cells. However, the dynamics of molecules within cells hamper their precise observation. We present here a detailed protocol for consecutive cycles of reversible cryo-arrest of living cells on a microscope that allows for a precise determination of the evolution of molecular patterns within individual living cells. The usefulness of this approach has been demonstrated by observing ligand-induced clustering of receptor tyrosine kinases as well as their activity patterns by SMLM and FLIM (Masip et al., 2016).

Keywords: Cryo-arrest(冷冻停滞), Fixation(固定), Superresolution(超分辨率), Single molecule localization(单分子定位), FLIM(FLIM)

Background

Understanding molecular processes in cells, e.g., the ligand-induced response of receptor-tyrosine kinases (RTKs), requires the precise spatio-temporal observation of molecular patterns. Due to variance in cellular states, this response needs to be monitored in individual cells rather than in cell populations (Snijder and Pelkmans, 2011). Using SMLM, individual molecules can be localized with high precision (Betzig et al., 2006). This allows, for instance, extracting information about the clustering of RTKs in the plasma membrane. Complementarily, confocal FLIM can unveil how molecules react as an ensemble within a diffraction-limited volume element. This can reveal interaction patterns of RTKs with downstream molecules, phosphorylation patterns as well as activity patterns by the use of conformational sensors (Offterdinger et al., 2004; Sabet et al., 2015). However, the acquisition time in FLIM and SMLM is in the order of minutes. Because many molecular arrangements in living cells–including those of RTKs–evolve on a much faster time scale, confocal FLIM images get blurred and spatial resolution is reduced severely (Masip et al., 2016). In SMLM, molecules are localized successively in consecutive frames. Performing these measurements on dynamic molecules in living cells will lead to a falsified image of localizations since molecules diffuse to different positions in the course of data acquisition. For instance, the epidermal growth factor receptor (EGFR) moves on average with a diffusion constant of 2-5 10-2 μm2/sec in the 2D environment of the membrane (Orr et al., 2005; Xiao et al., 2008). To allow for long acquisition times, irreversible chemical fixation with aldehydes is typically used. Yet, the use of chemical fixation agents is inefficient in immobilizing membranes and can lead to protein extraction or artificial clustering (Saffarian et al., 2007; Tanaka et al., 2010; Schnell et al., 2012). Further, the lethal fixation disrupts the out-of-equilibrium physiological state and prohibits the observation of the evolution of molecular patterns within an individual cell. We have therefore developed a reversible cryo-arrest that halts molecular movements for high-resolution imaging, yet maintains cells in a viable state. Upon cooling, the concentration of cryoprotective dimethyl sulfoxide (DMSO) is increased stepwise (Masip et al., 2016). This precludes the formation of ice crystals that can be lethal to cells while scattering light and altering cellular structure by displacing organic material (Dubochet et al., 1988 and 2012; Huebinger et al., 2016). At the same time, adding DMSO at low temperatures greatly reduces its toxicity (Farrant, 1965). A further advantage of the cryo-approach is that fluorophores become less reactive in the excited state, resulting in the emission of more photons before bleaching as well as a lowered production of cytotoxic radicals (Kaufmann et al., 2014), which can severely damage cells during acquisition at physiological temperatures (Wäldchen et al., 2015).

Materials and Reagents

  1. 10 μm thick double-sided adhesive tape (D80 19 x 50 mm) (Modulor, catalog number: 0332054 )
  2. Cover slides 21 x 26 mm (No.1) (Thermo Fisher scientific, Thermo scientificTM, catalog number: BBAD02100260#A* )
  3. 6-well plates for cell culture (SARSTEDT, catalog number: 83.3920 )
  4. 15-ml reaction tubes (e.g., SARSTEDT, catalog number: 62.554.502 )
  5. 200-μl pipette tips (e.g., Greiner Bio One International, catalog number: 739290 )
  6. Silicon tube
  7. Flexible silicon tubes (inner diameter [i.d.] 2 mm; outer diameter [o.d.] 4 mm and i.d. 1.5 mm; o.d. 3 mm) (e.g., VWR, catalog numbers: 228-0704 and 228-0702 )
  8. Cell line of interest and suitable cell culture medium
    Note: The protocol has so far been tested for the adherently growing cell lines HeLa (ATCC, catalog number: CCL-2 ), MDCK (ATCC, catalog number: CCL-34 ), COS-7 (ATCC, catalog number: CRL-1651 ), MCF7 cells (ATCC, catalog number: HTB-22 ) and HCT116 (ATCC, catalog number: CCL-2 47). Other cell lines may be used after proper testing for the reversibility of the cryo-arrest.
  9. Ethanol, absolute (e.g., Fisher Scientific, catalog number: 10342652 )
  10. Sterile water (sterilized by filtration)
  11. DMSO (min 99%) (e.g., Serva Electrophoresis, catalog number: 20385 )
  12. Liquid nitrogen
  13. Immersion oil (e.g., Olympus, catalog number: IMMOIL-F30CC )
  14. Transfection reagents and plasmid expressing fluorescent proteins of interest or cell lines stably expressing a fluorescent protein of interest. The protocol has been successfully used for confocal FLIM with a conformational activity sensor for EphrinA2 and EGFR tagged to mCitrine in combination with a phosphotyrosine binding domain tagged to mCherry. Single molecule localization has been performed with EGFR-mEos2 and with Vinculin tagged to a SNAP-Tag and labelled with SNAP-Cell TMR-star (New England Biolabs, catalog number: S9105S ). Requests about these plasmids can be addressed to the corresponding author
  15. HEPES or phosphate buffered imaging medium without phenol red (e.g., DMEM, PAN-Biotech, catalog number: P04-01163 )
  16. 10% fetal bovine serum (FBS) (PAN-Biotech, catalog number: P30-1505 )
  17. Streptomycin/penicillin (PAN-Biotech, catalog number: P06-07100 )
  18. L-glutamine (200 mM) (PAN-Biotech, catalog number: P04-80100 )
  19. 1% nonessential amino acids (PAN-Biotech, catalog number: P08-32100 )
  20. Different DMSO solutions (see Recipes)
  21. Cell culture medium (see Recipes)

