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Jul 2018

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Super-resolution Microscopy at Cryogenic Temperatures Using Solid Immersion Lenses
固体浸没透镜应用于低温条件下的超分辨率显微镜   

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Abstract

Our mechanistic understanding of cell function depends on imaging biological processes in cells with molecular resolution. Super-resolution fluorescence microscopy plays a crucial role by reporting cellular ultrastructure with 20-30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions, which substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixatives. Cryogenic conditions are, however, underutilized because of the lack of compatible high numerical aperture (NA) objectives. Here we describe a protocol for the use of super-hemispherical solid immersion lenses (superSILs) to achieve super-resolution imaging at cryogenic temperatures with an effective NA of 2.17 and resolution of ~10 nm.

Keywords: Super-resolution microscopy (超高分辨显微镜), Cryogenic microscopy (低温显微镜), Solid immersion lens (固体浸没透镜), Biological imaging (生物成像), Sample vitrification (样品玻璃化), Fluorescence microscopy (荧光显微镜)

Background

Fluorescence microscopy has for many years been one of the most important tools for understanding how biological systems are organized and how they function. Over the last decade, fluorescence microscopy has been revolutionized by the development of “super-resolution” methods that extend the limit of optical microscopy beyond the diffraction limit, providing unprecedented levels of information on the organization of molecular networks in cells. These methods include Structured Illumination Microscopy (SIM) (Gustafsson, 2000), Stimulated Emission Depletion Microscopy (STED) (Hell and Wichmann, 1994), and techniques based on the localization of individual fluorescent molecules with much better precision than the diffraction limit. The latter includes Photo-activated Localization Microscopy (PALM) (Betzig et al., 2006) and Stochastic Optical Reconstruction Microscopy (STORM) (Rust et al., 2006), referred to collectively in this protocol as Single Molecule Localization Microscopy (SMLM) techniques. Because of a requirement to stop motion within the cell when aiming for high resolution, super-resolution methods usually involve the use of chemical fixation, which has the potential to introduce artefacts that can be visible at the resolutions achieved (Whelan and Bell, 2015). This problem has long been recognized in the field of electron microscopy, where a different method is now routinely used to fix cells and preserve their structure: vitrification at cryogenic temperatures. In this method, samples are frozen using a cryogen, cooling rapidly to temperatures of -150 °C or below, resulting in the formation of amorphous ice, with the high rate of cooling preventing the formation of ice crystals (Dubochet and McDowall, 1981). SMLM techniques in particular should benefit from the use of cryogenic conditions. This is because the localization precision of fluorescent molecules is dependent on the number of photons emitted by the fluorophores (Thompson et al., 2002), and at cryogenic temperatures significantly more photons can be collected. Conventional widefield fluorescence microscopy at cryogenic temperatures is already used in Correlative Light and Electron Microscopy (CLEM) (de Boer et al., 2015) workflows, but the mismatch in resolution between optical microscopy and EM limits the value of the fluorescence data, which is usually restricted to locating general areas of interest. Hence, enabling cryogenic super-resolution fluorescence microscopy would be a significant advance for cryogenic CLEM workflows.

Given the advantages of vitrification over chemical fixation, it would be advantageous to apply it to super-resolution optical microscopy methods. However, its use has been severely limited for one major reason: super-resolution methods require the use of high numerical aperture (NA) objective lenses (NA > 1), and achieving this NA typically requires the use of oil immersion objectives, incompatible with cryogenic microscopy. A number of approaches have been taken to avoid this problem, including using non-immersion objectives with extremely bright fluorophores (Liu et al., 2015) and using special cryogenic immersion fluids and custom sample stages (Nahmani et al., 2017). However, these approaches have not been generally applied to the imaging of biological samples.

We have developed a low-cost, generally applicable method for super-resolution cryo-fluorescence microscopy that uses commercially available optical components and cryo-stage (Wang et al., 2019). For this, we use super-hemispherical solid-immersion lenses (superSILs). These lenses are made of high refractive index materials, and take the form of truncated spheres. In our application, their role is to take the place of immersion oil, and fill the gap between the sample and a conventional non-immersion objective. The combination of superSIL and air objective gives a high numerical aperture, dependent on the refractive index of the lens material (Terris et al., 1994; Zhang et al., 2007; Chen et al., 2013).

We have characterized the performance of superSILs for cryo-SMLM, showing that we are able to achieve a point spread function (PSF) size of around 150 nm, compared with around 330 nm for a cryogenic microscope with a conventional low NA air objective. This is because the combination of superSIL and objective achieves a NA of 2.17, much higher than the maximum NA of ~1.4 of oil-immersion objectives. The superSIL microscope achieves single molecule localization precisions of < 10 nm, and a lateral resolution of 12 nm (Wang et al., 2019). A major advantage of the superSIL method is that it can be achieved using readily available off-the-shelf components, with the possibility of easily adding the cryostage to microscopes commonly found in cell biology research laboratories. Moreover, superSIL assemblies can be cleaned and reused multiple times, making them an inexpensive resource. In this protocol, we focus on use of the superSIL system for super-resolution microscopy of bacteria, but also indicate how it might be applied to imaging of mammalian cells. A similar protocol could also be used to image other samples, such as purified protein complexes or vesicles. Used as described, the protocol provides a straightforward method for super-resolution imaging of biological samples at cryogenic temperatures, with the advantage of avoiding potential fixation artefacts. The resolution and localization precision achieved are significantly better than those routinely obtained in conventional room temperature SMLM microscopes. These advantages alone should make superSIL SMLM microscopy a valuable addition to the toolbox of cell biology researchers.

It is also possible to consider other potential advantages of the superSIL microscopy protocol. Firstly, because of the ultra-high NA, the resolution that can be achieved even at room temperature is significantly better than that obtained in conventional SMLM microscopes. The superSIL protocol therefore provides a simple low-cost method by which a basic fluorescence microscope could be converted to a high-performance super-resolution system. Our protocol describes use of the superSIL in an upright microscope, but the method could also be used in an inverted microscope by using a modified version of the superSIL assembly.

We believe that superSIL microscopy could have an important role to play in CLEM, helping to bridge the resolution gap between cryo-fluorescence microscopy and EM. We do not describe any CLEM approaches in this protocol, but possible methodologies could involve the transferring and imaging of thin lamellae produced by focused ion beam (FIB) milling under both superSIL and transmission EM modes, or the correlation of superSIL SMLM images with serial block-face scanning EM. Given the small size of the lens assembly, it may also be possible to incorporate a superSIL into the stage of an electron microscope, potentially permitting simultaneous or near-simultaneous imaging of the same sample with both light and electrons.

Materials and Reagents

  1. superSIL assembly
    1. Hyper Hemispheric Ball lens, Cubic Zirconia, 0.89 mm diameter x 0.73 mm thickness, λ\4 flatness (Knight Optical, LBB2018-C)
    2. Platinum Foil (Goodfellow, catalog number: PT000264 876-861-73)
    3. Cryo-compatible adhesive (Loctite Stycast, 2850 FT)

  2. Preparation of bacterial and fiducial solutions
    1. Flat cap centrifuge tubes, 50 ml (Fisherbrand, catalog number: 05-539-13)
    2. Eppendorf tubes, 2 ml (Eppendorf, catalog number: 0030 123.344)
    3. Tetraspeck 0.1 μm blue/green/orange/dark red beads (Molecular probes, Thermo Fisher Scientific, catalog number: T7279)
    4. Agar plate(s) with colonies of E.Coli expressing plasmid of interest
    5. LB medium granular (e.g., Melford, L24400-5000.0)
    6. 1 M HCl solution in water for pH adjustment (made up from HCl 37% ACS grade, 320331, Sigma-Aldrich)
    7. Selection antibiotics stock solutions in water (e.g., Kanamycin 25 mg/ml from powder-Sigma-Aldrich, K1377; Carbenicillin 100 mg/ml ready-made solution, Sigma-Aldrich, C1613)
    8. IPTG 1 M stock solution in water (made up from powder e.g., Fluorochem, M02726)
    9. PBS 1x pH 7.4 (Gibco, catalog number: 10010-015)
    10. Ultrapure deionized water (MilliQ or equivalent)
    11. LB medium (see Recipes)

  3. Cleaning of superSIL assemblies
    1. Clean glass slides (e.g., Fischer Scientific 16 x 38 mm microscope slides 244879)
    2. Filter paper (e.g., qualitative filter paper grade A, SLS 2036)
    3. Clean glass scintillation vials (e.g., Thermo Chromacol 20 ml vials 20-EPSVCA)
    4. superSIL assemblies (produced as described in Procedure A), cubic zirconia crystal NA 2.17
    5. Sulphuric acid ≥ 95% pure (e.g., Fischer Scientific S/9231/PB15)
    6. 30% Hydrogen Peroxide with inhibitor, ACS grade (e.g., Sigma-Aldrich, catalog number: 216763)
    7. At least 100 ml of MilliQ (or equivalent) water
    8. Piranha Solution (see Recipes)

