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Structured Illumination Microscopy (SIM) and Photoactivated Localization Microscopy (PALM) to Analyze the Abundance and Distribution of RNA Polymerase II Molecules on Flow-sorted Arabidopsis Nuclei
使用结构照明显微镜(SIM)和光敏定位显微镜(PALM)分析RNA聚合酶II分子在流式分选的拟南芥细胞核中的丰度及分布   

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Abstract

RNA polymerase II (RNAPII) is the enzyme transcribing most of the eukaryotic protein-coding genes. Analysing the distribution and quantification of RNAPII can help understanding its function in interphase nuclei. Although several investigations in mammals indicate the organization of RNAPII in so-called ‘transcription factories’ (Jackson et al., 1993; Rieder et al., 2012; Papantonis and Cook, 2013), their existence is still controversially discussed (Zhao et al., 2014).
Recently, based on super-resolution microscopy the presence of transcription factories was also suggested in plants. Applying structured illumination microscopy (SIM) and photoactivated localization microscopy (PALM) the distribution and number of RNAPII molecules in Arabidopsis nuclei were analysed and a positive correlation between RNAPII abundance and endopolyploidy was found (Schubert, 2014; Schubert and Weisshart, 2015).
Here, we present a protocol describing the isolation of Arabidopsis thaliana interphase nuclei via flow-sorting according to their endopolyploidy level, followed by a double immunostaining using antibodies specific for different RNAPII modifications and the subsequent evaluation by spatial SIM and PALM to achieve results regarding the abundance, distribution and co-localization of single inactive and active RNAPII molecules.

Keywords: Super-resolution microscopy(超高分辨率显微镜), Cell nucleus(细胞核), Transcription(转录), RNA polymerase(RNA聚合酶), Single molecule localization(单分子定位)


Part I. Flow sorting of Arabidopsis interphase nuclei

Materials and Reagents

  1. 22x22 mm high precision coverslips (170±5 µm, no. 1.5H) (Marienfeld-Superior) Rubber cement (Marabu Australia, catalog number: 290110000 )
  2. 5 ml Polystyrene Round-Bottom Tubes with 35 µm cell strainer cap (BD Biosciences, catalog number: 352235 ) or alternatively, 5 ml Polystyrene Round-Bottom Tube (BD Biosciences, catalog number: 352063 ) in combination with disposable filters CellTrics, 30 or 50 µm (Sysmex-Partec, catalog numbers: 04-0042-2316 or 04-0042-2317 )
    Note: Currently, it is "Corning, Falcon®, catalog number: 352235” and "Corning, Falcon®, catalog number: 352063”.
  3. 0.22 µm filter
  4. A. thaliana rosette leaves
  5. Calibration beads [SpheroTM Rainbow Calibration Particles (8 peaks)] (BD Biosciences, catalog number: 559123 )
  6. Accudrop Beads (BD FACSTM Accudrop Beads) (BD Biosciences, catalog number: 345249 )
  7. Formaldehyde solution 37% (Carl Roth GmbH + Co, catalog number: 7398.1 )
  8. 4', 6-diamidino-2-phenylindole (DAPI) (Life Technologies, Molecular ProbesTM, catalog number: D-1306 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D-1306”.
  9. Milli-Q water
  10. NaCl
  11. Na2HPO4.2H2O
  12. NaH2PO4
  13. Tris
  14. Na2EDTA
  15. Spermin
  16. KCl
  17. Triton X-100
  18. NaOH
  19. β-mercaptoethanol
  20. MgCl2
  21. Sucrose
  22. Tween 20
  23. HCl
  24. BD FACSFlow sheath fluid (BD Biosciences, catalog number: 342003 ) or alternatively, 1 x PBS (see Recipes)
  25. Formaldehyde fixative (see Recipes)
  26. 500 ml Tris-Buffer (see Recipes)
  27. 200 ml lysis buffer LB01 (Dolezel et al., 1989) (see Recipes)
  28. 100 µg/ml DAPI stock solution (see Recipes)
  29. 25 ml sucrose buffer (see Recipes)

Equipment

  1. FACSAria IIu (BD Biosciences)

Software

  1. FACSDiva software

Procedure

  1. Nuclei isolation
    1. Harvest 2-3 rosette leaves of four to six weeks old plants of A. thaliana into distilled (or deionized) water.
    2. Fix the plant tissue by transferring it into the formaldehyde fixative and incubate it for 20 min in a small glass beaker on ice under vacuum using a desiccator.
    3. Dry off the plant material shortly on filter paper.
    4. Wash in Tris-buffer 2x 10 min in a new glass beaker on ice, and dry off the plant material shortly on filter paper between the washing steps.
    5. Transfer the plant tissue in a pre-cooled Petri dish, add roughly 250 µl of LB01 lysis buffer and chop it with a sharp razor blade until getting a fine suspension.
    6. Add additional 250-750 µl (depending on the amount of used plant tissue) of LB01 lysis buffer and filter the suspension through a 30-50 µm mesh (using either tubes with cell strainer caps or other disposable filters) into a 5 ml Round-Bottom polystyrene tube and store it on ice. This suspension can either be used directly to prepare nuclei preparations suitable for immunostaining experiments (continue with procedure C ‘Preparation of coverslips’) or used for fluorescence activated cell sorting (FACS) before. The latter variant allows the separation of nuclei according to different ploidy levels and results in preparations of high purity that guarantee a minimum of background.
    7. Stain the suspension by adding DAPI stock solution to a final concentration of 1-2 µg/ml and store it for 10 min on ice.

  2. Sorting of nuclei using a FACSAria cell sorter
    1. Switch on the computer, the cytometer and the blue (488 nm) and violet (405 nm) or (Near-) UV (375 nm) laser and wait 30 min for them to warm up. Start the FACSDiva software, check the level of the fluidics in the cytometer window and refill sorting buffer (BD FACSFlow or 1x PBS) and empty the waste if needed. Perform fluidics startup, insert a 70 µm nozzle and switch on the stream. Control if the correct sort setup is selected (‘Sort’ - ‘Sort Setup’ - ’70 micron’). Switch on ‘Sweet Spot’ in the ‘Breakoff’ window.
    2. Run calibration beads to control the performance of the flow sorter. If 8 peaks are not displayed in corresponding log scale histograms for FITC and DAPI, clean the flow cell according to the FACSAria User’s Guide or the instructions of the operator.
    3. Run Accudrop Beads and determine the optimal drop delay using the ‘Auto Drop Delay’ function of the FACSAria according to FACSAria User’s Guide.
    4. Run nuclei sample, set up the trigger to DAPI fluorescence and select a proper threshold level that allows you to display the signals on a dot plot of side scatter (SSC) area (log scale) versus DAPI fluorescence area (log scale). Adjust the voltage of the photomultiplier for the DAPI fluorescence to achieve a displaying of all ploidy level populations (Figure 1A).
    5. Define a gate around the nuclei populations and select this to be displayed in a histogram of DAPI fluorescence area. Define sorting regions for the ploidy levels of interest (Figure 1B).
    6. Open the ‘Sort Layout’ window, select collection device ‘4 Tube’, precision mode ‘Purity’ and define the sort locations. Insert empty Eppendorf tubes into the collection device holders.
    7. Sort the required number of nuclei (10,000 to 50,000; depending on the availability of the individual ploidy levels and the desired number of coverslips) into collection tubes and store them afterwards on ice.


      Figure 1. Flow sorting of A. thaliana interphase nuclei. A. Dot plot of SSC area versus DAPI fluorescence area indicating populations of nuclei with different DAPI fluorescence intensities reflecting different levels of endopolyploidy. B. Histogram of DAPI fluorescence of particles gated as P1 in (A). The gates above of the individual peaks indicate the sorting gates of 2C, 4C, 8C and 16C nuclei.

  3. Preparation of coverslips
    1. Pipette 12 to 15 µl of sucrose buffer onto 22x22 mm high precision coverslips (Marienfeld) attached on slides by rubber cement (Marabu) (Figure 2A).
    2. Subsequently pipette equivalent volumes of sorted nuclei into the drop of sucrose buffer. Gently mix the solutions with a pipette tip.
    3. Air-dry coverslips over-night at room temperature.
    4. Coverslips can be used either immediately for immunostaining or stored for up to several months at -20 °C in the dark.

Recipes

  1. 1 L 1x PBS
    154 mM NaCl: 9 g
    7 mM Na2HPO4.2H2O: 1.246 g
    4 mM NaH2PO4: 0.48 g
    Dilute in A.dest. and adjust the pH to 7.0 using 1 N NaOH
  2. Formaldehyde fixative
    4% formaldehyde in Tris buffer (dilute 6 ml formaldehyde solution 37%, with 50 ml Tris buffer), the fixative should be prepared just before use. Attend to work with formaldehyde under a hood and dispose waste properly.
  3. 500 ml Tris-buffer
    10 mM Tris: 0.605 g
    10 mM Na2EDTA: 1.861 g
    100 mM NaCl: 2.922 g
    0.1% Triton X-100: 500 µl
    Dilute in A.dest. and adjust the pH to 7.5 using 1 N NaOH
  4. 200 ml Lysis buffer LB01 (Dolezel et al., 1989)
    15 mM Tris: 363 mg
    2 mM Na2EDTA: 148.9 mg
    0.5 mM Spermin: 20.2 mg
    80 mM KCl: 1.193 g
    20 mM NaCl: 233.8 mg
    0.1% Triton X-100: 200 µl
    Adjust pH to 7.5 using 1 N NaOH and filter through a 0.22 µm filter to remove small particles. Add 220 µl β–mercaptoethanol and store in 10 ml aliquots at -20 °C. Attend to work with β-mercaptoethanol under a hood and dispose waste properly.
  5. 100 µg/ml DAPI stock solution
    Dissolve 5 mg 4', 6-diamidino-2-phenylindole (DAPI) in 50 ml deionized water by stirring for 60 min and filter through a 0.22 µm filter to remove small particles. Store in 0.5 ml aliquots at -20 °C.
  6. 25 ml Sucrose buffer
    10 mM Tris: 30.3 mg
    50 mM KCl: 93.2 mg
    2 mM MgCl2·6H2O: 10.2 mg
    5% sucrose: 1.25g
    0.05% Tween 20: 12.5 µl
    Adjust pH to 8.0 using 5 N HCl and store in 1 ml aliquots at -20 °C


    Figure 2. Specimen preparation, immunostaining procedure and coverslip handling for microscopy. A. Fixing the coverslip at a slide by applying rubber cement; B. Adding of the blocking solution; C-D. Covering with parafilm. E. Slide placed in a humidity chamber; F. Cutting the rubber cement with a razor blade; G. Lifting the coverslip; H. Pipetting 1% 2-mercaptoethanol in 1x PBS into the coverslip containing coverslip chamber.