Equipment

  1. Scalpel
  2. Laminar flow hood
  3. Incubator (e.g., Nuaire, model: NU-5510/E )
  4. Fine forceps (e.g., Electron Microscopy Sciences, catalog number: 0203-7-PO )
  5. Anodized (black) aluminum flow-through chamber (custom-built; see Figure 1)
    Note: Anodizing the aluminum reduces its corrosion and thereby solvation of aluminum ions, which might otherwise influence cellular reactions. Anodizing with black color reduces reflection of light, which might interfere with the microscopic imaging.


    Figure 1. Design of the flow-through chamber. Photographic representation (A, B) and technical drawings (C-E) of the self-built flow-through chamber made out of aluminum. A. Top view on the flow through chamber with pipette tips inserted into the in- and outlet (1) and a drill hole (2; depth: 15 mm; diameter: 1 mm) for the insertion of a thermocouple. B. Bottom view with the cover slide glued to the flow-through chamber (3); C. Top view of the flow-through chamber; D. Side view section through the flow-through chamber (section a:a in C); E. Detail of the side view section (detail b in D). All dimensions in c-e are in mm.

  6. Polyvinyl chloride (PVC) insert for the microscope table (custom-built; see Figure 2), with 2 threaded holes to fix the stage using a metal clamp (see Figure 3B)


    Figure 2. Technical, drawings of the PVC-insert for the microscope stage. The technical drawings show a mounting that fits into Scan IM stages (Märzhäuser Wetzlar GmbH & Co. KG, Wetzlar, Germany). A. Bottom view; B. Side view section (a:a in A); C. Side view detail of the central part (b in B). All dimensions are in mm.

  7. Low-pressure syringe pump with a computer interface (Cetoni, model: neMESYS low pressure syringe pump )
  8. Cryo-stage with temperature control (Linkam Scientific, model: MDS600 , other models with similar cooling heads may also work)
  9. Thermocouple (e.g., PTFE-insulated type T with 0.08 mm wires; Omega Engineering, Deckenpfronn, Germany) with a data acquisition device connected to a computer for continuous temperature recording (e.g., OMEGA Engineering, model: OMB-DAQ-2408-2AO )
  10. For confocal FLIM: Confocal laser scanning microscope equipped with a time correlated single photon counting unit and a ps-pulsed laser (e.g., PicoQuant, model: MicroTime 200 )
  11. For SMLM: Microscope equipped with sensitive cameras (EMCCD or sCMOS) as well as lasers with adequate wavelength and power to image, switch on and bleach fluorophores
  12. Inverted microscope (Olympus, model: IX-81 )


    Figure 3. Cryo-stage mounted on a microscope. Photographic representation of the cryo-stage mounted on an inverted microscope ( IX-81 ; Olympus GmbH, Hamburg, Germany). A. Overview of the cryo-stage with peripheral instruments on the microscope. 1: Liquid nitrogen pump with control unit (Note: This should normally be disconnected from the optical table, since it may cause vibrations of the sample); 2: 15-ml tubes with medium of different DMSO-concentrations, inlet tube of the low-pressure syringe pump is inserted via a lid with a hole; 3: Low-pressure syringe pump; 4: Data acquisition device for thermocouple; 5: Central part of the cryo-stage as detailed in (B); 6: Liquid nitrogen reservoir. B. Detailed image of the central part of the microscope. 1: Tube connected to nitrogen pump; 2: Metal clamp to fix the stage to the PVC-insert; 3: Thermocouple to measure the temperature inside the silver block; 4: Medium inlet connected to the low pressure syringe pump; 5: Aluminum flow-through chamber (compare Figure 1); 6: PVC-insert for microscope stage (compare Figure 2); 7: Tube connected to the nitrogen reservoir; 8: Temperature controlled silver block with electrical counter heater; 9: Medium outlet tube; 10: Thermocouple to measure temperature of the aluminum block.

Software

  1. ThunderSTORM Plugin (Ovesny, Bioinformatics, 2014)
  2. ImageJ (Rasband, ImageJ, National Institutes of Health, Bethesda, Maryland, USA)

Procedure

  1. Culturing of cells
    Note: The following steps should be performed under a laminar flow hood. All instruments should be sterilized before. The adhesive tape should be taken out of its packing only under a laminar flow hood.
    1. Cut double-sided adhesive tape into 26-mm long pieces using a scalpel (Figure 4A).
      Note: Use one layer of adhesive tape with release liner as support. Then, add a second layer on top and cut this into size. This facilitates working with the adhesive tape, since it does not stick to the release liner.