  4. Plunge-freezing
    1. Ethane gas bottle
    2. Cleaned and glow discharged superSILs (prepared as described in Procedure D)
    3. Bacterial suspension (prepared as described in Procedure B)
    4. Fiducial suspension (prepared as described in Procedure C)
    5. Liquid nitrogen (at least a full 4 L dewar)
    6. 70% ethanol
    7. MilliQ water

Equipment

  1. superSIL assembly and microscope
    1. Ceramic hot plate (VWR, catalog number: 444-0624)
    2. Coordinate measuring machine (OGP, SmartScope ZIP 250)

    Microscope stand
    1. Cerna Microscope Body with Epi-Illumination Arm (Thorlabs, catalog number: CEA1350)
    2. Nikon D-FL Epi-Fluorescence Illuminator (Thorlabs, catalog number: CSE1000)
    3. Microscope cube assembly for Nikon TE2000 (Thorlabs, catalog number: TLV-TE2000)
    4. Single objective arm (Thorlabs, catalog number: CSA1100)
    5. Objective focusing module (Thorlabs, catalog number: ZFM2020)
    6. 3-axis controller and knob box for 1" Cerna stages (Thorlabs, catalog number: MCM3001)
    7. TIRF module comprising of X/Y translator and right-angle mirror mount (Thorlabs, ST1XY-D and KCB1/M)
    8. Translation stage (Prior, H117P2IX/G)
    9. Translation stage controller (Prior, ProScan III H31XYZEF)
    10. Image splitting module (Cairn, Twincam)

    Optics, sample stage, and cameras
    The following components are used in the microscope schematic illustrated in Figure 1. In principle, most popular commercial microscope stands could be modified to create a similar configuration since various adapter plates for the cryo-stage are available from Linkam Scientific Ltd and the Cairn Twincam can be purchased with c-mount threads on the input and output ports to suit most types of scientific camera.
    Note: Check the emission spectra of your fluorophore or dye of choice at cryogenic temperature and select dichroic and emission filters to suit. The chromatic aberration between channels can be compensated for by changing the strength of lens l6 and axial translation of sCMOS camera 2.
    1. l1: f = 30 mm achromatic doublet lens (Thorlabs, catalog number: AC254-030-A-ML)
    2. l2: f = 150 mm achromatic doublet lens (Thorlabs, catalog number: AC254-150-A-ML)
    3. l3: f = 100 mm achromatic doublet lens (Thorlabs, catalog number: AC254-100-A-ML)
    4. l4: f = 150 mm achromatic doublet lens (Thorlabs, catalog number: AC254-150-A-ML)
    5. l5: f = 200 mm 1x camera tube lens (Thorlabs, catalog number: WFA4100 )
    6. l6: f = 500 mm B-BK7 plano-convex lens (Thorlabs, catalog number: LA1908)
    7. l7: 50x, 0.75 NA Microscope objective lens (Mitutoyo, M Plan Apo HR 378-814-4)
    8. p1: 30 μm pinhole (Thorlabs, P30D)
    9. t1: Top-hat beam shaping optic (TOPAG Lasertechnik GmbH, GTH-5-250-4-VIS)
    10. d1: Dichroic beam splitter (Semrock, Di-R405/488/561/635)
    11. d2: Dichroic beam splitter (Chroma, t565lpxr)
    12. f1: Quad-band emission filter (Semrock, FF01-446/523/600/677-25)
    13. f2: Green channel band-pass filter (Semrock, catalog number: FF03-525/50-25)
    14. f3: Red channel band-pass filter (Semrock, catalog number: FF01-593/40-25)
    15. q1: Achromatic quarter wave-plate (Thorlabs, AHWP10M-600)
    16. m1: Broadband dielectric mirror (Thorlabs, BB1-E02)
    17. Omicron LightHUB-6 containing four laser modules
      1. 405 nm 60 mW Phoxx diode laser
      2. 488 nm 200 mW Phoxx diode laser
      3. 561 nm 150 mW Cobolt Jive DPSS laser
      4. 642 nm 140 mW Phoxx diode laser
    18. Two back-illuminated sCMOS cameras with 18.7 mm chip sensor size (Teledyne Photometrics Ltd, 18.7 mm Prime 95B)
    19. Cryo-stage with autofill Dewar (Linkam, CMS196M and AUTOFILL)


      Figure 1. Microscope schematic detailing optics layout

  2. Preparation of bacterial and fiducial solutions
    1. Swinging bucket centrifuge (e.g., Beckman Coulter Allegra X-15R centrifuge)
    2. P1000, P100 and P10 pipettes and tips
    3. Autoclave (e.g., Prestige Medical Classic tabletop autoclave)
    4. Spectrophotometer (e.g., Amersham Biosciences Ultrospec 10 cell density meter)
    5. MSC cabinet (e.g., Scanlaf Mars Safety Class 2)
    6. Temperature-controlled, agitating incubator (e.g., New Brunswick Scientific Innova 44R)
    7. Vortex (e.g., IKA MS2 minishaker)
    8. Immersion probe pH meter (e.g., Hanna PH21)

  3. Cleaning of superSIL assemblies
    1. Chemical fume hood
    2. P1000 pipette and tips
    3. Glow discharger (e.g., Quorum GloQube)
    4. Inverted tweezers (e.g., Dumont N1)
    5. Timer

  4. Plunge-freezing
    1. FEI Vitrobot or equivalent plunge-freezer
    2. Flammable gas regulator (e.g., Air Liquide HBD-240-4-2.S)
    3. Gas leak detector (e.g., Everbuild Plumber’s P18 Gas Leak Detector)
    4. Inverted tweezers (e.g., Dumont N1)
    5. Curved tweezers (e.g., Dumont 7, 0130-7-PO)
    6. Blotting paper (e.g., Agar round filter paper for Vitrobot, 47000-100)
    7. P10 pipette and tips
    8. 1 L Thermos flask
    9. Funnel
    10. Tissue paper
    11. Grid boxes (e.g., SwissCI cryo-EM grid box MD16-104, Molecular Dimensions Limited)
    12. A 50 ml centrifuge tube attached to a piece of synthetic, non-absorbent twine
    13. Transport cryodewar (e.g., Statebourne Cryogenics OD-1)
    14. Handheld liquid nitrogen dewar (e.g., Worthington LD4)
    15. Storage cryodewar (e.g., Worthington VHC35)
    16. Hot plate or hair dryer
    17. Adequate personal protective equipment (PPE) for work with cryogenics (refer to local health and safety regulations at your facility)
    18. Optional: PFTE thread seal tape (e.g., Buffalo 12 m x 12 mm x 0.075 mm BS7786: 2006 Grade L)
    19. Optional: Local Exhaust Ventilation (LEV) inlet
    20. Optional: orientable tabletop lamp
    21. FEI Vitrobot or equivalent humidity controller

Software

  1. Micro-Manager v1.4.23 20161114 (Edelstein et al., 2014)
  2. Omicron Control Centre v2.1.4
  3. ImageJ version v1.52h (Schindelin et al., 2015)
  4. ThunderSTORM v1.3-2014-11-08 plugin (Ovesný et al., 2014)

Procedure

  1. Manufacturing superSIL assemblies
    Note: This step assumes the use of a mechanical workshop equipped with a laser cutter and drying oven.


    Figure 2. superSIL assembly manufacturing process. A. A 3 mm diameter platinum disc with a 1 mm diameter hole laser-cut in the center. B. the assembled disc with superSIL glued in place. Scale bar: 1 mm.

    1. Cut a 3 mm diameter circle from the platinum foil using the laser cutter. Drill a 1 mm diameter hole in the center (as shown in Figure 2A).
    2. Glue the superSIL into the hole using the cryo-compatible adhesive (as shown in Figure 2B).
    3. Cure the assembly in the oven for 24 h at 45 °C.
    4. Measure the angle between the flat surface of the superSIL and the flat surface of the platinum disk using the coordinate measuring machine. A flatness of < 1° is essential to minimize imaging aberrations.