Part II. Immunolabeling of sorted nuclei

Materials and Reagents

  1. Primary antibodies
    1. Mouse monoclonal anti-RNAPII CTD repeat YSPTSPS (1:300) (Abcam plc., catalog number: ab817 ) to detect inactive RNAPII molecules
    2. Rat monoclonal anti-RNAPII (phospho CTD Ser-2) (1:200) (Merck Millipore Corporation, catalog number: 04-1571 ) to detect active RNAPII molecules
  2. Secondary antibodies
    1. Goat anti-mouse-Cy5 (1:300) (Jackson ImmunoResearch Inc., catalog number: 715-175-151 )
    2. Goat anti-rat Alexa488 (1:200) (Jackson ImmunoResearch Inc., catalog number: 112-545-167 )
  3. Milli-Q water
  4. PIPES
  5. MgSO4
  6. EGTA (pH 6.9)
  7. BSA
  8. Triton X-100
  9. 1x MTSB (microtubule stabilizing) buffer (see Recipes)
  10. Blocking solution (see Recipes)
  11. Dilution buffer for antibodies (see Recipes)

Equipment

  1. Humidity chamber
  2. Slide cuvettes [e.g. vertical glass cuvettes (Electron Microscopy Sciences, catalog number: 70318-04 )]

Procedure

  1. After storage at -20 °C keep coverslips (fixed on slides by rubber cement) 10 min at room temperature (RT); then, place them in suitable racks and dry in an incubator at 60 °C for 45 min.
  2. Wash 5 min in 1x MTSB buffer in a slide cuvette.
  3. Add 60 µl of blocking solution to each sample, cover with parafilm and incubate 1 h at RT in a humidity chamber (Figures 2B-E).
  4. Dilute the primary antibodies in dilution buffer.
  5. Remove the parafilm and blocking solution, then add 40 µl of the primary antibody solution to each coverslip, cover with parafilm and incubate overnight at 10 °C in a humidity chamber.
  6. Wash the coverslips 3x 5 min in 1x MTSB buffer by placing the slide into a cuvette.
  7. Dilute the secondary antibodies, add 40 µl of this dilution to each coverslip, cover with parafilm and incubate overnight at 10 °C in a humidity chamber.
  8. Wash the coverslips 1x 5 min in 1x MTSB buffer in a slide cuvette.
  9. Dehydrate in ethanol series (70%, 90%, 96%) for 5 min each.
  10. Air dry in dark.

Recipes

  1. 1x MTSB (microtubule stabilizing buffer)
    50 mM PIPES
    5 mM MgSO4
    5 mM EGTA (pH 6.9)
  2. Blocking solution
    8% BSA
    0.1% Triton X-100 in 1x MTSB
  3. Dilution buffer for antibodies
    1% BSA in 1x MTSB buffer
    Store in 1 ml aliquots at -20 °C.

Part III. SIM and PALM evaluation

This protocol is based on using the ELYRA PS.1 system with the software ZEN 2012. Systems from other suppliers have similar functions. Therefore, the protocol can well serve also for other commercial systems including the processing steps. Being trained on such systems the resemblance of specific instructions to those given here should become obvious.

Materials and Reagents

  1. 200 nm TetraSpeck™ Fluorescent Microspheres slide (Carl Zeiss Microscopy GmbH) 
    Note: TetraSpeck™ Fluorescent Microspheres is a trademark of Thermo Fisher Scientific.
  2. 40 nm FluoSpheres® Carboxylate-Modified Microspheres slide (Carl Zeiss Microscopy GmbH, catalog number: 1783-456 )
  3. 40 nm FluoSpheres® Carboxylate-Modified Microspheres (Thermo Fisher Scientific)
  4. 200 nm TetraSpeckTM microspheres (Thermo Fisher Scientific, catalog number: T-7280 )
  5. 2-mercaptoethanol (Sigma-Aldrich, catalog number: 63689 )
  6. NaCl
  7. Na2HPO4.2H2O
  8. NaH2PO4
  9. NaOH
  10. 1x Phosphate-buffered saline (PBS) (see Recipes)
  11. Phosphate buffered saline solution 10x concentrate (10x PBS) (Sigma-Aldrich, catalog number: P5493 ) (see Recipes)

Equipment

  1. Coverslip chamber 'Chamlide' for 22x22 mm coverslips (Live Cell Instruments, catalogue number: CM-S22-1 )
  2. Microscope system Elyra PS.1 (Carl Zeiss Microscopy GmbH)

Software

  1. Software ZEN 2012 (Carl Zeiss Microscopy GmbH)

Procedure

  1. Sample mounting and nuclei selection
    1. Remove the coverslip from the slide by carefully cutting the rubber cement with a razor blade at one side and lifting it from the slide (Figures 2F-G). Place the coverslip into a 22x22 mm coverslip chamber and cover with 1% 2-mercaptoethanol in 1x PBS (Figure 2H). Close with the lid.
    2. Select favorable nuclei using the 63x/1.4 Oil Plan-Apochromat objective, X-Cite (LED) illumination, the eyepieces and apply the ZEN tool 'Locate' (Figure 3).


      Figure 3. Microscope control by 'Locate'. Settings for reflected fluorescence light ocular detection are activated (yellow line).

  2. Raw data acquisition for 3D SIM
    Using the same objective, first acquire the SIM image and then the PALM image (as described in the next section) by exciting with the 642 nm and 488 nm lasers subsequently (51 µm grid for 642 nm and 34 µm grid for 488 nm excitations; 5 rotations). Starting with the longer wavelength minimizes bleaching.
    1. Switch from tool 'Locate' to the tool 'Acquisition' and select in the ‘Imaging Setup’ tool ‘SIM’ to acquire SIM raw data image stacks (Figure 4).


      Figure 4. ‘Imaging Setup’ tool of ZEN ‘Acquisition’ for SIM. ‘SIM’ is selected and a two track experiment with track 1 using the 642 nm laser is configured.

    2. In the ‘Channels’ tool define a multi-track experiment using the 642 nm laser for the first channel and the 488 nm laser for the second channel. Adjust laser power to ~10 %, camera gain to 30 V and exposure time of the Andor iXon885 camera to 100 ms to view your sample. Adjust laser power and/or gain if necessary. But use the 'Range Indicator' to avoid overexposure. In the ‘Acquisition Mode’ select the appropriate frame size and set the number of rotations to 5. Adjust frame size further by cropping if needed. If using crop, only crop centrally and do not use panning, otherwise PALM and SIM images will not match later on (Figure 5).


      Figure 5. Example of central cropping without panning

    3. Focus on the sample and define the focus as the central slice.
    4. Using one of the laser lines, define the stack in the ‘Z-stack’ tool using the ‘Center’ method. Use the ‘Optimal’ button to meet Nyquist criteria for the Z sectioning. We recommend setting the slice number to an uneven number to have the focal plane later as the central slice (Figure 6).


      Figure 6. ‘Z-Stack’ tool of ZEN ‘Acquisition’. ‘The ‘Center’ Method is selected. The focus is centered by activating the ‘Center’ button and the optimal slice thickness (0.110 µm) to meet the Nyquist criteria is chosen.

    5. Acquire the image stacks using first the 642 nm line followed by the 488 nm line, which keeps bleaching at a minimum (Figure 7).


      Figure 7. SIM raw data image. Shown is one of the 21 SIM raw slices (5 phases at 5 rotations) from the z-stack of the RNAPIISer2ph sample. The grid projection is visible as stripes on the nucleus.

    6. Save the SIM raw data image stack.

  3. Raw data acquisition for 3D PALM
    1. Select in the ‘Imaging Setup’ tool ‘Laser WF’ to acquire PALM raw data image stacks and activate the ‘TV1: EM CCD Andor PALM’ button (Figure 8).


      Figure 8. ‘Imaging Setup’ tool of ZEN ‘Acquisition’ for PALM. The experiment is set up for imaging with the 642 nm laser line using the ultra-high power field (uHP). The Andor iXon897 camera is selected by ‘choosing the ‘TV1: EM CCD Andor PALM’ button.

    2. Select the appropriate frame size in the ‘Acquisition Mode’. Since the Andor iXon897 camera to be used for PALM has 512 x 512 active pixels with a pixel size of 16 µm x 16 µm and the Andor iXon885 camera used for SIM has1004 x 1002 active pixels with a pixel size of 8 µm x 8 µm, the frame size in x and y has to be half of that compared to SIM imaging to get finally the same image size. For example, if the SIM image was acquired with 512 x 512 pixels, the PALM image needs to be sampled at 256 x 256 pixels. Do not bin pixels.
    3. Start with the 642 nm laser line. In the ‘Channel’ tool use a low laser power (up to 5%) and high EMCCD gain (200) to re-focus on the sample using 100 ms integration time. Prominent structures might help in refocusing.
    4. Switch to the ultrahigh power field (uHP) to increase power density.
    5. Slide in the 3D PALM slider. Then, activate the ‘Definite Focus’ with continuous stabilization to counteract any z-drift on the touch screen display of the microscope`s stand (Figure 9).