      Figure 4. Preparation of culture dish with cover slides. A. A layer of double-sided adhesive tape (1) is glued on a smooth surface (e.g., an anodized aluminum plate). A second layer of double-sided adhesive tape is glued on top (2) and cut to length of a cover slide (left image). Afterwards, a rectangular of app. 21 x 5 mm is cut out in the center and removed (right image). B. The piece of double-sided adhesive tape is glued on a cover slide (4; left image). Mild pressing ensures tight contact and prevents leakage (right image). C. Photographic representation of a 6-well cell culture dish with cover slides with double sided sticky tape (arrowheads) in cell culture medium with phenol red. The double-sided sticky tapes have cut-outs fitting the cavity of the flow through chamber (arrows; compare Figure 1B).

    2. Cut out a 21 x 5 mm rectangular in the middle of each piece of adhesive tape using a scalpel and remove the inner rectangular (Figure 4A).
    3. Glue the adhesive tape on a 21 x 26 mm cover slide. Mildly press to ensure a tight contact without breaking the cover slide (Figure 4B).
    4. Sterilize the cover slides by dipping them in ethanol and wash them with sterile, distilled water.
    5. Place the cover slides in the 6-well-plate (Figure 4C).
    6. Detach cultured cells and plate them in the 6-well dish in regular cell culture medium (see Figure 4). The adhesive tape is not affected by the cell culture medium and remains sticky.
      Note: Let the cover slides dry or wash with cell culture medium before adding the cells, since distilled water is harmful to the cells.
    7. Incubate at 37 °C/5% CO2 at least until they are firmly attached. Cells can be cultured under this condition for days and transfected with the respective constructs of interest.

  2. Assembly of the cryo-stage on the microscope
    1. Prepare 10%, 20%, 30% and 50% (vol/vol) solutions of DMSO in imaging medium in 15-ml reaction tubes.
      Note: The inlet tube of the low temperature syringe pump can be inserted into the 15-ml reaction tubes through a hole in the lid (Figure 3A).
    2. Take the silver cooling head out of the casing of the cryo-stage.
    3. Connect the nitrogen pump, the liquid nitrogen reservoir using flexible silicon tube (i.d. 2 mm; o.d. 4 mm) and the electronic temperature control to the silver cooling head (see Figure 3B).
    4. Set the temperature of the silver block to 37 °C, using the control unit for the liquid nitrogen pump (Figure 3A-1).
    5. Attach a piece of flexible silicon tube (i.d. 1.5 mm; o.d. 3.5 mm) to the outlet of the low-pressure syringe pump.
      Note: The volume of the tube connected to the syringe pump should not exceed 200 μl. Otherwise, this would increase the amount of medium that needs to be exchanged during every step of the cryo-arrest.
    6. Fill the syringe and the tube with imaging medium without DMSO using the software of the automated syringe. Avoid air bubbles.
      Note: For the first filling, small amounts of medium have to be filled in the syringe and pressed through its outlet to remove air bubbles in the syringe and tubes before filling the syringe.
    7. Assemble the PVC-insert into the microscope table and center the objective below the opening for the flow through chamber (Figure 3B).
    8. Press 200-μl pipette tips through the in- and outlet of the flow-through chamber from the bottom. Remove the parts sticking out of the bottom with a scalpel. Also, cut of a few millimeters of the tapered end of the pipette tip with scissors (compare Figures 1A and 1B).

  3. Mounting of cells to the cryo-stage
    Note: Steps C1-C4 have to be performed quickly to protect the cells from drying out.
    1. Take the cover slide with adhesive tape and cells grown on it out of the 6-well plate
    2. Remove the release liner with fine forceps.
    3. Glue the cover slide to the flow-through chamber so that the cut-out in the middle fits to the cavity of the flow through chamber (Figure 1B).
    4. Fill the flow-through chamber with regular cell culture medium by pipetting through one of the pipet tips attached to the flow-through chamber. Fill the chamber and both pipet tips completely. Avoid air bubbles.
    5. Place the flow through chamber in the cavity of the PVC-insert (Figures 3B-5 and 3B-6).
    6. Connect the silicon tube from the syringe pump to one of the pipette tips of the flow through chamber (Figure 3B-4).
    7. Connect a piece of silicon tube (i.d. 1.5 mm; o.d. 3.5 mm; length ≤ 100 mm) to the other pipet tip and place an empty petri dish below the open end of the silicon tube (Figure 3B-9).
    8. Put a small drop of oil (e.g., immersion oil) on top of the aluminum chamber and mount the cooling head on it using the metal clamp (Figures 3B-2 and 3B-9)
    9. Connect the thermocouple into the drilling hole of the aluminum stage. Start the temperature measurement. The measured temperature should be above 36 °C, if the thermocouple is in reasonably good contact with the aluminum chamber.