  2. Preparation of bacterial suspensions
    Note: Preserve sterility of your cultures by performing all operations until Step B6 under an MSC or next to a Bunsen burner.
    The procedure assumes that the selected bacterial strain is expressing a suitable fluorescent protein for SMLM. We have imaged successfully using Enhanced GFP (EGFP) (Wang et al., 2019) and there have been reports of cryo-SMLM using monomeric EGFP and mVenus (Kaufmann et al., 2014).
    1. Inoculate one colony of the bacterial strain of interest in 10 ml of LB agar.
    2. Add selecting antibiotics depending on selection markers present on plasmid of interest.
    3. Grow at 37 °C 220 rpm for 3 h, then check O.D. 660 on a spectrophotometer, using LB medium (Recipe 1) as a blank.
    4. If cultures have reached O.D. ~0.6, induce with IPTG 0.2-1 mM (exact amounts will have to be optimized in a case-by-case basis) and culture for 18 h at 25 °C.
    5. Keeping the culture sterile, take 2 ml of culture and transfer them to a 2 ml Eppendorf.
    6. Centrifuge at 3,000 x g 10 min, discard supernatant.
    7. Add 2 ml PBS, resuspend and centrifuge at 3,000 x g 10 min, then discard supernatant.
    8. Resuspend in 100 μl PBS 1x, keep on ice.

  3. Preparation of fiducial suspensions
    1. Vortex Tetraspeck vial at maximum setting for at least 10 s.
    2. Dilute Tetraspeck 1:10 in MilliQ (or equivalent purity) water.
    3. Vortex diluted bead suspension at maximum setting for at least 10 s.
    4. Keep on ice.

  4. Cleaning of superSIL assemblies
    This procedure should be used to clean superSILs after each use, before applying a new sample. This makes superSIL assemblies re-usable for a few cycles. Cleaning degrades the glue that attaches the lens to the platinum disk. When the superSIL becomes loose it can be re-attached as described in Procedure A.

    Important safety advice
    1. Piranha solution is highly acidic, oxidizing and exothermic. Do not make more than you need, do not cap the vial where you are making it, and do not store. Always use appropriate PPE. Prepare and use Piranha exclusively under a fume hood.
    2. DO NOT mix with organic solvents as this is likely to cause an explosion.
    3. Do not use hydrogen peroxide at concentrations higher than 30% or this may cause an explosion.
    4. After use, dilute leftovers with plenty of MilliQ water and leave it to outgas and degrade in a fume hood overnight, and then dispose down the drain with plenty of cold water.
    5. This procedure is visually demonstrated in Video 1.

      Video 1. SuperSIL cleaning process

    1. Prepare Piranha solution (Recipe 2) as described in Recipes.
    2. Spot ~100 μl of Piranha solution of a clean glass slide. Prepare a spot for each superSIL to be cleaned.
    3. Using a pair of inverted tweezers, place each superSIL on a spot of Piranha solution, flat face down.
    4. Incubate for 7 min at RT.
    5. Using a pair of inverted tweezers, rinse each superSIL thoroughly by dunking it in a vial of MilliQ water and blotting it on clean filter paper. Repeat the dunking and blotting at least three times.
    6. Repeat Step D5 once more using a clean vial of MilliQ water.
    7. Leave superSILs to dry on a piece of clean filter paper at least 1 h.
    8. Glow discharge superSILs flat face up for 120 s at 40 mA in a glow discharger.

  5. Plunge-freezing
    Important safety notice
    1. This step of the protocol entails works with cryogenics and with flammable gases, which can cause cryogenic burns, explosions and suffocation by oxygen displacement. Always wear appropriate PPE and make sure you are working in an area with adequate ventilation and oxygen depletion alarms. Always refer to the health and safety guidance on working with cryogenics and with flammable gases available at your facility. Obtain adequate training and/or supervision before commencing work.
    2. Procedures will be described assuming the use of a Vitrobot plunge freezer (FEI). Please refer to manufacturers’ instructions for how to translate these for use with equivalent equipment from other manufacturers.


      Figure 3. The cryo-bath of the Vitrobot plunge-freezer

    This procedure is visually demonstrated in Video 2.
    1. Turn on the Vitrobot at the main switch and install the humidity controller.
    2. Set humidity at 70% and temperature at 22 °C. Wait until they equilibrate (usually 25-30 min).
    3. In the options menu, set blot number to zero and allow the use of footpedal.
      Note: Automated blotting needs to be disabled to preserve the structural integrity of the superSILs.
    4. Fill the outer chamber of the cryo-bath with approximately 1 L of liquid nitrogen. Make sure the “spider” is in place (as shown in Figure 3).
      Note: Do not fill straight from the handheld dewar, rather use it to fill a smaller thermos flask. Use the funnel and tissue paper to filter out macroscopic ice crystals that might contaminate your sample. 
    5. Wait 10-15 min for the inner chamber to cool down and place grid boxes in the dedicated slots (max 4 boxes, so max 16 samples at any one time).
    6. Fit the regulator on the ethane bottle and check for leaks using the leak detector spray.
      Note: If leaks are detected, close all valves and restart. Use PVDF tape to wrap regulator thread if necessary. Abide by local safety regulations regarding the use of flammable gases.

      Video 2. SuperSIL plunge freezing process

    7. Slowly dispense the ethane into the inner cup and wait until it is almost full to remove the spider. Ethane should be in liquid state for plunge freezing. If necessary, melt solidified ethane by inserting a warm metal instrument into the ethane inner cup.
      Notes: 
      1. Orienting a local exhaust ventilation (LEV) head close to the cryo-bath is quite helpful in clearing cold ethane vapor and gauging levels more accurately.
      2. he spider needs to be removed for the following passages. Failing to do so might cause significant damage to the machine.
    8. Take up a superSIL with the Vitrobot-compatible tweezers, making sure that the flat face is facing the side of your dominant/favorite hand and load it on the shaft.
      Note: Make sure the tweezers are not proud of the shaft on either side as this can cause serious damage to the machine as the shaft is retracted.
    9. Press pedal to retract tweezers into waiting position.
    10. Place cryo-bath on the pedestal and press pedal to lift.
    11. Once cryo-bath has finished moving, press pedal to lower the tweezers into sample loading position.
    12. Apply 2.5 μl of bacterial suspension to the flat side of the superSIL through the side port.
    13. Open the front door and blot away most liquid using a piece of filter paper.
    14. Repeat sample application and blotting.
    15. Apply 2.5 μl of fiducial solution to the flat side of the superSIL through the side port.
    16. Repeat Step E13.
    17. Press pedal to start plunging sequence.
    18. Once the shaft and the cryo-bath have reached the end of the course, top up liquid nitrogen in the outer chamber, unload the tweezers from the shaft and move the superSIL from the ethane cup into the nitrogen bath.
      Notes:
      1. Extra light from an orientable tabletop lamp is quite helpful at this stage.
      2. Take the tweezers out of the ethane slowly to prevent liquid ethane from sticking to the superSIL surface, then transfer quickly in liquid nitrogen.
    19. Carefully release the catch on the tweezers and depose the superSIL in one of the slots of the grid box.
    20. Warm and dry all instruments using the heated plate or the hair dryer.
      Note: After each sample, wash the Vitrobot tweezers with 70% ethanol and let dry to prevent cross-contamination of the superSILs.
    21. Once all samples have been plunged, fill the transport dewar with liquid nitrogen and allow to settle.
    22. Cool down the tube on a string inside the transport dewar.
    23. Using cold tweezers, quickly transfer the grid boxes from the outer nitrogen bath of the Vitrobot cryo-bath into the tube.
    24. Store samples in a storage dewar filled with liquid nitrogen until ready to image (and no longer than a month).

  6. Sample transfer to the cryo-stage


    Figure 4. Linkam cryo-stage layout with lid removed

    1. Switch on the Linkam CMS196M cryo-stage and perform a bake-out cycle.
    2. Fill the Linkam Autofill Dewar with liquid nitrogen and couple to the CMS196M stage. Initiate cooling of the cryo-stage.
    3. Once the temperature inside the cryo-chamber has equilibrated to -196 °C, insert the Linkam sample transfer puck into one of the empty receptacles inside the cryo-chamber and allow to cool. Insert a blank sample cassette (as shown in Figure 4).
    4. Transfer a storage puck containing your superSIL samples into the remaining receptacle inside the cryo-chamber.
    5. Transfer the superSIL assemblies from the storage puck to the sample cassette using tweezers, paying close attention to the orientation; the curved surface of the superSIL needs to point towards the microscope objective once mounted.
    6. Once the superSILs have been mounted, close the cassette and transfer it to the brass bridge using the magnetic transfer tool. Remove the storage and transfer pucks from the cryo-chamber to minimize vibration during imaging.
    Note: It is essential to minimize the likelihood of ice contamination and de-vitrification of your sample during the sample transfer process. Ice crystals may form in humid environments, as a result of condensation from breath and from tools that have not been pre-dried using a heater plate.

  7. Finding focus


    Figure 5. Finding focus under bright field illumination. The top of the superSIL curved surface is shown focused in (A), viewed under the 50x, 0.75 NA objective lens. Viewing through the superSIL, characteristic scratches on the flat surface should become visible under bright field illumination, shown in (B), indicating that the sample plane is in focus. Scale bar: 10 µm.