      Figure 9. Set up Definite Focus for continuous operation via the touch screen display of the microscope stand. Press ‘Home’ and select ‘Settings’. On the new screen press ‘Components’ and select ‘Focus’. Press the ‘Continuous’ button to set up continuous operation (A). Move in the 3D PALM slider. Then, activate the ‘Definite Focus’ for continuous stabilization to counteract any z-drift by pressing ‘Home’ and then ‘Microscope’ followed by selecting ‘XYC’ and activating the Definite Focus by pressing the ‘ON' button (B).

    6. Define a time series in the ‘Time Series” tool for at least 20,000 frames. This frame number is mostly sufficient to bleach all available molecules. If then blinking does not mainly disappear, increase frame rate correspondingly. Conversely, if all molecules are bleached earlier, you can stop the experiment with fewer frames.
    7. Decrease the EMCCD gain of the Andor iXon897 camera to 10 V or below. Set exposure time to 30-50 ms. Increase laser power stepwise to 100% to avoid overexposure of the camera.
    8. Once the molecules are brought continuously to their dark states, increase the EMCCD gain until you reach 200 V without overexposure. You should now see individual molecules blinking. If molecules overlap heavily, wait with these settings until you can distinguish single molecules before recording. If the sample is too dim, increase to 300 V. However, you should be aware that >200 V will increase camera noise as well.
    9. Start recording the time series. If the sample does not contain fiducials control the temperature. The ELYRA PS.1 has a large incubator box in which approximately 30 °C. will be reached after 2 h. For 2D-PALM experiments in TIRF mode fiducial markers like Tetraspeck beads at a 1:1,000 dilution can be added conveniently to the solution as they settle to the glass surface and can serve as markers for drift correction. For 3D-PALM experiments their use is less helpful because the focus is not near the glass surface. Therefore, the temperature should be as stable as possible to minimize lateral drift. It is recommended to incubate the sample at least 1 h in the Elyra incubator before running the experiment. Save the 642 nm PALM time series (Figure 10).


      Figure 10. PALM raw data image. Shown is one of the 20,000 PALM raw images of the RNAPIISer2ph sample. Two events, identified as bi-lobed PRLIM PSF (Phase Ramp Localization Imaging Microscopy Point Spread Function) are encircled.

    10. Repeat steps 3-9 for the 488 nm laser line. Save the 488 nm PALM time series.

  4. SIM data processing
    1. Load the SIM image stacks into the ‘SIM’ Processing tool.
    2. Select ‘Manual’ processing and within the menu 'Auto Noise Filter', 'Baseline Cut' and 'Theoretical' PSF'. Leave parameters at default values: 'SE Frequency Weighting' at 1.0 and 'Sectioning' at 100/83/83. As output images select 'SR-SIM + Wide-field' to compare the resolution improvement. Otherwise just select SIM image (Figure 11).


      Figure 11. ‘SIM’ processing tool. Display for ‘Manual’ processing with ‘Auto noise filter’ and ‘Baseline Cut’ active and all parameters at default values. The deconvolution will use a theoretical PSF and the output will be a SIM and a Wide-field image stack.

    3. Process the stack for SIM by activating the ‘Apply’ button. Adjust brightness and contrast for each channel and save. Brightness and Contrast are best adjusted by setting the limits to the lower and upper boundaries of the histogram.

  5. Determination of the SIM resolution
    1. If a fine sub-resolution structure is available in the image, you can use it to define the SIM resolution in the sample. Alternatively, TetraSpeck™ Fluorescent Microsphere beads, ideally embedded in the sample, can be used.
    2. Load the SIM image together with the wide-field image and switch to the ‘Profile’ view tab. Make sure that under ‘Maintain’ in ‘Systems Options’ the ‘Apply brightness and contrast to diagrams and tables’ is selected in the ‘Image display’ tab.
    3. Identify a fine structure and draw a line across the structure.
    4. Adjust contrast and brightness to have the baseline at zero and the peak at 40,000 or 60,000 grey values as is convenient. For dim samples the adjustment of the peak will be at lower grey values.
    5. Export the table of graph to Microsoft Excel and determine the full width at half maximum (FWHM), which gives the resolution (Figure 12).


      Figure 12. Determination of SIM resolution. Shown is an area of RNAPIISer2ph staining in structured illumination (SIM) and wide-field (WF). The graph below shows a profile through a sub-resolution structure having a size of the expected ~120 nm for SIM compared to ~250 nm in WF, corresponding to a doubling of resolution.

  6. Channel Alignment for SIM
    1. Acquire a z-Stack of 200 nm TetraSpeckTM microspheres spread onto #1.5 coverslips and embedded in a hardening resin of your choice with a step size of 0.11 µm and 41 slices in the relevant colors with SIM and process the data with the ‘SIM’ processing tool. Alternatively, use the 200 nm TetraSpeckTM microspheres slide from Carl Zeiss Microscopy GmbH. This acquired stack will be used to calibrate the color offset of the system. Offsets will be stored in a template that later on can be used to correct the experimental data. Calibrated color offsets are specific for the objective and filters. Therefore, a new calibration is required when objectives and filters are exchanged.
    2. Load processed SIM data of the beads into the ‘Channel Alignment’ processing tool (Figure 13). Select the ‘Fit’ checkbox in order to align all channels to the first channel. Select either lateral (in x, y and z) or affine (lateral x, y, z & rotational x, y & stretching x, y) correction as needed.


      Figure 13. ‘Channel Alignment’ tool of ZEN ‘Processing’. The offset (in pixels) of channel 2 (488 nm) to channel 1 (642 nm) can be stored as a .bin file.

    3. Execute the alignment. The offsets in pixels of all channels in reference to the first channel are displayed. Press ‘Save’ to store the created correction template as a .bin file for later use in order to compensate for any chromatic aberrations and offsets between channels in SIM. Since the ‘Channel Alignment’ tool recognizes the image size as well as zoom and offset position, a template acquired in full frame can be used for cropped sample images.
    4. Assess the quality of the channel alignment by comparing the multi-color (TetraSpeckTM) beads before and after alignment. Using a highly corrected Zeiss Plan Apochromat 63x oil/1.4 objective chromatic aberrations are low. Nevertheless, slight offsets can be compensated to less than 20 nm deviation between the 488 nm and 642 nm excitations (Figure 14).


      Figure 14. TetraSpeckTM microspheres recorded with 488 nm and 642 nm excitation using a Zeiss Plan Apochromat 63x /1.4 Oil objective after SIM processing before and after channel alignment. The slight lateral and even smaller axial offsets were corrected to less than 20 nm.

    5. Load the SIM image stack of your sample into the ‘Channel Alignment’ processing tool as ‘Input’ (Figure 15). Most conveniently the image stack might contain the same channels in the same order as the correction template that is selected for correction. In this way, the determined offsets stored in the template can be applied without the need to assign the correct laser identity (ID) to the channels of the SIM image. If the order is not the same, correct IDs have to be assigned to each channel.


      Figure 15. ‘Channel Alignment’ tool of ZEN ‘Processing’. The 2 channel image stacks (excitation with 488 nm and 642 nm) are selected and the correction template with the same lasers in identical order is loaded. In this case the laser IDs of the image stack matches the laser IDs in the template. If this is not the case, the correct laser IDs have to be selected.

    6. Unselect the ‘Fit’ check box and ‘Load’ the correction template (.bin file) to correct the image channels via the values in the correction template. The ‘Fit’ check box should not be selected as otherwise the correction template will not be applied.
    7. Execute the alignment by activating the button ‘Apply’ (Figure 16). The table will show the offsets between the channels in reference to the first channel. Note, that since the beads used for the correction template are on the coverslip, refractive index mismatches do not play a role. If the sample is also fixed onto the coverslip the obtained correction is reliable and differences in the embedding would not matter. One should be aware, however, that if the sample is fixed on the slide, refractive index mismatches might have an influence. Thus, the correction may be less accurate. In these cases, the beads should be embedded in the same medium as the sample.


      Figure 16. Inactive RNAPII (false colored in magenta) and RNAPIISer2ph (false colored in green) before and after channel alignment using a template. The correction is visible especially at the boundaries of the nucleolus (n) devoid of fluorescence and by the lack of overlap in the left and upper area of the overlay.

  7. PSF simulation for 3D PALM data processing
    This procedure describes the PRLIM method. For bi-plane, astigmatism and double-helix approaches different procedures apply. The reader has to refer in those cases to specific protocols.
    1. Use an embedded 200 nm TetraSpeckTM microsphere slide at low density as provided by the manufacturer.
    2. Focus on the beads and use the focal position as the center of the stack. Slide in the 3D-PALM slider. You should see now two equally intense lobes at 45 degrees to each other (Figure 17). If necessary, refocus. Apply the ‘Center’ procedure in the ‘Z-Stack’ tool. Obtain a stack of the beads with the 3D-PALM slider set at 10 nm step size and 401 slices with the respective laser lines. This will ensure sufficient Nyquist sampling.


      Figure 17. PRLIM PSF. Appearance of the bi-lobed PSF generated by PRLIM at the focal plane (0 nm), 500 nm below (-500 nm) and above (+500 nm). The shape of the PSF encodes the z-position of the emitter.

    3. Load the stack into the ‘Experimental PSF’ of the ‘Processing tool’ and execute to create an average PSF (Figure 18). When you slide through the stack, only the two lobes should be visible in the focus. If other signals are visible at the edges of the PSF, you must discard it and acquire a new stack at a different position.


      Figure. 18. Experimental PSF. Averaging of many single PSFs in the XY, XZ and YZ view (A). The experimental PSF will be used for the simulations to create the ‘Localization precision table’. In case of two closely lying beads, a part of the second bead might be taken into account (B, red circle) leading to an impaired experimental PSF (C, red circle). Such an Experimental PSF should not be used. Instead, a new stack with less densely lying beads should be acquired. Alternatively, a region excluding such closely lying beads can be cut off from the original image.