  4. Reversible cryo-arrest
    1. Focus and image cells at 37 °C.
      Note: During this step it might be useful to supply the cells with fresh medium with a constant flow of 1 μl sec-1 using the automated syringe.
    2. For cryo-arrest: empty the syringe pump and load it with 10% DMSO solution, using the software control of the syringe pump.
    3. Set the temperature in the cryo-stage to 4 °C, using the control unit for the liquid nitrogen pump (Figure 3A-1).
    4. When the measured temperature in the aluminum chamber is stable, exchange the medium by applying a flow of 3 μl sec-1 for a total of 300 μl using the syringe pump.
    5. Empty the syringe and load it with 20% DMSO solution.
    6. Apply a flow of 3 μl sec-1 for a total of 300 μl using the syringe pump.
    7. Set temperature to -5 °C.
    8. Empty the syringe and load it with 30% DMSO solution.
    9. When the measured temperature in the aluminum chamber is stable, apply a flow of 3 μl sec-1 for a total of 300 μl using the syringe pump.
    10. Set temperature to -10 °C.
    11. Empty the syringe and load it with 50% DMSO solution.
    12. When the measured temperature in the aluminum chamber is stable, apply a flow of 3 μl sec-1 for a total of 600 μl using the syringe pump.
    13. Set temperature to -45 °C.
    14. After the temperature in the aluminum chamber is stable, cells can be imaged in the arrested stage.
    15. To go back to physiological temperature: Set temperature to -10 °C first.
    16. Empty the syringe and load it with 30% DMSO solution.
    17. When the measured temperature in the aluminum chamber is stable, apply a flow of 3 μl sec-1 for a total of 300 μl using the syringe pump.
    18. Set temperature to -5 °C.
    19. Empty the syringe and load it with 20% DMSO solution.
    20. When the measured temperature in the aluminum chamber is stable, apply a flow of 3 μl sec-1 for a total of 300 μl using the syringe pump.
    21. Set temperature to 4 °C.
    22. Empty the syringe and load it with 10% DMSO solution.
    23. Apply a flow of 3 μl sec-1 for a total of 300 μl using the syringe pump.
    24. Empty the syringe and load it with 0% DMSO solution.
    25. Apply a flow of 3 μl sec-1 for a total of 300 μl using the syringe pump.
    26. Set temperature to 37 °C.
    27. At 37 °C cells can be stimulated, e.g., with ligands for RTKs, light, etc.
    28. To arrest cells at a specific time after stimulation, start again with the cryo-arrest cycle. An example of EGFR localized by photoactivated localization microscopy in HeLa cells before and after stimulation with EGF is shown in Figure 5. More examples are shown in the original publication of the method (Masip et al., 2016).


      Figure 5. Photoactivated localization microscopy of EGFR in the same HeLa cell before and after stimulation. Shown are localization maps of a HeLa cell transfected with EGFR-mEos2 that was cryo-arrested before (left image) and 5 min after (right image) stimulation with 200 ng/ml EGF. Under reversible cryo-arrest EGFR molecules were localized by photo-activated localization microscopy with a precision of 21 ± 6 nm (mean ± SD). Scale bars = 5 μm

Data analysis

Reversible cryo-arrest is compatible with almost every fluorescence microscopy technique. Therefore, the data analysis will depend on the microscopy performed under cryo-arrest. So far reversible cryo-arrest has been applied with SMLM and FRET-FLIM. Details about the analysis of this can be found in the freely available authors version of Masip et al., 2016 (Europe PubMed Central plus, Manuscript #69708). In brief, SMLM images may be reconstructed using the ThunderSTORM Plugin (Ovesny, Bioinformatics, 2014) for ImageJ (Rasband, ImageJ, National Institutes of Health, Bethesda, Maryland, USA). For FRET-FLIM data it is recommended to quantify the fraction of fluorescent molecules that undergo FRET in each pixel on the image by global analysis (Grecco et al., 2009).

Notes

  1. In this protocol, we focus on the application of the cryo-arrest to study RTK-activation in adherently growing mammalian cells. In the tested cells, general morphology was preserved. They did not induce expression of stress response genes and growth factor induced signaling proceeded normally after the cryo-arrest (Masip et al., 2016). The protocol is likely adaptable to various types of biological samples and processes. However, when reversible cryo-arrest is established in a new laboratory or a different cellular system is used, it is recommended that controls be performed to ensure that the cryo-arrest does not influence the produced data. At least three different experiments with fluorescently labeled cells (e.g., by transfecting a fluorescent protein such as EGFP) should be performed to judge their morphology. After the cryo-arrest, cells should be labeled with a dye that stains dead cells (e.g., propidium iodide). Dead cells can be quantified by dividing the number of fluorescently labeled cells that are positive for the dead cell marker by the total number of fluorescently labeled cells. Additionally, the expression of cold and heat stress proteins should be tested and the reaction under investigation should be quantified with and without cryo-arrest to exclude influences of the cryo-arrest procedure on the process under investigation (Masip et al., 2016).

Recipes

  1. Different DMSO solutions (10%, 20%, 30% and 50% [vol/vol])
    Different DMSO solutions were prepared by pipetting DMSO and HEPES-buffered medium without phenol red in the corresponding ratios into 15-ml reaction tubes and inverting the tube several times
  2. Cell culture medium (for the cell line indicated in the equipment section)
    Dulbecco’s modified Eagle medium (DMEM) with phenol red supplemented with 10% fetal bovine serum (FBS), 100 μg ml-1 streptomycin plus 100 U ml-1 penicillin, 1% L-glutamine (200 mM) and 1% nonessential amino acids

Acknowledgments

The authors would like to thank Michael Reichl and Petra Glitz for excellent technical support, the workshop of the Max-Planck Institute of Molecular Physiology for construction of custom parts, Astrid Krämer for critical proofreading of the manuscript and Sven Müller for photographic images of the setup. This study was funded by the Fraunhofer Society and the Max-Planck Society for the Promotion of Science in the joint initiative ‘CryoSystems’ and the European Research Council (ERC AdG 322637). This protocol is based on a previously published protocol (Masip et al., 2016).