    1. Start Micro-Manager and bring up a live view of the camera(s).
    2. Switch on the bright field condenser LED built-in to the Linkam CMS196M.
    3. Lower the microscope objective in to the Linkam chamber towards the superSIL. Monitor the green channel camera output. Pay close attention to the distance between the objective and superSIL; a collision will likely destroy the Linkam brass bridge leading to large repair costs and delays.
      Note: Light from the built-in LED condenser is unlikely to appear in the red channel.
    4. As the objective approaches the superSIL, you will notice the brightness of the green channel increase. Adjust the microscope stage laterally to ensure the brightest area remains in the center of the camera’s field of view as you lower the objective further.
    5. The curved surface of the superSIL will appear in focus. Translate the microscope stage laterally so the very top of the curved surface is in the center of the camera’s field of view (Figure 5A) and the periphery blurs out of focus.
    6. Now lower the objective approximately 1 mm to find the virtual imaging plane that is located below the flat surface of the superSIL. You will notice inherent scratches in the flat surface of the superSIL appearing into focus first (Figure 5B). If the ice that has been produced in the plunge-freezing procedure is relatively thin, the plane at which the sample is located should be very close to this flat surface of the superSIL.

  8. Imaging
    Note: You have the option to store the collected images in memory or to stream them straight to hard disk. sCMOS cameras produce large quantities of data so make sure you have enough available space before continuing otherwise the system may become unresponsive and crash. A typical dual-color STORM experiment comprising of 10,000 full-frames per channel consumes approximately 10 GB of data. Computer memory equal to or greater than 32 GB and a solid-state hard drive of capacity equal to or greater than 1 TB is recommended as a minimum requirement.
    1. Take an optional bright field image snapshot. Switch off the LED condenser.
    2. Switch on the required lasers once they have warmed up sufficiently.
    3. Open the laser shutter on the microscope to illuminate your sample.
    4. Fine-tune the objective height until the fiducial markers are in focus.
    5. Optional: Adjust the micrometer on the TIRF module to increase the contrast from your sample.
    6. Start with a low laser power density (~50 W/cm2). Wait for the sample to photo-bleach sufficiently until you see clear well-separated single molecules blinking. Increase the laser power density if necessary until you observe the desired blinking characteristics for your fluorophore of choice. The time that the molecule spends in its emitting bright state should be as low as possible compared to the time spent in the dark state.
    7. Adjust the camera exposure time and acquire an appropriate number of frames for your experiment based on your fluorophore of choice and sample conditions. For E. Coli containing eGFP, we chose to acquire 10,000 frames with a 50 ms exposure time.
    8. Save the acquired frames as 16-bit unsigned integer TIF stacks.
    9. Reverse the “Finding Focus” steps to safely remove your samples from the cryo-microscope stage.

Data analysis

  1. Single molecule localization
    1. Open ImageJ and load a TIF stack of interest.
    2. Run the ThunderSTORM plugin. Verify your camera settings are correct under “Camera setup” and you have entered appropriate fitting and sub-pixel localization settings based on your theoretical point spread function size (Figure 6).
    3. Select a desired method for visualizing the reconstructed image.
    4. Click “Preview” to see how the analysis handles the current frame of data and verify that it detects all of the visible single molecules.
    5. Click “OK” to run the analysis for the entire dataset.
    6. Once the analysis has completed, you will notice a table of results appears on the screen alongside a reconstructed image. Save the table of results as a .csv file using the “Export” feature as this can be used to re-generate the reconstructed image at a later time.


      Figure 6. Typical ThunderSTORM settings based on the effective point spread function and pixel size at the imaging plane of the superSIL

  2. Drift correction
    1. You can apply drift correction to your dataset if fiducial markers are present and in focus. Click the “Drift Correction” tab and load the sub-menu by clicking on the double arrow icon. We have found that setting a maximum distance of 500 nm, minimum marker ratio of 0.9 and trajectory smoothing factor of 0.001 under the drift correction options produce satisfactory results given the nature of the drift in our particular cryo-stage.
    2. Once the drift correction has been calculated and applied, a new reconstruction appears reflecting the changes. Zoom in to inspect the fiducial markers using the magnifier tool from the ImageJ toolbar; if there is still a visible drift then you may need to adjust the variables discussed in the previous step.
    3. Save the revised results table as a .csv file using the “Export” function.

  3. Filtering
    Note: The fitting algorithms within ThunderSTORM can be used to remove unwanted localization artifacts arising from sources such as ice crystals, aggregated fiducials, sample auto-fluorescence or scatter.
    1. On the “Filter” tab, you can define your desired filters using Boolean algebra expressions.
    2. For example, if your system has a theoretical point spread function sigma of 125 nm, you could choose to include only those localizations with a sigma in the range 100-150 nm with the expression “sigma > 100 & sigma < 150”.
    3. Your sample of interest may have an intensity range of 100-1000 ADU counts with the fiducial markers having an intensity over 6000 ADU counts. In this scenario, you can exclude the fiducial markers by using expression “intensity < 6000”.

  4. Visualization and measurements
    1. It is possible to apply color look up tables (LUTs) to your reconstructed image by selecting “File > Import > LUT…”.
    2. The pixel size needs to be correctly defined in order to make measurements in your reconstructed image. This can be verified by selecting the reconstructed image window and navigating to “Image > Properties”. The pixel size should be set to that of the camera divided by the final magnification used in the visualization settings of ThunderSTORM. For instance, if you have a pixel size of 46.8 nm and chose to have a 5x final magnification, then your reconstructed pixel size is 9.36 nm.
    3. You can now measure approximate distances between features using the ruler tool located on the main ImageJ toolbar.

  5. Advanced data analysis
    The .csv data table generated by ThunderSTORM can be imported into other software packages for advanced data analysis, including single-molecule clustering, stoichiometry and co-localization between channels.

Recipes

  1. LB medium
    1. Weigh out 25 g of granules
    2. Add 1 L of MilliQ water
    3. Adjust pH to 7.2 using HCl
    4. Autoclave to sterilize
  2. Piranha Solution
    1. Add 900 μl of concentrated sulphuric acid to a glass scintillation vial
    2. Add 300 μl of 30% hydrogen peroxide to the vial and quickly mix
      Note: The vial will heat up considerably.
    3. Use immediately. Do not cap the vial

    Important safety advice
    1. Piranha solution is highly acidic, oxidizing and exothermic. Do not make more than you need, do not cap the vial where you are making it, and do not store. Always use appropriate PPE. Prepare and use Piranha exclusively under a fume hood.
    2. Do not mix with organic solvents as this is likely to cause an explosion.
    3. Do not use hydrogen peroxide at concentrations higher than 30% or this may cause an explosion.
    4. After use, dilute leftovers with plenty of MilliQ water and leave it to outgas and degrade in a fume hood overnight, and then dispose down the drain with plenty of cold water.
    5. This recipe makes enough Piranha solution to clean 20+ superSILs with the method described above. Note that the hazard associated with preparation of Piranha solution increases if larger volumes are made. For volumes larger than 5 ml, the solution should always be prepared on ice to minimize heating.

Acknowledgments

This work has been funded by Medical Research Council grant MR/K015591/1 to MMF. The protocol is based on work published in Wang et al. (2019).

Competing interests

The authors declare no competing interests.