    4. Load the average PSF into the ‘Localization Precision’ processing function under the PALM processing menu and execute to obtain a simulated PSF file with z-localization in dependence of the PSF shape.

  8. PALM data processing, single-molecule counting and distance measurements
    This protocol describes the procedure available in the ZEN 2012 software. Software from other suppliers or freely available software might contain identical or similar possibilities. Please follow in these cases the instructions of the supplier.
    1. Load the PALM time series into the ‘PALM’ processing tool (Figure 19).


      Figure 19. PALM processing tool. A 3D PALM data set was loaded and the parameters set to the displayed values. By activating the ‘PSF File’ button, the PSF with attached 'Localization precision table’ was loaded.

    2. ‘3D’ processing should be automatically selected by the software for a 3D PALM data set. It will show the identified emitters and circles them. Choose ‘Account for Overlap’ for multi-emitter fitting whenever you see intersecting circles, which in 3D-PALM is mostly the case, because its PSF is more spread than a conventional PSF. Use the default value of 6 for ‘Peak Intensity to Noise’ and decrease ‘Peak Mask Size’ from default 19 to 15 to minimize overlapping circles. The 19 is fail-safe, but often the two lobes will fit in a circle of a size of 15. Load the respective simulated PSF file and execute processing.
    3. With the resulting vector map (PALM image) perform a model-based drift correction in the ‘Automatic’ mode (Figure 20).


      Figure 20. PAL-Drift view tab. Model-based drift correction in 'Automatic' mode selected.

    4. Group the detected events with the ‘Capture Radius’ set to 2 pixels and the 'Max On Time’ and the ‘Off Gap’ sets to 4 and 10 frames, respectively (Figure 21). It is unlikely for most of the dyes that the same molecule is on for more than 4 frames or off for more than 10 consecutive frames under the applied imaging conditions. Also, if two events are not within a distance of two pixels, it is unlikely that the two events are caused by the same molecule.


      Figure 21. PAL-Grouping view tab. Definition of filters that determine, which events are considered to be derived from the same emitter.

    5. Render the image at 10 nm x, y pixel size and at 40 nm in z to obtain a Nyquist sampling with a resolution of 20 nm laterally and 80 nm axially, corresponding to the maximal resolution which can be obtained by 3D-PALM (Figure 22). If needed, you can filter the data. For example, if you want to display data which show a localization precision between 10 nm and 40 nm laterally and between 10 nm and 80 nm axially, set the boundaries accordingly for the 'Localization precision' and the 'Localization precision z' in the PAL-Filter tool (Figure 23). If there are enough well localized molecules, this yields a higher resolved image. Store the PALM vector map.


      Figure 22. PAL-Rendering view tab. Selection of `Pixel Resolution’ and ‘Display Mode.’

    6. Define a region of interest using the ‘Graphics’ tool view and select the ‘Use Region(s)’ in the 'PAL-Filter’ view tab to define x, and y dimensions. Set additionally the ‘PositionZ’ range (Figure 23).


      Figure 23. PAL-Filter tab view. The active check-boxes will apply the displayed settings to the image.

    7. Activate ‘Table’ in the ‘PAL-Statistics’ view tab (Figure 24) and scroll down the table to the last entry showing the number of the detected molecules (Figure 25). Please note, that grouping is advisable to avoid counting molecules twice and more. The histogram (Figure 26) can be assessed to estimate the distribution of the parameter values, it can help to set the filters and to check the quality of the data. For example, the histogram for the localization precision will inform about the achievable resolution in the image. In the example shown in Figure 26, the peak is around 40 nm, which is about 10-fold better than the diffraction limited resolution. Furthermore, the histogram indicates that there are few molecules of very high localization precision values, which can be filtered out by setting the respective limits in the PAL-Filter tab view (Figure 23).


      Figure 24. PAL-Statistics view tab. Pressing the ‘Table’ and ‘Statistic Plot’ tabs will display the molecule table with parameters and histograms / scatter plots of the selected parameters.


      Figure 25. Statistics table. The table lists all events with the respective parameters. The last index indicates the total number of detected molecules.


      Figure 26. Histogram. The histogram shows the value distribution of the selected parameter. In this example the localization precision is displayed.

    8. To measure distances, display the localized molecules as centroid or Gaus-render with 1 pixel in the ‘PAL-render’ view tab. Use the ‘Graphics’ line tool to measure distances between two localized molecules (Figure 27).


      Figure 27. Distance measurement of molecules. PALM vector map with Gaus + cross displayed. Distances are measured by drawing a line between two crosses using the ‘Graphics’ tool. Then, the length is displayed.

    9. If clustering is observed (best seen with a Rainbow look up table when molecules are Gaus rendered), draw a circle or ellipsoid using the graphics tool to approximate the outlines of the cluster. Please ensure that the outline is approximated as best as possible. To our experience the measurement error stays within ±10%. Determine the mass of center by the intersection of two perpendicular diameters (circle) or the two axes (ellipsoid). Use the centers to determine the distance between clusters (Figure 28).


      Figure 28. Distance measurements between clusters. Clusters can be most conveniently identified by a Rainbow look up table. They can be approximated by circles or ellipses as shown (in black) for small (small circles) or big (large circle and ellipse) clusters. Their mass center can be determined by the intersection of two diameters (circles) or axes (ellipse). Connecting the centers by a line from the ‘Graphics’ tool will display the length of their distance.

  9. Channel Alignment for PALM and co-localization of molecules
    1. Load the PALM vector maps (tables) into the ‘Convert to Image’ processing tool of the PALM processing functions and execute the conversion of the vector map into a ‘czi’ image format. 'Convert to Image' preserves the voxel resolution as defined in the PALM data set. Please note that the ‘Channel Alignment’ processing function will work on PALM vector maps only if fiducial markers are present.
    2. To merge the two PALM channels use either the ‘Copy’Channels’ or the ‘Channel Alignment’ processing tool. In the latter case, load the first converted PALM image as ‘Input’ and the second one as ‘Input 2’. If the 'Fit' check box is unselected and the 'Input 2' check box active, the two channels will be just copied together without a fit application after activating 'Apply' (Figure 29).


    Figure 29. ‘Channel Alignment’ tool of ZEN ‘Processing’. Two channels are loaded as ‘Input Image’ and ‘Input Image 2’. If the ‘Fit’ box is not selected and the ‘Input 2 box’ selected, then the two channels will be copied together into a merged two-channel image.

    1. Load the merged channels into the ‘Channel Alignment' tool. Select the 'Fit’ button to align the second channel to the first channel. Please note that drift between the two consecutive PALM images is also interpreted as a color offset. When no fiducials are used it is essential that drift is kept at a minimum to avoid any contribution to color offset. Z-drift was compensated with the ‘Definite Focus’, whereas lateral drift was largely minimized by pre-incubation of the sample for at least 1 h in the incubator and keeping the temperature in the incubator constant. Thus, the lateral drift was less than 50 nm over a period of 25 min (Figure 30). If significant structures, like nucleoli devoid of fluorescence, are present, they can be used for channel alignment by drawing a region of interest around them.


    Figure 30. Drifts obtained with ‘Definite Focus’ and pre-incubation of the samples. Frame time was 50 ms.

    1. Fine adjust color correction by using 'Channel Shift' of the ‘Adjust’ processing tool under ‘visual inspection’, if significant structures can be used as landmarks for the alignment (Figure 31). Alternatively, if drift was minimal (≤100 nm), the 'Channel Alignment' tool with the correction template loaded can be used as described for SIM images.


    Figure 31. Inactive RNAPII (false colored in magenta) and RNAPIISer2ph (false colored in green) before and after drift correction and channel alignment followed by visual inspection based on structural landmarks (e. g. nucleolus = n)

    1. Pseudo-color channels, conveniently in green and magenta for co-localization. Look for co-localization (appears in yellow). But any colors with a distinct mixed color will do. For quantitation use the 'Coloc' tool of ZEN.

  10. Alignment of SIM and PALM images
    1. Set the slice thickness of the PAL vector map identical to the slice thickness of the SIM image in the Render view tab (Figure 22).
    2. Convert the PALM vector map into a 'czi' format image using the ‘Convert to Image’ PALM processing tool.
    3. Copy the SIM and converted PALM image together using the ‘Copy Channels’ tool and load into the ‘Channel Alignment’ tool (Figure 32). Alternatively load the SIM and converted PALM images as ‘Input’ and ‘Input 2’ into the ‘Channel Alignment tool’.


      Figure 32. 'Channel Alignment' tool of ZEN ‘Processing’ to align SIM and PALM images representing the same structures

    4. In the latter case, when the SIM and PALM channels represent the same structures, the alignment tool can be used to fit the channels together if the 'Fit' and 'Input 2' boxes are selected. For the ‘Copy channel’ image only the ‘Fit’ box has to be selected. If prominent structures are present, define a region of interest for the alignment. Execute the alignment (Figure 33).


      Figure 33. Overlay of the PALM (color coded in green) on SIM image (color coded in grey) of RNAPIISer2ph before and after channel alignment. The inset on the right upper corner is a magnified view of the boxed area and displays the localization of RNAPIISer2ph molecules Gaus rendered within the reticulate structures revealed by SIM.

    5. If necessary, employ prominent structures for fine adjustment by visual inspection using the 'Channel Shift' of the ‘Adjust’ processing tool group.