References

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  2. Dubochet, J. (2012). Cryo-EM-the first thirty years. J microsc 245(3): 221-224.
  3. Dubochet, J., Adrian, M., Chang, J. J., Homo, J. C., Lepault, J., McDowall, A. W. and Schultz, P. (1988). Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21(2): 129-228.
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  5. Farrant, J. (1965). Mechanism of cell damage during freezing and thawing and its prevention. Nature 205(4978): 1284-1287.
  6. Grecco, H. E., Roda-Navarro, P. and Verveer, P. J. (2009). Global analysis of time-correlated single photon counting FRET-FLIM data. Opt Express 17(8): 6493-6508.
  7. Huebinger, J., Han, H. M., Hofnagel, O., Vetter, I. R., Bastiaens, P. I. and Grabenbauer, M. (2016). Direct measurement of water states in cryopreserved cells reveals tolerance toward Ice crystallization. Biophys J 110(4): 840-849.
  8. Kaufmann, R., Hagen, C. and Grünewald, K. (2014). Fluorescence cryo-microscopy: current challenges and prospects. Curr Opin Chem Biol 20: 86-91.
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  10. Offterdinger, M., Georget, V., Girod, A. and Bastiaens, P. I. (2004). Imaging phosphorylation dynamics of the epidermal growth factor receptor. J Biol Chem 279(35): 36972-36981.
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  12. Sabet, O., Stockert, R., Xouri, G., Bruggemann, Y., Stanoev, A. and Bastiaens, P. I. (2015). Ubiquitination switches EphA2 vesicular traffic from a continuous safeguard to a finite signalling mode. Nat Commun 6: 8047.
  13. Saffarian, S., Li, Y., Elson, E. L. and Pike, L. J. (2007). Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis. Biophys J 93(3): 1021-1031.
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简介

通过单分子定位显微镜(SMLM)或荧光寿命成像显微镜(FLIM)的荧光活细胞成像原理上允许在个体,活细胞中的分子模式的时空观察。然而,细胞内分子的动力学阻碍了它们的精确观察。我们在这里介绍一个详细的方案,用于显微镜上活细胞可逆冷冻停滞的连续循环,允许精确测定各个活细胞内分子模式的演变。通过观察受体酪氨酸激酶的配体诱导的聚集以及SMLM和FLIM的活性模式已经证明了该方法的有用性(Masip等人,2016)。

了解细胞中的分子过程,例如受体 - 酪氨酸激酶(RTK)的配体诱导反应需要精确的时空观察分子模式。由于细胞状态的差异,这种反应需要在个体细胞而不是细胞群体中进行监测(Snijder和Pelkmans,2011)。使用SMLM,各个分子可以以高精度进行定位(Betzig等人,2006)。这允许例如提取关于质膜中的RTK聚类的信息。互补地,共焦FLIM可以揭示分子如何在衍射受限体积元素内作为整体反应。这可以通过使用构象传感器揭示RTK与下游分子的相互作用模式,磷酸化模式以及活性模式(Offterdinger等人,2004; Sabet等人)。 ,2015)。但是,FLIM和SMLM的收购时间是几分钟。由于活细胞(包括RTKs)中的许多分子排列以更快的时间尺度演化,所以共焦FLIM图像变得模糊,并且空间分辨率被严重降低(Masip等人,2016)。在SMLM中,分子在连续的帧中连续定位。在活细胞中对动态分子进行这些测量将导致局部化的伪造图像,因为分子在数据采集过程中扩散到不同的位置。例如,在膜的2D环境中,表皮生长因子受体(EGFR)平均移动的扩散常数为2-5×10 -2 -2 / sup 2 / sup / sec (Orr等人,2005; Xiao等人,2008)。为了允许长的采集时间,通常使用与醛类不可逆的化学固定。然而,化学固定剂的使用在固定膜方面是低效的,并且可以导致蛋白质提取或人工聚集(Saffarian等人,2007; Tanaka等人,2010) ; Schnell等人,2012)。此外,致命的固定物破坏失衡的生理状态,并且禁止观察个体细胞内分子模式的演变。因此,我们开发了一种可逆的冷冻抑制措施,可以阻止高分辨率成像的分子运动,同时保持细胞处于可行状态。冷却后,冷冻保护性二甲基亚砜(DMSO)的浓度逐步增加(Masip等人,2016)。这导致冰晶的形成,其可以通过置换有机材料散射光并改变细胞结构而致死细胞(Dubochet等人,1988和2012; Huebinger等人。,2016)。同时,在低温下加入DMSO大大降低了其毒性(Farrant,1965)。低温方法的另一个优点是荧光团在激发态下变得较少反应,导致在漂白之前发射更多的光子以及细胞毒性基团的降低的产生(Kaufmann等人, 2014),其可以在生理温度下获取期间严重损伤细胞(Wäldchen等人,2015)。