References

  1. Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, O. W., Olenych, S., Bonifacino, J. S., Davidson, M. W., Lippincott-Schwartz, J. and Hess, H. F. (2006). Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793): 1642-1645.
  2. Chen, R., Agarwal, K., Sheppard, C. J., Phang, J. C. and Chen, X. (2013). A complete and computationally efficient numerical model of aplanatic solid immersion lens scanning microscope. Opt Express 21(12): 14316-14330.
  3. de Boer, P., Hoogenboom, J. P. and Giepmans, B. N. (2015). Correlated light and electron microscopy: ultrastructure lights up! Nat Methods 12(6): 503-513.
  4. Dubochet, J. and McDowall, A. W. (1981). Vitrification of pure water for electron-microscopy. J Microsc-Oxford 124(3): Rp3-Rp4.
  5. Edelstein, A. D., Tsuchida, M. A., Amodaj, N., Pinkard, H., Vale, R. D. and Stuurman, N. (2014). Advanced methods of microscope control using muManager software. J Biol Methods 1(2).
  6. Gustafsson, M. G. (2000). Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198(Pt 2): 82-87.
  7. Hell, S. W. and Wichmann, J. (1994). Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19(11): 780-782.
  8. Kaufmann, R., Schellenberger, P., Seiradake, E., Dobbie, I. M., Jones, E. Y., Davis, I., Hagen, C. and Grünewald, K. (2014). Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions. Nano Lett 14(7): 4171-4175.
  9. Liu, B., Xue, Y., Zhao, W., Chen, Y., Fan, C., Gu, L., Zhang, Y., Zhang, X., Sun, L., Huang, X., Ding, W., Sun, F., Ji, W. and Xu, T. (2015). Three-dimensional super-resolution protein localization correlated with vitrified cellular context. Sci Rep 5: 13017.
  10. Nahmani, M., Lanahan, C., DeRosier, D. and Turrigiano, G. G. (2017). High-numerical-aperture cryogenic light microscopy for increased precision of superresolution reconstructions. Proc Natl Acad Sci U S A 114(15): 3832-3836.
  11. Ovesný, M., Křížek, P., Borkovec, J., Svindrych, Z. and Hagen, G. M. (2014). ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30(16): 2389-2390.
  12. Rust, M. J., Bates, M. and Zhuang, X. (2006). Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10): 793-795.
  13. Schindelin, J., Rueden, C. T., Hiner, M. C. and Eliceiri, K. W. (2015). The ImageJ ecosystem: An open platform for biomedical image analysis. Mol Reprod Dev 82(7-8): 518-529.
  14. Terris, B. D., Mamin, H. J. and Rugar, D. (1994). Near-field optical data storage using a solid immersion lens. Appl Phys Lett 65(4): 388-390.
  15. Thompson, R. E., Larson, D. R. and Webb, W. W. (2002). Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82(5): 2775-2783.
  16. Wang, L., Bateman, B., Zanetti-Domingues, L. C., Moores, A. N., Astbury, S., Spindloe, C., Darrow, M. C., Romano, M., Needham, S. R., Beis, K., Rolfe, D. J., Clarke, D. T. and Martin-Fernandez, M. L. (2019). Solid immersion microscopy images cells under cryogenic conditions with 12 nm resolution. Commun Biol 2: 74.
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简介

我们对细胞功能的机械理解取决于以分子分辨率对细胞中的生物过程进行成像。超分辨率荧光显微镜通过报告具有20-30 nm分辨率的细胞超微结构起着至关重要的作用。但是,该分辨率不足以使工作中的大分子机械成像。一种提高分辨率的途径是在低温条件下成像,这大大提高了大多数荧光团的亮度,并比化学固定剂更好地保留了天然超微结构。但是,由于缺乏兼容的高数值孔径(NA)物镜,因此未充分利用低温条件。在这里,我们描述了一种使用超半球形固体浸没透镜( super SILs)的协议,可在低温下以2.17的有效NA和〜10 nm的分辨率实现超分辨率成像。
【背景】荧光显微镜多年来一直是了解生物系统如何组织及其功能的最重要工具之一。在过去的十年中,荧光显微镜通过“超分辨率”方法的发展而发生了革命,该方法将光学显微镜的范围扩展到了衍射极限之外,从而提供了有关细胞分子网络组织的前所未有的信息水平。这些方法包括结构照明显微镜(SIM)(Gustafsson,2000),受激发射损耗显微镜(STED)(Hell和Wichmann,1994),以及基于单个荧光分子定位的技术,其精确度远高于衍射极限。后者包括光激活定位显微镜(PALM)(Betzig等,2006)和随机光学重建显微镜(STORM)(等,2006),在本协议中统称为单分子定位显微镜(SMLM)技术。由于在寻求高分辨率时需要停止细胞内的运动,因此超分辨率方法通常涉及化学固定,这有可能引入在达到的分辨率下可见的伪像(Whelan和Bell,2015年) 。这个问题早在电子显微镜领域就已被认识到,在电子显微镜领域,现在常规使用一种不同的方法来固定细胞并保存其结构:在低温下玻璃化。在这种方法中,使用冷冻剂将样品冷冻,迅速冷却至-150°C或更低的温度,导致形成无定形冰,且高冷却速率阻止了冰晶的形成(Dubochet和McDowall,1981年)。 。 SMLM技术尤其应从低温条件的使用中受益。这是因为荧光分子的定位精度取决于荧光团发出的光子数量(Thompson等人,2002),并且在低温下,可以收集到更多的光子。相关光和电子显微镜(CLEM)(de Boer et al。,2015)工作流程中已经使用了低温下的常规宽视野荧光显微镜,但光学显微镜和EM之间的分辨率不匹配限制了该值。荧光数据,通常仅限于定位感兴趣的一般区域。因此,对于超低温CLEM工作流程而言,启用超低温超高分辨率荧光显微镜将是一项重大进步。

鉴于玻璃化优于化学固定的优势,将其应用于超分辨率光学显微镜方法将是有利的。但是,其使用受到严格限制的一个主要原因是:超分辨率方法需要使用高数值孔径(NA)物镜(NA> 1),并且要达到此NA通常需要使用与油不兼容的油浸物镜。低温显微镜。为了避免这个问题,已经采取了许多方法,包括使用具有超亮荧光团的非浸没物镜(Liu et al。,2015),以及使用特殊的低温浸没液和定制的样品台(Nahmani <等等。,2017年)。然而,这些方法通常没有被应用于生物样品的成像。

我们已经开发了一种低成本,普遍适用的超分辨率冷冻荧光显微镜方法,该方法使用了商用光学组件和冷冻台(Wang et al。,2019)。为此,我们使用了超半球形固体浸没透镜( super SILs)。这些透镜由高折射率材料制成,并呈截球形的形式。在我们的应用中,它们的作用是代替浸油,并填补样品与常规非浸没物镜之间的间隙。 super SIL和空气物镜的组合提供了较高的数值孔径,取决于透镜材料的折射率(Terris et al。,1994; Zhang et等人,2007年;陈等人,2013年)。

我们已经表征了 super SILs在低温SMLM上的性能,表明我们能够实现约150 nm的点扩散函数(PSF)大小,而具有常规的低NA空气物镜。这是因为 super SIL和物镜的组合获得的NA为2.17,远高于油浸物镜的最大NA〜1.4。 super SIL显微镜的单分子定位精度<10 nm,横向分辨率为12 nm(Wang et al。,2019)。 super SIL方法的主要优点是可以使用现成的现成组件来实现,并且可以轻松地将冷冻台添加到细胞生物学研究实验室常用的显微镜中。而且, super SIL组件可以清洗并重复使用多次,从而使它们成为廉价的资源。在此协议中,我们着重于将 super SIL系统用于细菌的超分辨率显微镜检查,还指出了如何将其应用于哺乳动物细胞成像。类似的方案也可用于使其他样品成像,例如纯化的蛋白质复合物或囊泡。如所描述的那样使用,该协议提供了一种在低温下对生物样品进行超分辨率成像的直接方法,其优点是避免了潜在的固定假象。获得的分辨率和定位精度明显优于常规室温SMLM显微镜中常规获得的分辨率和定位精度。仅凭这些优点,就可以使 super SIL SMLM显微镜成为细胞生物学研究人员工具箱的宝贵补充。

也有可能考虑 super SIL显微镜协议的其他潜在优势。首先,由于NA的超高值,即使在室温下也能获得比传统SMLM显微镜更高的分辨率。因此, super SIL协议提供了一种简单的低成本方法,通过该方法可以将基本的荧光显微镜转换为高性能的超分辨率系统。我们的协议描述了在直立显微镜中使用 super SIL的方法,但是通过使用 super SIL组件的改进版,该方法也可以在倒置显微镜中使用。

我们相信, super SIL显微镜可能在CLEM中发挥重要作用,有助于弥合冷冻荧光显微镜和EM之间的分辨率差距。我们没有在此协议中描述任何CLEM方法,但是可能的方法可能涉及在 super SIL模式和透射EM模式下通过聚焦离子束(FIB)铣削产生的薄薄片的转移和成像,或者超 SIL SMLM图像与串行块面扫描EM的相关性。鉴于透镜组件的尺寸很小,也有可能将 super SIL集成到电子显微镜的工作台中,从而有可能允许使用光和光对同一样品进行同时或接近同时的成像。电子。

关键字:超高分辨显微镜, 低温显微镜, 固体浸没透镜, 生物成像, 样品玻璃化, 荧光显微镜

材料和试剂

  1. super SIL组件
    1. 超半球球形透镜,立方氧化锆,直径0.89毫米x厚度0.73毫米,λ\ 4平坦度(Knight Optical,LBB2018-C)
    2. 铂箔(Goodfellow,目录号:PT000264 876-861-73)
    3. 低温兼容粘合剂(乐泰Stycast,2850英尺)