Notes

  1. The alignment of PALM and SIM images in 3D needs careful adjustment of the instrument for data acquisition. You have to ensure that the image sizes are the same. If you are using the Andor iXon 885 camera with its 8 µm pixel sized chip for SIM and the Andor iXon 897 camera with its 16 µm pixel for PALM, the frame size for PALM has to be half (8 µm/16 µm) compared to SIM. Thus, if the SIM image was recorded with a frame size of 512 x 512, the PALM image must be taken with a frame size of 256 x 256. This implies that the frame size of SIM must be dividable by 2 in our case, to obtain integer numbers for the frame size of PALM.
  2. The central slice of the SIM image stack must be the focal plane later in the 3D PALM experiment. Any drift in z that occurs between the SIM acquisition and the following PALM recording must be compensated. In course of the long PALM acquisition, active drift control is essential. When matching both images in z, the PALM slice thickness must be exactly adapted to the SIM slice thickness via the 'PALM-Rendering' view tab of the ZEN software. Even then, one should be aware that in SIM the assignment of light contribution by deconvolution (DCV) will be less precise than the localization of molecules. Hence there is the chance of non-matching structures. Aligning SIM and PALM images for one plane is more reliable. In this case the SIM image plane is used for PALM recording and all localized events can be assigned to one plane.
  3. SIM raw data acquisition should start with the longer wavelength to minimize bleaching and cross-excitation.
  4. If cropping is needed during 3D-SIM raw data acquisition, crop only centrally and do not use panning, otherwise PALM and SIM images will not match later on.
  5. During 3D-SIM raw data acquisition the slice number has to be set to an uneven number to have the focal plane later as the central slice.
  6. If beads used for the correction template are on the coverslip, refractive index mismatches do not appear. If the sample is also fixed onto the coverslip the obtained correction is reliable. However, if the sample is fixed on the slide, refractive index mismatches might have an influence. Thus, the correction may be less accurate and the beads should be embedded in the same medium as the sample.
  7. To perform channel alignment for PALM and the co-localization of molecules, please regard that the ‘Channel Alignment’ processing function will work on PALM vector maps only if fiducial markers are present.

Recipes

  1. Phosphate buffered saline solution 10x concentrate (10x PBS)
    1x solution is prepared by 10-fold dilution in ddH2O

Acknowledgments

We thank Andrea Kunze and Joachim Bruder for excellent technical assistance. The protocol was developed based on previously published work of Schubert (2014), Schubert and Weisshart (2015).

References

  1. Doležel, J., Binarová, P. and Lucretti, S. (1989). Analysis of nuclear DNA content in plant cells by flow cytometry. Biol Plant 31: 113-120.
  2. Jackson, D. A., Hassan, A. B., Errington, R. J. and Cook, P. R. (1993). Visualization of focal sites of transcription within human nuclei. EMBO J 12(3): 1059-1065.
  3. Papantonis, A. and Cook, P. R. (2013). Transcription factories: genome organization and gene regulation. Chem Rev 113(11): 8683-8705.
  4. Rieder, D., Trajanoski, Z. and McNally, J. G. (2012). Transcription factories. Front Genet 3: 221.
  5. Schubert, V. (2014). RNA polymerase II forms transcription networks in rye and Arabidopsis nuclei and its amount increases with endopolyploidy. Cytogenet Genome Res 143(1-3): 69-77.
  6. Schubert, V. and Weisshart, K. (2015). Abundance and distribution of RNA polymerase II in Arabidopsis interphase nuclei. J Exp Bot 66(6): 1687-1698.
  7. Zhao, Z. W., Roy, R., Gebhardt, J. C., Suter, D. M., Chapman, A. R. and Xie, X. S. (2014). Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy. Proc Natl Acad Sci U S A 111(2): 681-686.

简介

RNA聚合酶II(RNAPII)是转录大多数真核蛋白编码基因的酶。分析RNAPII的分布和定量可以帮助理解其在间期核中的功能。虽然哺乳动物中的几个研究表明RNAPII在所谓的"转录工厂"中的组织(Jackson等人,1993; Rieder等人,2012; Papantonis和Cook ,2013),但是它们的存在仍然存在争议(Zhao等人,2014)。
近来,基于超分辨率显微镜,在植物中也提出了转录工厂的存在。应用结构化照明显微镜(SIM)和光活化定位显微镜(PALM),分析拟南芥核中的RNAPII分子的分布和数目,并且发现RNAPII丰度和内源多倍体之间的正相关性(Schubert,2014; Schubert和Weisshart,2015)。
这里,我们提出了描述根据它们的内源多倍体水平通过流式分选分离拟南芥间期核的方案,随后是使用特异性抗体的双重免疫染色不同的RNAPII修饰,随后通过空间SIM和PALM评估以获得关于单一无活性和活性RNAPII分子的丰度,分布和共定位的结果。

关键字:超高分辨率显微镜, 细胞核, 转录, RNA聚合酶, 单分子定位

第一部分拟南芥间期细胞核的流式细胞分选

材料和试剂

  1. 22x22 mm高精度盖玻片(170±5μm,编号1.5H)(Marienfeld-Superior)橡胶胶泥(Marabu Australia,目录号:290110000)
  2. 将具有35μm细胞过滤帽(BD Bioscienes,目录号:352235)的5ml聚苯乙烯圆底管或可选地,与一次性过滤器CellTrics,30或5的组合的5ml聚苯乙烯圆底管(BD Biosciences,目录号:352063) 50μm(Sysmex-Partec,目录号:04-0042-2316或04-0042-2317)
    注意:目前,"Corning,Falcon ?目录号:352235"和"Corning,Falcon ?,目录号:352063" >
  3. 0.22μm过滤器
  4. A。 thaliana 玫瑰花叶
  5. 校准珠[SpheroTM彩虹校准颗粒(8个峰)](BD Biosciences,目录号:559123)
  6. Accudrop Beads(BD FACS Accudrop Beads)(BD Biosciences,目录号:345249)
  7. 甲醛溶液37%(Carl Roth GmbH + Co,目录号:7398.1)
  8. 4',6-二脒基-2-苯基吲哚(DAPI)(Life Technologies,Molecular Probes ,目录号:D-1306)
    注意:目前,它是"Thermo Fisher Scientific,Molecular Probes TM ,目录号:D-1306"。
  9. Milli-Q水
  10. NaCl
  11. 2
  12. NaH 2 PO 4 sub
  13. Tris
  14. Na 2 EDTA
  15. Spermin
  16. KCl
  17. Triton X-100
  18. NaOH
  19. β-巯基乙醇
  20. MgCl 2
  21. 蔗糖
  22. 吐温20
  23. HCl
  24. BD FACSFlow鞘液(BD Biosciences,目录号:342003)或者1×PBS(参见Recipes)
  25. 甲醛固定剂(参见配方)
  26. 500 ml Tris缓冲液(见配方)
  27. 200ml裂解缓冲液LB01(Dolezel等人,1989)(参见Recipes)
  28. 100μg/ml DAPI储备溶液(见配方)
  29. 25ml蔗糖缓冲液(见Recipes)

设备

  1. FACSAria IIu(BD Biosciences)

软件

  1. FACSDiva软件

程序

  1. 核隔离
    1. 收获2-3周龄的4至6周龄的植物的玫瑰花叶。 thaliana 转换成蒸馏水(或去离子水)
    2. 将植物组织转移到甲醛中固定 固定剂并在冰上在小玻璃烧杯中孵育20分钟 使用干燥器抽真空
    3. 在滤纸上短暂干燥植物材料。
    4. 在冰上用新的玻璃烧杯在Tris-缓冲液中洗涤2x 10分钟,并干燥 ?关闭植物材料不久在滤纸之间的洗涤 步骤。
    5. 转移植物组织在预冷却的培养皿中,添加 大约250微升的LB01裂解缓冲液,并用锋利的刀片切割,直到得到精细的悬浮液
    6. 添加额外的250-750微升(取决于 关于使用的植物组织的量)的LB01裂解缓冲液并过滤 悬浮液通过30-50μm网(使用带有细胞的管) 过滤器帽或其他一次性过滤器)装入5ml圆底瓶中 聚苯乙烯管并将其存储在冰上。可以使用该悬浮液 直接制备适于免疫染色的核制剂 实验(继续程序C'制备盖玻片)或用于荧光激活 细胞分选(FACS)。后一种变体允许分离 核根据不同的倍性水平并导致制备 的高纯度,保证最低的背景
    7. 通过添加DAPI储备溶液至1-2μg/ml的最终浓度并在冰上储存10分钟来染色悬浮液。

  2. 使用FACSAria细胞分选仪对核进行分选
    1. 打开计算机,细胞仪和蓝色(488 nm)和 紫色(405nm)或(近)UV(375nm)激光并等待30分钟 他们热身。启动FACSDiva软件,检查级别 流式细胞仪窗口和再填充分选缓冲液(BD FACSFlow 或1x PBS),如果需要,清空废物。执行流体启动, 插入一个70μm的喷嘴并打开气流。控制是否正确 选择排序设置('排序' - '排序设置' - '70微米')。打开 '断点'窗口中的'甜点'。
    2. 运行校准珠 控制分流器的性能。如果不显示8个峰 ?在FITC和DAPI的相应对数标度直方图中,清洁流 ?细胞根据FACSAria用户指南或的说明 操作员
    3. 运行Accudrop Beads并确定最佳液滴延迟 ?使用FACSAria的"自动丢弃延迟"功能 FACSAria用户指南。
    4. 运行核心示例,设置触发器 DAPI荧光,并选择一个适当的阈值水平,允许您 ?在侧散射(SSC)面积的点图上显示信号 尺度)对DAPI荧光面积(对数尺度)。调整电压 光电倍增管为DAPI荧光实现显示 ?所有倍性水平群体(图1A)
    5. 定义一个门 核子群体并选择这将显示在直方图中 ?DAPI荧光面积。定义倍性水平的分类区域 ?兴趣(图1B)。
    6. 打开"排序布局"窗口,选择 收集装置'4 Tube',精密模式'纯度'并定义排序 位置。将空的Eppendorf管插入收集装置 持有人
    7. 排序所需的核数(10,000到50,000; 取决于个体倍性水平的可用性和 所需数量的盖玻片)装入收集管并储存 之后在冰上