关键字:冷冻停滞, 固定, 超分辨率, 单分子定位, FLIM

材料和试剂

  1. 10μm厚双面胶带(D80 19 x 50 mm)(Modulor,目录号:0332054)
  2. 盖板21 x 26 mm(No.1)(Thermo Fisher科学,Thermo Scientific TM ,目录号:BBAD02100260#A *)
  3. 用于细胞培养的6孔板(SARSTEDT,目录号:83.3920)
  4. 15ml反应管(例如,SARSTEDT,目录号:62.554.502)
  5. 200微升移液器吸头(例如,Greiner Bio One International,目录号:739290)
  6. 硅管
  7. 柔性硅管(内径[id] 2mm;外径[od] 4mm和标准1.5mm; od 3mm)(例如,VWR,目录号:228-0704和228- 0702)
  8. 感兴趣的细胞系和合适的细胞培养基
    注意:该方案迄今为止已经测试了附着增长的细胞系HeLa(ATCC,目录号:CCL-2),MDCK(ATCC,目录号:CCL-34),COS-7(ATCC,目录号:CRL-1651),MCF7细胞(ATCC,目录号:HTB-22)和HCT116(ATCC,目录号:CCL-247)。其他细胞系可以在适当测试冷冻阻滞的可逆性之后使用。
  9. 乙醇,绝对(例如,Fisher Scientific,目录号:10342652)
  10. 无菌水(通过过滤灭菌)
  11. DMSO(最小99%)(例如,Serva Electrophoresis,目录号:20385)
  12. 液氮
  13. 浸油(例如,奥林巴斯,目录号:IMMOIL-F30CC)
  14. 表达感兴趣的荧光蛋白的转染试剂和质粒稳定表达感兴趣的荧光蛋白的细胞系。该协议已经成功地用于具有构象活性传感器的共聚焦FLIM,用于EphrinA2和EGFR标记的mCitrine与与mCherry标记的磷酸酪氨酸结合结构域结合。已经用EGFR-mEos2和标记为SNAP-Tag的Vinculin进行单分子定位,并用SNAP细胞TMR-星(New England Biolabs,目录号:S9105S)标记。有关这些质粒的请求可以发送给相应的作者
  15. HEPES或不含酚红的磷酸盐缓冲成像介质(例如,DMEM,PAN-Biotech,目录号:P04-01163)
  16. 10%胎牛血清(FBS)(PAN-Biotech,目录号:P30-1505)
  17. 链霉素/青霉素(PAN-Biotech,目录号:P06-07100)
  18. L-谷氨酰胺(200mM)(PAN-Biotech,目录号:P04-80100)
  19. 1%非必需氨基酸(PAN-Biotech,目录号:P08-32100)
  20. 不同的DMSO溶液(参见食谱)
  21. 细胞培养基(参见食谱)

设备

  1. Scalpel
  2. 层流罩
  3. 孵化器(例如,,Nuaire,型号:NU-5510/E)
  4. 细镊子(例如,电子显微镜科学,目录号:0203-7-PO)
  5. 阳极氧化(黑色)铝流通室(定制;见图1)
    注意:阳极氧化铝减少其腐蚀,从而降低铝离子的溶解度,否则可能会影响细胞反应。黑色阳极氧化可减少光的反射,这可能会影响显微成像。


    图1.流通室的设计。由铝制成的自建式流通室的摄影表示(A,B)和技术图纸(C-E)。 A.流通室的俯视图,其中移液管插头插入入口(1)和钻孔(2;深度:15mm;直径:1mm),用于插入热电偶。 B.底视图,盖玻片胶合到流通室(3); C.流通室的俯视图; D.通过流通室的侧视图部分(C节中的a节:a); E.侧视图部分的详细信息(D中的详细信息b)。 c-e中的所有尺寸均为mm。

  6. 用于显微镜台的聚氯乙烯(PVC)插件(定制;见图2),带有2个螺纹孔,以使用金属夹固定平台(参见图3B)


    图2.用于显微镜平台的PVC插入件的技术图纸。技术图纸显示了适用于Scan IM阶段的安装(MärzhäuserWetzlar GmbH& Co. KG,Wetzlar,Germany)。 A.底视图B.侧视图(a:a in A); C.中央部分的侧视图(B中的b)。所有尺寸均为mm。

  7. 具有计算机接口的低压注射泵(Cetoni,型号:neMESYS低压注射泵)
  8. 具有温度控制的冷冻阶段(Linkam Scientific,型号:MDS600,具有类似冷却头的其他型号也可以工作)
  9. 具有连接到计算机用于连续温度记录的数据采集设备(例如,,具有0.08mm电线的PTFE绝缘型T;欧米茄工程公司,Deckenpfronn,德国)的热电偶(例如, >,OMEGA工程,型号:OMB-DAQ-2408-2AO)
  10. 对于共焦FLIM:配备时间相关单光子计数单元和ps脉冲激光(例如,PicoQuant,型号:MicroTime 200)的共焦激光扫描显微镜
  11. 对于SMLM:配有敏感摄像机(EMCCD或sCMOS)的显微镜以及具有足够波长和功率的激光器进行成像,打开和漂白荧光团
  12. 倒置显微镜(Olympus,型号:IX-81)


    图3.安装在显微镜上的冷冻阶段安装在倒置显微镜(IX-81; Olympus GmbH,Hamburg,Germany)上的低温阶段的照片表示。 A.在显微镜下使用周边仪器进行低温阶段的概述。 1:具有控制单元的液氮泵(注意:这通常与光学平台断开,因为它可能会导致样品的振动); 2:具有不同DMSO浓度的培养基的15ml管,低压注射泵的入口管经由具有孔的盖插入; 3:低压注射泵; 4:热电偶数据采集装置; 5(B)中详细说明的低温阶段的中部; 6:液氮储层。 B.显微镜中央部分的详细图像。 1:管连接氮气泵; 2:将金属夹固定在PVC插件上; 3:热电偶测量银块内的温度; 4:中等入口连接低压注射泵; 5:铝流通室(比较图1); 6:显微镜载物台用PVC插片(比较图2); 7:管连接到氮气池; 8:带电加热器的温控银块; 9:中出口管; 10:测量铝块温度的热电偶。