  2. 制备细菌和基准溶液
    1. 平盖离心管,50毫升(Fisherbrand,目录号:05-539-13)
    2. Eppendorf管,2 ml(Eppendorf,目录号:0030 123.344)
    3. Tetraspeck 0.1μm蓝色/绿色/橙色/深红色小珠(分子探针,Thermo Fisher Scientific,目录号:T7279)
    4. 带有表达 E.Coli 克隆的目的质粒的琼脂平板
    5. LB中等颗粒(例如,梅尔福德,L24400-5000.0)
    6. 用于水中pH调节的1 M HCl溶液(由HCl 37%ACS级320331,Sigma-Aldrich制成)
    7. 水中选择的抗生素储备溶液(例如,卡那霉素25 mg / ml,来自Sigma-Aldrich粉末,K1377;羧苄青霉素100 mg / ml现成的溶液,Sigma-Aldrich,C1613)
    8. IPTG 1 M储备水溶液(由粉末例如,Fluorochem,M02726组成)
    9. PBS 1x pH 7.4(Gibco,目录号:10010-015)
    10. 超纯去离子水(MilliQ或同等水平)
    11. LB培养基(请参阅食谱)

  3. 清洁 super SIL组件
    1. 清洁载玻片(例如,Fischer Scientific 16 x 38 mm显微镜载玻片244879)
    2. 滤纸(例如,定性滤纸A级,SLS 2036)
    3. 干净的玻璃闪烁瓶(例如,Thermo Chromacol 20 ml瓶20-EPSVCA)
    4. super SIL组件(按程序A所述生产),立方氧化锆晶体NA 2.17
    5. 硫酸纯度≥95%(例如,Fischer Scientific S / 9231 / PB15)
    6. 30%过氧化氢与抑制剂,ACS级( e.g。,Sigma-Aldrich,目录号:216763)
    7. 至少100毫升MilliQ(或同等水平)水
    8. 食人鱼解决方案(请参阅食谱)

  4. 急冻
    1. 乙烷气瓶
    2. 清洁并排放辉光的 super SIL(按程序D所述进行制备)
    3. 细菌悬浮液(按照步骤B所述制备)
    4. 基准悬架(如程序C中所述进行制备)
    5. 液氮(至少4升杜瓦瓶)
    6. 70%乙醇
    7. MilliQ水

设备

  1. super SIL组件和显微镜
    1. 陶瓷热板(VWR,目录号:444-0624)
    2. 坐标测量机(OGP,SmartScope ZIP 250)

    显微镜支架
    1. 带有Epi照明臂的Cerna显微镜镜身(Thorlabs,目录号:CEA1350)
    2. 尼康D-FL Epi荧光照明器(Thorlabs,目录号:CSE1000)
    3. 尼康TE2000的显微镜立方体组件(Thorlabs,目录号:TLV-TE2000)
    4. 单物镜臂(Thorlabs,目录号:CSA1100)
    5. 物镜聚焦模块(Thorlabs,目录号:ZFM2020)
    6. 用于1“ Cerna位移台的3轴控制器和旋钮盒(Thorlabs,目录号:MCM3001)
    7. TIRF模块,包括X / Y转换器和直角镜安装架(Thorlabs,ST1XY-D和KCB1 / M)
    8. 翻译阶段(以前的H117P2IX / G)
    9. 平移台控制器(PriScan,ProScan III H31XYZEF)
    10. 图像分割模块(Cairn,Twincam)

    光学元件,样品台和照相机
    在图1所示的显微镜示意图中使用了以下组件。原则上,可以从Linkam Scientific Ltd获得各种用于冷冻台的转接板,并且可以从Cairn Twincam罐上改装大多数流行的商用显微镜支架以创建类似的配置。可以在输入和输出端口上使用C型安装螺纹购买,以适合大多数类型的科学相机。
    注意:在低温下检查所选荧光团或染料的发射光谱,并选择适合的二向色和发射滤光片。通道之间的色差可以通过更改镜头的强度16和sCMOS相机2的轴向平移来补偿。
    1. l 1 :f = 30毫米消色差双合透镜(Thorlabs,目录号:AC254-030-A-ML)
    2. l 2 :f = 150毫米消色差双合透镜(Thorlabs,目录号:AC254-150-A-ML)
    3. l 3 :f = 100毫米消色差双合透镜(Thorlabs,目录号:AC254-100-A-ML)
    4. l 4 :f = 150毫米消色差双合透镜(Thorlabs,目录号:AC254-150-A-ML)
    5. l 5 :f = 200毫米1倍镜筒镜头(Thorlabs,目录号:WFA4100)
    6. l 6 :f = 500 mm B-BK7平凸透镜(Thorlabs,目录号:LA1908)
    7. l 7 :50x,0.75 NA显微镜物镜(Mitutoyo,M Plan Apo HR 378-814-4)
    8. p 1 :30μm针孔(Thorlabs,P30D)
    9. t 1 :高顶光束成形光学器件(TOPAG Lasertechnik GmbH,GTH-5-250-4-VIS)
    10. d 1 :二向色分束镜(Semrock,Di-R405 / 488/561/635)
    11. d 2 :二向色分束器(色度,t565lpxr)
    12. f 1 :四频发射滤波器(Semrock,FF01-446 / 523/600 / 677-25)
    13. f 2 :绿色通道带通滤波器(Semrock,目录号:FF03-525 / 50-25)
    14. f 3 :红色通道带通滤波器(Semrock,目录号:FF01-593 / 40-25)
    15. q 1 :消色差四分之一波片(Thorlabs,AHWP10M-600)
    16. m 1 :宽带介电镜(Thorlabs,BB1-E02)
    17. 包含四个激光模块的Omicron LightHUB-6
      1. 405 nm 60 mW福克斯二极管激光器
      2. 488 nm 200 mW福克斯二极管激光器
      3. 561 nm 150 mW Cobolt Jive DPSS激光器
      4. 642 nm 140 mW福克斯二极管激光器
    18. 两个具有18.7 mm芯片传感器尺寸的背照式sCMOS相机(Teledyne Photometrics Ltd,18.7 mm Prime 95B)
    19. 具有自动填充杜瓦瓶(Linkam,CMS196M和AUTOFILL)的低温台


      图1.显微镜原理图,详细说明光学器件布局

  2. 制备细菌和基准溶液
    1. 摆桶式离心机(例如,贝克曼库尔特Allegra X-15R离心机)
    2. P1000,P100和P10移液器和吸头
    3. 高压灭菌器(例如,Prestige Medical Classic台式高压灭菌器)
    4. 分光光度计(例如,Amersham Biosciences Ultrospec 10细胞密度仪)
    5. MSC机柜(例如,Scanlaf Mars安全等级2)
    6. 温度控制的搅拌培养箱(例如,New Brunswick Scientific Innova 44R)
    7. 涡流(例如,IKA MS2 minishaker)
    8. 浸入式pH计(例如,Hanna PH21)

  3. 清洁 super SIL组件
    1. 化学通风柜
    2. P1000移液器和吸头
    3. 辉光放电器(例如,Quorum GloQube)
    4. 倒镊子(例如,Dumont N1)
    5. 计时器

  4. 急冻
    1. FEI Vitrobot或同等的柱塞式冷冻机
    2. 易燃气体调节器(例如,液化空气HBD-240-4-2.S)
    3. 气体泄漏检测器(例如,Everbuild Plumber的P18气体泄漏检测器)
    4. 倒镊子(例如,Dumont N1)
    5. 弯曲镊子(例如,Dumont 7,0130-7-PO)
    6. 印迹纸(例如,Vitrobot的Agar圆形滤纸,47000-100)
    7. P10移液器和吸头
    8. 1 L保温瓶
    9. 漏斗
    10. 卫生纸
    11. 网格箱(例如,SwissCI cryo-EM网格箱MD16-104,Molecular Dimensions Limited)
    12. 50毫升离心管连接到一块合成的非吸收性麻线上
    13. 运输低温战(例如,Statebourne低温OD-1)
    14. 手持式液氮杜瓦瓶(例如,沃辛顿LD4)
    15. 存储孤岛战(例如,沃辛顿VHC35)
    16. 电热板或吹风机
    17. 足够用于低温工作的个人防护设备(PPE)(请参阅您所在机构的当地健康和安全法规)
    18. 可选:PFTE螺纹密封胶带(例如,布法罗12 m x 12 mm x 0.075 mm BS7786:2006 Grade L)
    19. 可选:局部排气通风(LEV)进口
    20. 可选:可定向的台灯
    21. FEI Vitrobot或同等湿度控制器

软件

  1. Micro-Manager v1.4.23 20161114(Edelstein等,2014)
  2. Omicron控制中心v2.1.4
  3. ImageJ版本v1.52h(Schindelin等,2015)
  4. ThunderSTORM v1.3-2014-11-08插件(Ovesny et al。,2014)