      图1. A的流分类。拟南芥间期核。 A. SSC面积与DAPI荧光面积的点图 ?表明具有不同DAPI荧光的核的群体 反映不同水平的内源多倍体的强度。 B.直方图 的DAPI荧光在(A)中作为P1门控的颗粒。各个峰上方的门表示2C,4C,8C和16C的??分选门 核。

  3. 盖玻片的制备
    1. 移取12至15微升的蔗糖缓冲液,22x22毫米高精度 盖玻片(Marienfeld)通过橡胶水泥(Marabu)附着在载玻片上 (图2A)。
    2. 随后移液器等同体积的分选 核进入蔗糖缓冲液滴。轻轻地将溶液与a混合 移液器尖端
    3. 空气干燥的盖玻片在室温下过夜。
    4. 盖玻片可立即用于免疫染色或在黑暗中在-20℃下储存长达几个月。

食谱

  1. 1 L 1 x PBS
    154mM NaCl:9g
    7mM Na 2 HPO 4 sup 2 H 2 2H 2 O:1.246g
    4mM NaH 2 PO 4:0.48g
    在A.dest稀释。并用1N NaOH将pH调节至7.0
  2. 甲醛固定剂
    4%甲醛的Tris缓冲液(稀释6ml甲醛溶液37%,用50ml Tris缓冲液),固定剂应在即将使用前制备。参加在敞篷下使用甲醛的工作,并妥善处理废物
  3. 500ml Tris缓冲液
    10mM Tris:0.605g
    10mM Na 2 EDTA:1.861g
    100mM NaCl:2.922g
    0.1%Triton X-100:500μl
    在A.dest稀释。并用1N NaOH将pH调节至7.5
  4. 200ml裂解缓冲液LB01(Dolezel et al。,1989)
    15mM Tris:363mg
    2mM Na 2 EDTA:148.9mg
    0.5mM Spermin:20.2mg
    80mM KCl:1.193g
    20mM NaCl:233.8mg
    0.1%Triton X-100:200μl
    使用1N NaOH将pH调节至7.5,并通过0.22μm过滤器过滤以除去小颗粒。加入220微升β-巯基乙醇,并存储在10毫升等分在-20°C。参加在发动机罩下使用β-巯基乙醇,并妥善处理废物
  5. 100μg/ml DAPI储液
    通过搅拌60分钟溶解5mg 4',6-二脒基-2-苯基吲哚(DAPI)在50ml去离子水中,并通过0.22μm过滤器过滤以除去小颗粒。以0.5ml等分试样在-20℃下储存
  6. 25毫升蔗糖缓冲液
    10mM Tris:30.3mg
    50mM KCl:93.2mg
    2mM MgCl 2·6H 2 O:10.2mg/dm 2 5%蔗糖:1.25g
    0.05%吐温20:12.5μl
    使用5N HCl调节pH至8.0,并以1ml等分试样在-20℃下保存


    图2.样品制备,免疫染色程序和显微镜的盖玻片处理。A.通过应用橡胶水泥在载玻片上固定盖玻片; B.加入封闭溶液;光盘。覆盖用石蜡膜。 E.将载玻片置于湿度箱中; F.用刀片切割橡胶骨水泥; G.提起盖玻片; H.在1×PBS中将1%2-巯基乙醇吸移到含盖玻片室的盖玻片中

第二部分。分选的核的免疫标记

材料和试剂

  1. 一抗
    1. 小鼠单克隆抗RNAPII CTD重复YSPTSPS(1:300)(Abcam plc。, 目录号:ab817)以检测无活性的RNAPII分子
    2. 鼠 单克隆抗RNAPII(磷酸CTD Ser-2)(1:200)(Merck Millipore 公司,目录号:04-1571)以检测活性RNAPII分子
  2. 二抗
    1. 山羊抗小鼠-Cy5(1:300)(Jackson ImmunoResearch Inc.,目录号:715-175-151)
    2. 山羊抗大鼠Alexa488(1:200)(Jackson ImmunoResearch Inc.,目录号:112-545-167)
  3. Milli-Q水
  4. PIPES
  5. MgSO 4 4 /
  6. EGTA(pH 6.9)
  7. BSA
  8. Triton X-100
  9. 1x MTSB(微管稳定)缓冲液(见配方)
  10. 阻止解决方案(参见配方)
  11. 抗体的稀释缓冲液(参见配方)

设备

  1. 湿度室
  2. 幻灯片比色杯[例如垂直玻璃比色杯(Electron Microscopy Sciences,目录号:70318-04)]

程序

  1. 在-20°C储存后,保持盖玻片(在胶片上通过橡胶胶固定)在室温(RT)下10分钟;然后,将它们放在合适的架子上,在60℃的培养箱中干燥45分钟
  2. 在玻片比色杯中,在1x MTSB缓冲液中洗5分钟
  3. 向每个样品加入60μl封闭溶液,覆盖石蜡膜并在室温下在湿度室中孵育1小时(图2B-E)。
  4. 稀释缓冲液稀释一抗。
  5. 取出石蜡膜和封闭溶液,然后加入40μl一抗溶液到每个盖玻片,盖上石蜡膜,并在湿度箱中在10°C孵育过夜。
  6. 将玻片放入比色杯中,在1x MTSB缓冲液中洗涤盖玻片3x 5分钟
  7. 稀释第二抗体,加入40μl的稀释液到每个盖玻片,盖上石蜡膜,并在湿度箱中在10°C孵育过夜。
  8. 在玻片比色杯中的1x MTSB缓冲液中洗涤盖玻片1x 5分钟
  9. 在乙醇系列中脱水(70%,90%,96%),每次5分钟
  10. 空气在黑暗中干燥。

食谱

  1. 1x MTSB(微管稳定缓冲液)
    50 mM PIPES
    5mM MgSO 4 5mM EGTA(pH 6.9)
  2. 封锁解决方案
    8%BSA
    0.1%Triton X-100在1x MTSB中
  3. 抗体的稀释缓冲液
    1%BSA的1x MTSB缓冲液
    以1ml等分试样在-20℃下储存

第三部分。 SIM和PALM评估

该协议基于使用ELYRA PS.1系统与软件ZEN 2012. 2012年。来自其他供应商的系统具有类似的功能。因此,该协议也可以很好地用于包括处理步骤的其他商业系统。在这样的系统上训练时,与这里给出的特定指令的相似性应当变得明显。

材料和试剂

  1. 200nm TetraSpeck TM荧光微球幻灯片(Carl Zeiss Microscopy GmbH)注意:TetraSpeck TM荧光微球是Thermo Fisher Scientific的商标。
  2. 40nm FluoSpheres改进的微球幻灯片(Carl Zeiss Microscopy GmbH,目录号:1783-456)
  3. 40nm FluoSpheres 羧酸酯修饰的微球(Thermo Fisher Scientific)
  4. 200nm TetraSpeck TM 微球(Thermo Fisher Scientific,目录号:T-7280)
  5. 2-巯基乙醇(Sigma-Aldrich,目录号:63689)
  6. NaCl
  7. 2
  8. NaH 2 PO 4 sub
  9. NaOH
  10. 1x磷酸盐缓冲盐水(PBS)(参见配方)
  11. 磷酸盐缓冲盐溶液10x浓缩物(10x PBS)(Sigma-Aldrich,目录号:P5493)(参见Recipes)

设备

  1. 用于22x22mm盖玻片的盖玻片室'Chamlide'(Live Cell Instruments,目录号:CM-S22-1)
  2. 显微镜系统Elyra PS.1(Carl Zeiss Microscopy GmbH)

软件

  1. 软件ZEN 2012(Carl Zeiss Microscopy GmbH)

程序

  1. 样品安装和原子核选择
    1. 通过小心地切割橡胶,从幻灯片中取出盖玻片 水泥,在一侧具有剃刀刀片并将其从滑块提升 (图2F-G)。将盖玻片放入一个22x22毫米的盖玻片室 并用1x PBS中的1%2-巯基乙醇覆盖(图2H)。关闭 盖子
    2. 使用63x/1.4油选择有利的核 平面 - 消色差物镜,X-Cite(LED)照明,目镜和 应用ZEN工具"Locate"(图3)。


      图3。 "定位"显微镜控制。反射荧光的设置 光眼检测被激活(黄线)。

  2. 用于3D SIM的原始数据采集
    使用相同的目标,首先通过随后使用642nm和488nm激光器(用于642nm的51μm栅格和用于488nm激发的34μm栅格)激发,获得SIM图像,然后获得PALM图像(如下一部分所述) 5转)。从较长的波长开始,可最大限度地减少漂白
    1. 从工具"定位"切换到工具"采集",并在中选择 ?'成像设置'工具'SIM'来获取SIM原始数据图像堆栈(图 ?4)。


      图4. ZEN'Acquisition'的'Imaging Setup'工具 选择"SIM",使用轨道1进行双轨实验 ?配置642 nm激光
    2. 在"频道"工具中 定义了使用642 nm激光器作为第一个的多轨实验 和用于第二通道的488nm激光器。调整激光功率 到?10%,摄像机增益到30 V和Andor iXon885的曝光时间 相机到100毫秒查看您的样品。调整激光功率和/或增益if 必要。但使用"范围指示器"以避免过度曝光。在里面 "采集模式"选择适当的帧大小并设置数字 的旋转5.如果需要,通过裁剪进一步调整框架尺寸。如果 使用裁剪,只集中作业,不使用平移,否则PALM 和SIM卡映像以后将不匹配(图5)。


      图5.没有平移的中心裁剪示例

    3. 对样本进行对焦,并将焦点定义为中心切片
    4. 使用其中一条激光线,在"Z-stack"工具中定义堆叠 ?使用"中心"方法。使用'最佳'按钮满足奈奎斯特 Z切片的标准。我们建议将切片编号设置为 使得焦平面稍后作为中心切片的不均匀数量 (图6)。


      图6. ZEN"Acquisition"的"Z-Stack"工具。选择了"中心"方法。焦点以激活为中心 "中心"按钮和最佳切片厚度(0.110μm)来满足 选择奈奎斯特标准。