软件

  1. ThunderSTORM插件(Ovesny,Bioinformatics,2014)
  2. ImageJ(Rasband,ImageJ,National Institutes of Health,Bethesda,Maryland,USA)

程序

  1. 细胞培养
    注意:以下步骤应在层流罩下进行。所有仪器均应先消毒。胶带应仅在层流罩下方从其包装中取出。
    1. 使用手术刀将双面胶带切成26毫米长的片(图4A)。
      注意:使用一层带隔离衬垫的胶带作为支撑。然后,在顶部添加第二层,并将其切成大小。这样便于使用胶粘带,因为它不会粘附到剥离衬垫上。


      图4.具有盖玻片的培养皿的制备 A.将一层双面胶带(1)胶合在光滑表面上(例如,阳极氧化铝板)。将第二层双面胶带胶合在顶部(2)上并切割成盖玻片(左图)的长度。之后,一个矩形的应用程序21 x 5 mm在中心切出并取出(右图)。 B.双面胶带粘贴在封面滑块上(4;左图)。轻轻按压确保紧密接触并防止泄漏(右图)。 C.具有带有双面胶带(箭头)的带盖玻片的6孔细胞培养皿在具有酚红的细胞培养基中的照片表示。双面胶带具有穿过流通室的空腔的切口(箭头;比较图1B)。

    2. 使用手术刀在每片胶带的中间切出一个21 x 5毫米的矩形,并拆下内部矩形(图4A)。
    3. 将胶带粘在21 x 26 mm的盖子上。轻轻按下以确保紧密接触,而不会破坏盖板滑块(图4B)。
    4. 通过将盖子浸在乙醇中并用无菌蒸馏水清洗盖子来消毒。
    5. 将盖板放在6孔板中(图4C)。
    6. 分离培养的细胞并将其置于普通细胞培养基中的6孔培养皿中(参见图4)。胶带不受细胞培养基的影响,并保持粘稠 注意:在加入细胞之前,让盖子用细胞培养基干燥或洗涤,因为蒸馏水对细胞有害。
    7. 至少在37℃/5%CO 2下孵育直到它们牢固地附着。细胞可在此条件下培养数天,并用相关的感兴趣的构建体进行转染
  2. 在显微镜下组装低温阶段
    1. 在成像介质中,在15 ml反应管中制备10%,20%,30%和50%(体积/体积)的DMSO溶液。
      注意:低温注射泵的入口管可以通过盖子上的一个孔插入15ml反应管中(图3A)。
    2. 将银冷却头从低温阶段的外壳中取出。
    3. 使用柔性硅管(i.d. 2 mm; o.d. 4 mm)和电子温度控制将氮气泵,液氮储存器连接到银冷却头(见图3B)。
    4. 使用液氮泵的控制单元将银块的温度设置为37°C(图3A-1)。
    5. 将一块柔性硅管(i.d. 1.5 mm; o.d. 3.5 mm)连接到低压注射泵的出口。
      注意:连接到注射泵的管的体积不应超过200μl。否则,这将增加在每次冷冻逮捕的每一步中需要交换的媒体数量。
    6. 使用自动化注射器的软件,将注射器和管与成像介质无DMSO填充。避免气泡。
      注意:对于第一次灌装,必须在注射器中填充少量培养基,并将其压出其出口,以便在注射器内注入去除注射器和管内的气泡。
    7. 将PVC插入物组装到显微镜台上,将目标置于流通室的开口下方(图3B)。
    8. 从底部通过流通室的入口和出口按200微升移液管吸头。用手术刀清除从底部伸出的部位。此外,用剪刀切割移液管尖端的锥形端的几毫米(比较图1A和1B)。

  3. 将细胞安装到低温阶段
    注意:步骤C1-C4必须快速执行,以保护细胞免受干燥。
    1. 用胶带将盖子滑块和生长在其上的细胞从6孔板上取出
    2. 用精细镊子取下释放衬垫。
    3. 将封盖滑块胶合到流通室,使中间的切口适合流通室的空腔(图1B)。
    4. 通过移液通过连接到流通室的移液管尖端之一来将流通室填充到常规的细胞培养基中。填充房间和两个移液器吸头完全。避免气泡。
    5. 将流通室放置在PVC插件的空腔中(图3B-5和3B-6)。
    6. 将硅管从注射泵连接到流通腔的移液管尖端之一(图3B-4)。
    7. 将一块硅管(i.d. 1.5 mm; o.d. 3.5 mm;长度≤100 mm)连接到另一个吸头,并在硅管开口端下方放置一个空培养皿(图3B-9)。
    8. 在铝室顶部放一小滴油(例如浸油),并使用金属夹将冷却头安装在其上(图3B-2和3B-9) >
    9. 将热电偶连接到铝合金平台的钻孔中。开始温度测量。测量温度应高于36°C,如果热电偶与铝合金室接触良好。