程序

  1. 制造superSIL组件
    注意:此步骤假定使用配备有激光切割机和干燥箱的机械车间。


    图2. superSIL组件的制造过程。 A.直径3 mm的铂金圆盘,中心有一个直径为1 mm的激光切割孔。 B.装配好的 super SIL的光盘。比例尺:1毫米。

    1. 使用激光切割机从铂箔上切出一个直径为3毫米的圆。在中心钻一个直径为1毫米的孔(如图2A所示)。
    2. 使用低温兼容粘合剂将 super SIL胶粘到孔中(如图2B所示)。
    3. 在45°C下,将组件在烤箱中固化24小时。
    4. 使用坐标测量机测量 super SIL平面与铂金圆盘平面之间的角度。平坦度&lt; 1°对于最大程度地降低成像像差至关重要。

  2. 细菌悬浮液的制备
    注意:通过在MSC或本生灯旁边的所有步骤进行步骤B6之前,可以保持培养物的无菌性。
    该过程假定所选的细菌菌株表达适合SMLM的荧光蛋白。我们已经成功使用增强型GFP(EGFP)进行了成像(Wang等,2019),并且已有报道使用单体EGFP和mVenus进行冷冻SMLM(Kaufmann等)。 >,2014年)。
    1. 在10 ml LB琼脂中接种目标细菌菌株的一个菌落。
    2. 根据目标质粒上存在的选择标记添加选择抗生素。
    3. 在37°C 220 rpm下生长3小时,然后检查外径。在分光光度计上用660稀释,使用LB介质(配方1)作为空白。
    4. 如果文化达到了外径约0.6,用IPTG 0.2-1 mM诱导(具体情况必须根据具体情况进行优化),并在25°C下培养18 h。
    5. 保持培养物无菌,取2 ml培养物并将其转移到2 ml Eppendorf中。
    6. 于3,000 x g 离心10分钟,弃去上清液。
    7. 加入2 ml PBS,重悬并于3,000 x g 离心10分钟,然后弃去上清液。
    8. 重悬于100μlPBS 1x中,置于冰上。

  3. 基准悬架的制备
    1. 以最大设置涡旋Tetraspeck小瓶至少10 s。
    2. 用MilliQ(或同等纯度)的水将Tetraspeck 1:10稀释。
    3. 在最大设置下涡旋稀释的珠悬浮液至少10 s。
    4. 保持冰上。

  4. 清洁 super SIL组件
    每次使用后,应在使用新样品之前使用此程序清洁 super SIL。这使得 super SIL组件可重复使用几个周期。清洁会使将镜头附着在铂金盘上的胶水退化。当 super SIL松动时,可以按照过程A中的说明重新安装。

    重要的安全建议
    1. Piranha溶液是高度酸性的,会氧化并放热。不要制造超出所需的数量,不要在制造时盖住小瓶,也不要存放。始终使用适当的PPE。专门在通风橱下准备和使用食人鱼。
    2. 请勿与有机溶剂混合,否则可能会引起爆炸。
    3. 请勿使用浓度超过30%的过氧化氢,否则可能引起爆炸。
    4. 使用后,用大量MilliQ水稀释剩菜,将其放气并在通风橱中降解过夜,然后将其与大量冷水一起排入下水道。
    5. 此过程在视频1中得到了直观展示。


      视频1. SuperSIL清洁过程

    1. 按照食谱中所述准备食人鱼溶液(配方2)。
    2. 现货〜100μl干净玻璃载玻片上的食人鱼溶液。为每个要清洁的 super SIL准备一个斑点。
    3. 用一对镊子将每个 super SIL放在Piranha溶液的一个点上,面朝下。
    4. 在室温下孵育7分钟。
    5. 使用一对倒镊子,将每个 super SIL浸泡在MilliQ小瓶中,然后在干净的滤纸上吸干,以彻底冲洗。重复扣篮和吸墨至少三遍。
    6. 使用干净的MilliQ小瓶再次重复步骤D5。
    7. 让 super SILs在干净的滤纸上干燥至少1小时。
    8. 辉光放电器中的辉光放电 super SILs在40 mA下以120 s的平面朝上向上放电。

  5. 柱塞冻结
    重要安全声明
    1. 协议的这一步骤需要使用低温剂和易燃气体,它们可能会因氧气置换而引起低温灼伤,爆炸和窒息。始终佩戴适当的个人防护装备,并确保您在有足够通风和氧气耗尽警报的区域中工作。请始终参阅有关在您的工厂中使用低温和易燃气体的健康和安全指南。开始工作之前,请先进行适当的培训和/或监督。
    2. 假定使用Vitrobot浸入式冷冻机(FEI)进行说明。请参阅制造商的说明,以了解如何将其翻译为与其他制造商的等效设备一起使用。


      图3. Vitrobot浸入式冷冻机的冷冻浴

    视频2中直观地演示了此过程。
    1. 打开总开关上的Vitrobot并安装湿度控制器。
    2. 将湿度设置为70%,温度设置为22°C。等待直到它们平衡(通常25-30分钟)。
    3. 在选项菜单中,将印迹数设置为零,并允许使用脚踏板。
      注意:需要禁用自动印迹,以保持superSIL的结构完整性。
    4. 用约1 L的液氮填充冷冻浴的外腔。确保“蜘蛛”到位(如图3所示)。
      注意:请勿直接从手持式杜瓦瓶中加满水,而要用它来填充较小的热水瓶。使用漏斗和薄纸过滤掉可能污染样品的宏观冰晶。
    5. 等待10至15分钟,使内室冷却并将网格盒放入专用插槽中(最多4个盒,因此一次最多可容纳16个样品)。
    6. 将调节器安装在乙烷瓶上,并使用检漏仪喷雾器检查是否泄漏。
      注意:如果检测到泄漏,请关闭所有阀门并重新启动。如有必要,使用PVDF胶带包裹调节器螺纹。遵守有关可燃气体使用的地方安全法规。


      视频2. SuperSIL骤冷过程

    7. 将乙烷缓慢地分配到内杯中,然后等待直到乙烷几乎充满为止,以除去蜘蛛网。乙烷应处于液态,以进行骤冷。如有必要,可通过将热金属仪器插入乙烷内杯中来熔化固化的乙烷。
      注释:
      1. 将局部排气通风(LEV)头靠近冷冻浴放置,对于更准确地清除冷的乙烷蒸气和进行测量非常有帮助。
      2. 在以下段落中,需要将蜘蛛取下。否则可能会严重损坏计算机。
    8. 用兼容Vitrobot的镊子装上 super SIL,确保平坦的一面朝向您惯用或惯用的手的侧面,并将其加载到轴上。
      注意:请确保镊子不在轴的两侧,因为当轴缩回时,这可能会严重损坏机器。
    9. 踩踏板将镊子缩回等待位置。
    10. 将低温浴放在基座上,然后踩踏板以抬起。
    11. 冷冻浴完成移动后,按踏板将镊子降低到样品加载位置。
    12. 通过侧面端口将2.5μl细菌悬浮液施加到 super SIL的平坦侧。
    13. 打开前门,用一张滤纸将大部分液体吸干。
    14. 重复样品施加和印迹。
    15. 通过侧面端口将2.5μl基准溶液施加到 super SIL的平坦侧面。
    16. 重复步骤E13。
    17. 踩踏板开始跳入序列。
    18. 一旦轴和冷冻浴到达过程的终点,请在外腔中注满液氮,从轴上卸下镊子,然后将 super SIL从乙烷杯中移入氮气中洗澡。
      注释:
      1. 在这个阶段,可定向的台式灯产生的额外光非常有用。
      2. 将镊子从乙烷中缓慢取出,以防止液态乙烷粘附到superSIL表面,然后在液态氮中快速转移。
    19. 小心释放镊子的卡子,并将 super SIL放在网格盒的一个插槽中。
    20. 使用加热板或吹风机加热并干燥所有仪器。
      注意:每次取样后,用70%的乙醇洗涤Vitrobot镊子,并使其干燥以防止 super SILs交叉污染。
    21. 一旦所有样品都投入后,用液氮填充运输杜瓦瓶并使其沉降。
    22. 在运输杜瓦瓶内部的管子上冷却管子。
    23. 使用冷镊子,将格栅盒从Vitrobot低温浴的外部氮气浴快速转移到试管中。
    24. 将样品保存在充满液氮的杜瓦瓶中,直到准备成像(不超过一个月)。