    5. 获取映像堆栈 首先使用642nm线,然后是488nm线,这保持 漂白至少(图7)。


      图7. SIM原始数据 。显示的是21个SIM原始切片之一(5个旋转5阶段) 从RNAPIISer2ph样品的z-叠加。网格投影是 可看见作为在细胞核的条纹。

    6. 保存SIM原始数据图像堆栈。

  3. 用于3D PALM的原始数据采集
    1. 在"成像设置"工具中选择"激光WF"以采集PALM原始图像 数据图像堆栈并激活"TV1:EM CCD Andor PALM"按钮 (图8)。


      图8. ZEN的"映像设置"工具 的实验 642nm激光线使用超高功率场(uHP)。安道尔 iXon897相机选择'TV1:EM CCD Andor PALM' 按钮
    2. 在中选择适当的框架大小 "获取模式"。自从Andor iXon897相机用于PALM 具有512×512个有效像素,像素尺寸为16μm×16μm, 安卓iXon885相机用于SIM有1004 x 1002有源像素与a 像素大小为8μmx 8μm,则x和y中的帧大小必须为一半 相比于SIM成像最终得到相同的图像尺寸。对于 例如,如果SIM图像是用512×512像素采集的,PALM 图像需要以256 x 256像素进行采样。不要像素点。
    3. 从642 nm激光线开始。在"通道"工具中使用低激光 ?功率(高达5%)和高EMCCD增益(200),以重新聚焦样品 使用100 ms积分时间。突出的结构可能有帮助 重新聚焦。
    4. 切换到超高功率场(uHP)以增加功率密度
    5. 滑动3D PALM滑块。然后,激活"定焦" 具有连续稳定性以抵消触摸上的任何z漂移 屏幕显示(图9)。


      图9.通过触摸设置定焦连续操作 按"首页"并选择 '设置'。在新屏幕上按"组件",然后选择"焦点"。 按"连续"按钮设置连续操作(A)。移动 在3D PALM滑块。然后,激活"定焦" 连续稳定以抵消任何z漂移按"Home" 然后是"显微镜",然后选择"XYC"并激活 按"ON"按钮(B)确定焦点。

    6. 定义a 时间序列"工具中的时间序列至少20,000帧。这个 帧数主要足以漂白所有可用的分子。如果 ?那么闪烁不会主要消失,增加帧速率 相应地。相反,如果所有分子被更早漂白,你 可以用更少的帧停止实验
    7. 降低EMCCD 安东iXon897摄像机的增益至10 V或更低。将曝光时间设置为 30-50毫秒。将激光功率逐步增加到100%,以避免过度曝光 ?相机。
    8. 一旦分子连续进入 它们的暗态,增加EMCCD增益,直到你达到200V ?过度暴露。你现在应该看到个别分子闪烁。如果 分子重叠,等待这些设置,直到可以 在记录之前区分单个分子。如果样品太暗, ?增加到300 V.但是,您应该注意到> 200 V将 增加相机噪音。
    9. 开始记录时间系列。 如果样品不含有基准,控制温度。的 ELYRA PS.1有一个大型孵化箱,其中约30°C。将 2小时后达到。对于在TIRF模式基准中的2D-PALM实验 可以加入标记如1:1,000稀释的Tetraspeck珠 当它们沉降到玻璃表面并且可以时,方便地到溶液 ?用作漂移校正的标记。对于3D-PALM实验 使用不太有用,因为焦点没有靠近玻璃表面。 因此,温度应该尽可能稳定以最小化 横向漂移。建议孵育样品至少1小时 运行实验之前的Elyra孵化器。保存642 nm PALM 时间序列(图10)。


      图10. PALM原始数据图像显示的是RNAPIISer2ph样品的20,000个PALM原始图像之一。 两个事件,标识为双叶PRLIM PSF(相位边缘定位成像显微镜点扩散函数)被包围。

    10. 对488 nm激光线重复步骤3-9。保存488 nm PALM时间序列。

  4. SIM数据处理
    1. 将SIM映像堆栈加载到"SIM"处理工具中。
    2. 选择"手动"处理,并在菜单"自动噪声过滤器"中, '基线剪切'和'理论'PSF'。保留默认参数 值:'SE频率加权'在1.0和'切割'在100/83/83。 作为输出图像选择'SR-SIM + Wide-field'来比较分辨率 改进。否则只需选择SIM卡图像(图11)。


      图11."SIM"处理工具。显示"手动"处理 "自动噪声滤波器"和"基线切割"激活,所有参数均为 默认值。反卷积将使用理论PSF和 输出将是SIM和宽域图像堆栈。

    3. 处理 通过激活"应用"按钮的SIM的堆栈。调整亮度 和每个通道的对比度并保存。亮度和对比度是最好的 ?通过将限制设置为下限和上限来调整 ?柱状图。

  5. 确定SIM分辨率
    1. 如果图像中有精细的子分辨率结构,您可以 ?使用它来定义样本中的SIM分辨率。或者, TetraSpeck?荧光微球珠,理想地嵌入 样品,可以使用。
    2. 将SIM卡映像与 宽场图像并切换到"配置文件"视图选项卡。确保 在"系统选项"中的"保持"下,应用亮度和对比度 ?到图和表"在"图像显示"选项卡中选择。
    3. 识别精细结构,在结构上画一条线。
    4. 调整对比度和亮度以使基线为零 峰值在40,000或60,000灰度值,因为方便。对于暗淡的样品 峰值的调整将为较低的灰度值。
    5. 出口 将表的表转换为Microsoft Excel并确定全宽 半最大值(FWHM),给出分辨率(图12)。


      图12.确定SIM分辨率。显示的是一个区域 RNAPIISer2ph染色在结构化照明(SIM)和宽领域 (WF)。下图显示了通过子分辨率结构的配置文件 ?其具有对于SIM为?120nm的大小与?250nm in相比的大小 WF,对应于分辨率的一倍。

  6. SIM卡的通道对齐
    1. 获得铺在其上的200nm TetraSpeck TM微球的z-堆叠 #1.5盖玻片和嵌入在您选择的硬化树脂中 步长为0.11μm,用SIM和相关颜色的41个切片 使用'SIM'处理工具处理数据。或者,使用 来自Carl Zeiss Microscopy GmbH的200nm TetraSpeck TM微球。 这个获取的堆栈将用于校准的颜色偏移 系统。偏移将存储在稍后可以使用的模板中 以校正实验数据。校准的色偏是特定的 为目标和过滤器。因此,需要新的校准 当目标和过滤器交换时
    2. 加载处理的SIM卡 珠的数据进入"通道对齐"处理工具(图 13)。选择"适合"复选框,以便将所有通道对齐 第一通道。选择横向(在x,y和z)或仿射(横向 x,y,z&旋转x,y&拉伸x,y)校正为 需要

      图13. ZEN的"通道对齐"工具 "处理中"。通道2(488 nm)到通道1的偏移量(以像素为单位) (642 nm)可以存储为.bin文件
    3. 执行 对准。所有通道的偏移量(以像素为单位) 第一个通道。按"保存"保存创建的 校正模板作为.bin文件供以后使用以补偿 用于SIM中的通道之间的任何色差和偏移。以来 ?"通道对齐"工具可识别图像大小以及缩放 和偏移位置,可以使用以全帧获取的模板 裁剪的示例图片。
    4. 评估渠道的质量 通过比较多色(TetraSpeck TM )珠粒之前和之后的比对 对齐后。使用高度校正蔡司计划Apochromat 63x 油/1.4物镜色差低。然而,轻微 偏移可以补偿到488之间的小于20nm的偏差 nm和642nm激发(图14)

      图14。 用488nm和642nm激发记录的TetraSpeck TM TM 微球 使用Zeiss Plan Apochromat 63x/1.4物镜SIM后 处理通道对齐前后。轻微横向和 甚至更小的轴向偏移校正到小于20nm
    5. 将样品的SIM图像堆栈加载到"通道对齐" 处理工具作为"输入"(图15)。最方便的图像 堆栈可能包含相同顺序的相同通道 校正模板被选择用于校正。这样, 可以应用存储在模板中的预定偏移 需要为通道分配正确的激光识别(ID) SIM图像。如果订单不相同,则必须分配正确的ID ?到每个频道

      图15."渠道对齐"工具 ZEN'Processing'。 2通道图像堆栈(激发与488 nm和 ?642nm)和具有相同激光的校正模板 以相同的顺序加载。在这种情况下,图像的激光ID 堆栈匹配模板中的激光ID。如果不是这样, 必须选择正确的激光ID
    6. 取消选择 "合适"复选框和"加载"校正模板(.bin文件) 通过校正模板中的值来校正图像通道。 不应选择"适合"复选框作为校正 模板将不会应用。
    7. 通过执行对齐 激活按钮"应用"(图16)。该表将显示 参考第一通道的通道之间的偏移。注意, 因为用于校正模板的珠子在其上 盖玻片,折射率不匹配不起作用。如果样品 ?也固定在盖玻片上,获得的校正是可靠的 并且嵌入中的差异不重要。应该知道, 然而,如果样品固定在载玻片上,折射率 不匹配可能有影响。因此,校正可以更少 准确。在这些情况下,珠应该嵌入在其中 中等作为样本

      图16.非活性RNAPII(假 有色洋红色)和RNAPIISer2ph(假色为绿色) ?使用模板进行渠道对齐后。修正可见 特别是在没有荧光的核仁(n)的边界 ?以及由于在叠加的左侧和上部区域中缺少重叠。

  7. 用于3D PALM数据处理的PSF模拟
    此过程描述PRLIM方法。对于双平面,散光和双螺旋方法,应用不同的程序。读者在这些情况下必须参考具体的协议
    1. 使用由制造商提供的低密度的嵌入的200nm TetraSpeck TM Suppress TM微球载玻片。
    2. 聚焦在珠子上,使用焦点位置作为中心 堆栈。滑动3D-PALM滑块。你现在应该看到两个平等 强烈的叶瓣彼此成45度(图17)。如果需要, 重新聚焦。在"Z-Stack"工具中应用"中心"过程。获得a 堆叠的珠子与3D-PALM滑块设置在10纳米步长和 401切片与相应的激光线。这将确保足够 奈奎斯特采样