  4. 可逆冷冻
    1. 聚焦和图像细胞在37°C。
      注意:在此步骤过程中,使用自动注射器向细胞提供1μlsec -1 的恒定流量的新鲜培养基。
    2. 对于冷冻停止:使用注射泵的软件控制,将注射器泵清空并用10%DMSO溶液装载。
    3. 将冷冻阶段的温度设置为4°C,使用液氮泵的控制单元(图3A-1)。
    4. 当铝室中的测量温度稳定时,使用注射泵将总共300μl的3μlsec -1 的流量应用。
    5. 将温度设置为-10°C。
    6. 清空注射器并用50%DMSO溶液装载。
    7. 当铝室内的测量温度稳定时,使用注射泵将总共600μl的3μlsec -1的流量应用。
    8. 将温度设置为-45°C。
    9. 铝室温度稳定后,细胞可以在停滞阶段成像。
    10. 回到生理温度:首先将温度设定在-10°C
    11. 清空注射器并用30%DMSO溶液装载。
    12. 当铝室中的测量温度稳定时,使用注射泵将总共300μl的3μlsec -1 的流量应用。
    13. 将温度设置为-5°C。
    14. 清空注射器并用20%DMSO溶液装载。
    15. 当铝室中的测量温度稳定时,使用注射泵将总共300μl的3μlsec -1 的流量应用。
    16. 将温度设置为4°C。
    17. 清空注射器并用10%DMSO溶液装载。
    18. 使用注射泵,使用3μlsec -1 的流量总共为300μl
    19. 清空注射器并用0%DMSO溶液装载。
    20. 使用注射泵,使用3μlsec -1 的流量总共为300μl
    21. 将温度设置为37°C。
    22. 在37℃下,细胞可被刺激,例如与RTK,轻链,等等的配体有关。
    23. 在刺激后的特定时间止血细胞,再次用冷冻停止循环再次开始。在EGF刺激之前和之后,通过光激活定位显微镜在HeLa细胞中定位的EGFR的实例显示在图5中。更多的例子显示在方法的最初出版物(Masip等人,2016)中。 。


      图5.刺激前后相同HeLa细胞中EGFR的光激活定位显微镜。
      显示了在(左图)和5周前冷冻缓冲的EGFR-mEos2转染的HeLa细胞的定位图分钟后(右图)刺激200ng/ml EGF。在可逆冷冻抑制下,EGFR分子通过光激活定位显微镜定位,精确度为21±6nm(平均值±SD)。刻度棒=5μm

数据分析

几乎所有的荧光显微技术都可以兼容冷冻阻滞。因此,数据分析将取决于在冷冻停止下进行的显微镜检查。迄今为止,SMLM和FRET-FLIM已经应用了可逆的冷冻逮捕。关于分析的详细信息可以在免费提供的Masip 等的版本,2016(Europe PubMed Central plus,Manuscript#69708)中找到。简而言之,SMLM图像可以使用ImageJ(Rasband,ImageJ,National Institutes of Health,Bethesda,Maryland,USA)的ThunderSTORM插件(Ovesny,Bioinformatics,2014)来重建。对于FRET-FLIM数据,建议通过全局分析来量化图像中每个像素中经历FRET的荧光分子的分数(Grecco等人,2009)。

笔记

  1. 在本协议中,我们专注于冷冻停滞的应用研究贴壁生长哺乳动物细胞中的RTK激活。在测试细胞中,保留了一般形态。他们没有诱导应激反应基因的表达,并且生长因子诱导的信号通常在冷冻阻滞后进行(Masip等人,2016)。该方案可能适用于各种类型的生物样品和过程。然而,当在新的实验室中建立可逆的冷冻阻滞或使用不同的细胞系统时,建议进行控制以确保冷冻停止不会影响所产生的数据。应进行至少三次具有荧光标记细胞的不同实验(例如,通过转染荧光蛋白如EGFP),以判断其形态。冷冻停止后,细胞应用染色污染细胞(例如,碘化丙啶)的染料标记。死细胞可通过将死细胞标记物阳性的荧光标记细胞数除以荧光标记细胞的总数来量化。此外,应测试冷和热应激蛋白的表达,并且正在进行和不进行冷冻抑制来量化正在研究的反应以排除冷冻停滞程序对所研究的方法的影响(Masip等人, em>。,2016)。

食谱

  1. 不同的DMSO溶液(10%,20%,30%和50%[vol/vol])
    通过将不含酚红的DMSO和HEPES缓冲介质以相应的比例吸移到15-ml反应管中并反转管数次来制备不同的DMSO溶液
  2. 细胞培养基(用于设备部分所示的细胞系)
    含有补充有10%胎牛血清(FBS),100μg/ml链霉素加100μg/ml青霉素的酚红的Dulbecco改良Eagle培养基(DMEM),1 %L-谷氨酰胺(200mM)和1%非必需氨基酸

致谢

作者要感谢Michael Reichl和Petra Glitz的优秀技术支持,Max-Planck分子生理学研究所的研讨会,用于构建定制零件,AstridKrämer对原稿的严格校对和SvenMüller的设置摄影图像。这项研究由弗劳恩霍夫协会和马克斯普朗克科学促进会共同倡议"冷冻系统"和欧洲研究委员会(ERC AdG 322637)资助。该协议基于以前发布的协议(Masip 等人,2016)。

参考文献

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Huebinger, J., Masip, M. E., Christmann, J., Wehner, F. and Bastiaens, P. I. (2017). Reversible Cryo-arrests of Living Cells to Pause Molecular Movements for High-resolution Imaging. Bio-protocol 7(8): e2236. DOI: 10.21769/BioProtoc.2236.
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