  6. 样品转移至低温台


    图4.移除盖子的Linkam冷冻台布局

    1. 开启Linkam CMS196M冷冻台并执行烘烤周期。
    2. 用液氮填充Linkam Autofill杜瓦瓶,然后连接到CMS196M平台。启动冷却台的冷却。
    3. 冷冻室内的温度平衡到-196°C后,将Linkam样品转移圆盘插入冷冻室内的一个空容器中,然后冷却。插入空白样品盒(如图4所示)。
    4. 将包含您的 super SIL样品的储存圆盘转移到冷冻室中的其余容器中。
    5. 使用镊子将 super SIL组件从存储圆盘转移到样品盒中,要密切注意方向;安装后, super SIL的弯曲表面需要指向显微镜物镜。
    6. 一旦安装了 super SIL,请关闭盒式磁带,然后使用磁性传输工具将其传输到黄铜桥。从冷冻室中取出储存器和转移圆盘,以最大程度地减少成像过程中的振动。
    注意:在样品转移过程中,最大程度地减少样品被冰污染和去玻璃化的可能性至关重要。在潮湿的环境中,由于呼吸和未使用加热板进行预干燥的工具所凝结的结果,可能会形成冰晶。

  7. 寻找焦点


    图5.在明场照明下寻找焦点。在50x 0.75 NA物镜下, super SIL曲面的顶部显示为聚焦在(A)中。通过 super SIL观察,在(B)中所示的明场照明下,应该可以看到平坦表面上的特征性划痕,表明样品平面已对准焦点。比例尺:10μm。

    1. 启动Micro-Manager,并显示摄像机的实时视图。
    2. 打开Linkam CMS196M内置的明场电容器LED。
    3. 将显微镜物镜降低到 super SIL的Linkam室。监控绿色通道摄像机的输出。密切注意物镜与 super SIL之间的距离;碰撞可能会损坏Linkam黄铜桥,从而导致大量维修费用和延误。
      注意:红色LED通道不太可能出现内置LED聚光镜发出的光。
    4. 当物镜接近 super SIL时,您会注意到绿色通道的亮度增加。横向调整显微镜载物台,以确保在您进一步降低物镜时,最亮的区域保持在相机视野的中心。
    5. super SIL的曲面将聚焦。横向移动显微镜载物台,使曲面的最顶部位于相机视场的中心(图5A),并且外围模糊而无法聚焦。
    6. 现在将物镜降低大约1毫米,以找到位于 super SIL平面下方的虚拟成像平面。您会发现 super SIL平面上的固有划痕首先成为焦点(图5B)。如果在急冻过程中产生的冰相对较薄,则样品所在的平面应非常接近 super SIL的平面。

  8. 影像
    注意:您可以选择将收集的图像存储在内存中或直接将其流式传输到硬盘。 sCMOS相机会产生大量数据,因此请确保继续之前有足够的可用空间,否则系统可能会变得无响应并崩溃。每个通道包含10,000个全帧的典型双色STORM实验消耗大约10 GB的数据。最低要求建议使用等于或大于32 GB的计算机内存以及容量等于或大于1 TB的固态硬盘。
    1. 拍摄可选的明场图像快照。关闭LED电容器。
    2. 所需的激光充分加热后,再打开它们。
    3. 打开显微镜上的激光快门以照亮样品。
    4. 微调物镜高度,直到基准标记对准焦点。
    5. 可选:调整TIRF模块上的千分尺以增加样品的对比度。
    6. 从低激光功率密度(〜50 W / cm 2 )开始。等待样品充分的光漂白,直到看到清晰的分离良好的单分子闪烁。如有必要,请增加激光功率密度,直到观察到所选荧光团所需的闪烁特性为止。与在黑暗状态下花费的时间相比,分子在其发光状态下花费的时间应尽可能短。
    7. 根据您选择的荧光团和样品条件,调整相机的曝光时间并为实验获取适当数量的帧。对于 E。含有eGFP的Coli ,我们选择以50毫秒的曝光时间获取10,000帧。
    8. 将获取的帧另存为16位无符号整数TIF堆栈。
    9. 反向执行“查找焦点”步骤,以从冷冻显微镜载物台安全地取出样品。

数据分析

  1. 单分子定位
    1. 打开ImageJ并加载感兴趣的TIF堆栈。
    2. 运行ThunderSTORM插件。在“摄像机设置”下验证您的摄像机设置正确,并且已根据理论点扩展函数大小输入了合适的拟合和亚像素定位设置(图6)。
    3. 选择所需的方法以可视化重建的图像。
    4. 单击“预览”以查看分析如何处理当前数据帧并验证其检测到所有可见的单个分子。
    5. 单击“确定”以对整个数据集运行分析。
    6. 分析完成后,您会注意到结果表出现在屏幕上,与重建的图像一起。使用“导出”功能将结果表另存为.csv文件,因为此功能可在以后用于重新生成重建的图像。


      图6.基于有效点扩散函数和 super SIL
      成像平面上像素大小的典型ThunderSTORM设置
  2. 漂移校正
    1. 如果基准标记存在且处于焦点位置,则可以对数据集应用漂移校正。单击“漂移校正”选项卡,然后通过单击双箭头图标加载子菜单。我们已经发现,在我们的特定低温阶段,考虑到漂移的性质,在漂移校正选项下,将最大距离设置为500 nm,最小标记比设置为0.9,轨迹平滑因子设置为0.001可获得令人满意的结果。
    2. 一旦计算并应用了漂移校正,就会出现一个新的重构,以反映这些变化。使用ImageJ工具栏中的放大镜工具放大以检查基准标记;如果仍然有明显的漂移,则可能需要调整上一步中讨论的变量。
    3. 使用“导出”功能将修改后的结果表另存为.csv文件。

  3. 过滤
    注意:ThunderSTORM中的拟合算法可用于消除由冰晶,聚集基准点,样品自动荧光或散射等来源引起的不必要的定位伪影。
    1. 在“过滤器”选项卡上,您可以使用布尔代数表达式定义所需的过滤器。
    2. 例如,如果您的系统具有125 nm的理论点扩散函数sigma,则可以选择仅包括sigma在100-150 nm范围内的那些定位,并使用表达式“ sigma&gt; 100和sigma&lt; 150”。
    3. 您感兴趣的样品可能具有100-1000 ADU计数的强度范围,而基准标记的强度超过6000 ADU计数。在这种情况下,您可以使用表达式“ intensity&lt; 6000”。

  4. 可视化和测量
    1. 通过选择“文件”>“颜色”,可以将颜色查找表(LUT)应用于重建的图像。导入&gt; LUT…”。
    2. 需要正确定义像素大小,以便在重建的图像中进行测量。可以通过选择重建的图像窗口并导航到“ Image&gt;属性”。像素大小应设置为相机的像素大小,再除以ThunderSTORM可视化设置中使用的最终放大率。例如,如果您的像素大小为46.8 nm,并且选择了5倍的最终放大倍数,则您重构的像素大小为9.36 nm。
    3. 现在,您可以使用ImageJ主工具栏上的标尺工具测量要素之间的近似距离。

  5. 先进的数据分析
    ThunderSTORM生成的.csv数据表可以导入其他软件包中以进行高级数据分析,包括单分子聚类,化学计量和通道之间的共定位。

菜谱

  1. LB培养基
    1. 称出25克颗粒
    2. 加入1升MilliQ水
    3. 使用HCl将pH调整至7.2
    4. 高压灭菌器消毒
  2. 食人鱼解决方案
    1. 将900μl浓硫酸加入玻璃闪烁瓶中
    2. 向小瓶中加入300μl的30%过氧化氢并快速混合
      注意:小瓶会明显加热。
    3. 立即使用。不要盖上样品瓶

    重要的安全建议
    1. Piranha溶液是高度酸性的,会氧化并放热。不要制造超出所需的数量,不要在制造时盖住小瓶,也不要存放。始终使用适当的PPE。专门在通风橱下准备和使用食人鱼。
    2. 请勿与有机溶剂混合,否则可能会引起爆炸。
    3. 请勿使用浓度超过30%的过氧化氢,否则可能引起爆炸。
    4. 使用后,用大量MilliQ水稀释剩菜,使其脱气并在通风橱中降解过夜,然后将其与大量冷水一起排入下水道。
    5. 此配方可制成足够的Piranha解决方案,以上述方法清洁20多种superSIL。请注意,如果制备更大的量,与食人鱼溶液制备相关的危险会增加。对于大于5毫升的溶液,应始终在冰上制备溶液,以尽量减少热量。

致谢

这项工作由医学研究理事会授予MMF的MR / K015591 / 1资助。该协议基于Wang 等人 。(2019)中发表的工作。

利益争夺

作者宣称没有利益冲突。

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引用:Bateman, B. C., Zanetti-Domingues, L. C., Moores, A. N., Needham, S. R., Rolfe, D. J., Wang, L., Clarke, D. T. and Martin-Fernandez, M. L. (2019). Super-resolution Microscopy at Cryogenic Temperatures Using Solid Immersion Lenses. Bio-protocol 9(22): e3426. DOI: 10.21769/BioProtoc.3426.
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