      图17. PRLIM PSF。的外观 由在PRLIM在焦平面(0nm),500nm下方产生的双瓣PSF (-500nm)以上(+ 500nm)。 PSF的形状编码 发射器的z位置。

    3. 将堆栈加载到 "实验PSF"的"处理工具"并执行创建 平均PSF(图18)。当你滑过堆栈,只有两个 叶应该在焦点可见。如果其他信号可见 PSF的边缘,你必须丢弃它并在a获取一个新的堆栈 不同的位置

      图。 18.实验PSF。在XY,XZ和YZ视图中对许多单个PSF求平均值(A)。的 实验PSF将用于模拟创建 '本地化精度表'。在两个靠近的珠的情况下,a 可以考虑第二个胎圈的一部分(B,红色圆圈) 导致受损的实验性PSF(C,红色圆圈)。这样 不应使用实验PSF。相反,一个新的堆栈少 应该获得密集的珠子。或者,一个区域 不包括这种靠近的胎圈可以从原件上切掉 图片。

    4. 将平均PSF加载到'本地化 PALM处理菜单下的精度处理功能 执行以获得具有z定位的模拟PSF文件 ?的PSF形状
  8. PALM数据处理,单分子计数和距离测量
    此协议描述了ZEN 2012软件中提供的过程。来自其他供应商的软件或免费提供的软件可能包含相同或相似的可能性。请在这些情况下遵循供应商的说明
    1. 将PALM时间序列加载到'PALM'处理工具中(图19)。


      图19. PALM处理工具。加载了3D PALM数据集, 参数设置为显示的值。通过激活'PSF文件' 按钮,加载了带有"本地化精度表"的PSF
    2. "3D"处理应由软件自动选择 ?一个3D PALM数据集。它将显示识别的发射器和圆圈 他们。每当选择多发射器配件的"重叠帐户" 你看到相交的圆,在3D-PALM中大多数情况下, 因为它的PSF比传统的PSF更广泛。使用默认值 "峰值噪声强度"值为6,"峰值掩模尺寸" 从默认值19到15,以最小化重叠的圆。 19是 故障安全,但通常两个叶片将适合在大小为15的圆。 ?加载相应的模拟PSF文件并执行处理
    3. 使用结果矢量图(PALM图像)在"自动"模式下执行基于模型的漂移校正(图20)。


      图20. PAL-Drift视图选项卡。选择了"自动"模式下的基于模型的漂移校正。

    4. 将"捕获半径"设置为2像素,对检测到的事件进行分组 并且"最大开启时间"和"关闭间隙"设置为4和10帧, (图21)。这对于大多数染料来说是不可能的 相同的分子打开超过4帧或关闭超过10 在应用的成像条件下的连续帧。另外,如果两个 事件不在两个像素的距离内,则不太可能 这两个事件是由同一分子引起的

      图21. PAL分组视图选项卡定义过滤器, 哪些事件被认为是从同一发射器派生的
    5. 渲染图像在10 nm x,y像素大小和40 nm的z到 获得具有横向20nm和80nm的分辨率的奈奎斯特采样 ?轴向,对应于可以获得的最大分辨率 通过3D-PALM(图22)。如果需要,您可以过滤数据。例如, ?如果要显示显示定位精度的数据 在轴向上在10nm和40nm之间和在10nm和80nm之间, 在PAL-Filter工具(图23)中为'本地化精度'和'本地化精度z'设置相应的边界。如果有 是足够好的局部分子,这产生较高的分辨率 图片。存储PALM矢量图。


      图22. PAL-Rendering视图标签"像素分辨率"和"显示模式"的选择。

    6. 使用"图形"工具视图和定义感兴趣区域 选择"PAL-Filter"视图选项卡中的"使用区域"以定义x和 ?y尺寸。另外设置"PositionZ"范围(图23)。


      图23. PAL-Filter选项卡视图。活动复选框会将显示的设置应用于图像。

    7. 激活'PAL-Statistics'视图选项卡(图24)和 向下滚动表到最后一个条目显示的数字 (图25)。请注意,建议分组 以避免计数分子两次以上。直方图(图26) 可以评估估计参数值的分布,它可以帮助设置过滤器和检查数据的质量。对于 例如,定位精度的直方图将通知 在图像中可实现的分辨率。在图1所示的例子中 如图26所示,峰值为约40nm,这是约10倍 衍射极限分辨率。此外,直方图指示 存在极少的具有非常高的定位精度值的分子, ?它可以通过设置相应的限制过滤掉 PAL-Filter选项卡视图(图23)。


      图24. PAL统计 ?视图标签。将显示"表格"和"统计图"选项卡 分子表中的参数和直方图/散点图 ?所选参数

      图25.统计表。 表格列出了具有相应参数的所有事件。最后一个索引 表示检测到的分子的总数

      图 26.直方图。直方图显示的值分布 选择参数。在这个例子中,定位精度是 显示
    8. 要测量距离,请显示本地化 分子作为质心或高斯渲染具有1个像素的'PAL-render' 视图选项卡。使用"图形"线工具来测量两者之间的距离 局部分子(图27)

      图27.距离 测量分子。显示高斯+交叉的PALM矢量图。 通过在两个十字之间使用线绘制线来测量距离 '图形'工具。然后,显示长度。

    9. 如果 观察聚类(最好用彩虹查找表查看时间 分子是高斯渲染),使用绘制圆或椭圆 图形工具来近似集群的轮廓。请确保 轮廓被尽可能最佳地近似。我们的经验 测量误差保持在±10%以内。确定中心的质量 ?两个垂直直径(圆)或两个 轴(椭圆体)。使用中心来确定之间的距离 簇(图28)。


      图28.距离测量 。群集可以最方便地由a标识 彩虹查寻表。它们可以由圆或椭圆近似 如图所示(黑色)为小(小圆圈)或大(大圆圈和 椭圆)簇。它们的质心可以由 两个直径(圆)或轴(椭圆)的交点。连接 中心由"图形"工具的一条线显示长度 的距离。

  9. PALM的通道比对和分子的共定位
    1. 将PALM矢量映射(表)加载到"转换为图像" 处理工具的PALM处理功能并执行 将矢量图转换为"czi"图像格式。 '转换成 图像"保留如在PALM数据集中定义的体素分辨率。 请注意,"通道对齐"处理功能将起作用 仅在存在基准标记时在PALM矢量图上
    2. 至 合并两个PALM通道使用'Copy'Channels'或 '通道对齐'处理工具。在后一种情况下,加载第一个 转换的PALM图像作为"输入",第二个作为"输入2"。如果未选中"适合"复选框,并且"输入2"复选框处于活动状态, 两个通道将只是复制在一起,没有一个合适的应用程序 激活"应用"(图29)。


    图29. ZEN"处理"的"通道对齐"工具。两个通道作为"输入图像"和"输入图像2"加载。如果未选择"适合"框并选择"输入2框",则两个通道将一起复制为合并的双通道图像。

    1. 将合并的通道加载到"通道对齐"工具中。选择 "Fit"按钮将第二个通道与第一个通道对齐。 请注意,两个连续PALM图像之间的漂移也是 解释为颜色偏移。当没有使用基准时 至关重要的是,漂移被保持在最小,以避免任何贡献 颜色偏移。 Z-漂移用"确定焦点"补偿,而 ?通过样品的预温育大大减小了侧向漂移 ?在孵育器中保持温度至少1小时 孵化器常数。因此,横向漂移小于50nm 期间25分钟(图30)。如果显着结构,如核仁 没有荧光,存在,它们可以用于通道 通过绘制围绕它们的感兴趣区域来进行对准。


    图30.通过"确定焦点"和样品预孵育获得的漂移。帧时间为50 ms。

    1. 使用"调整"的"频道偏移"精细调整颜色校正 ?加工工具在"目视检查"下,如果有意义 结构可以用作对齐的界标(图31)。 或者,如果漂移最小(≤100nm),"通道对准" 工具与加载的校正模板可以按照所述使用 SIM图像。


    图31.在漂移校正和通道对准之前和之后的非活性RNAPII(洋红假色)和RNAPIISer2ph(假色),然后基于结构界标(例如核仁= n )

    1. 伪彩色通道,方便地为绿色和品红色 共定位。寻找共定位(以黄色显示)。但任何 颜色有明显的混合颜色。对于定量使用 ZEN的"Coloc"工具。

  10. SIM和PALM图像的对齐
    1. 设置PAL向量图的切片厚度与切片相同 渲染视图选项卡中的SIM图像的厚度(图22)。
    2. 使用"转换为图像"PALM处理工具将PALM矢量地图转换为"czi"格式图像。
    3. 使用"复制通道"工具将SIM和转换的PALM图像复制在一起,并加载到"通道对齐"工具中(图32)。 或者,将SIM和转换的PALM图像加载为"输入"和 "输入2"到"渠道对齐"工具中。


      图32. ZEN的"通道对齐"工具处理以对齐表示相同结构的SIM和PALM图像

    4. 在后一种情况下,当SIM和PALM信道代表 相同的结构,对准工具可以用于安装通道 一起,如果选择'适合'和'输入2'框。对于"复制" 通道"图像,则只需选择"适合"框。如果突出 结构,定义用于比对的感兴趣区域。 执行对齐(图33)。


      图33.的叠加 在SIM图像上的PALM(颜色编码为绿色)(颜色编码为灰色) RNAPIISer2ph通道对齐之前和之后。右侧的插图 上部角落是装箱区域的放大视图并显示 RNAPIISer2ph分子的定位 网状结构由SIM显示。

    5. 如有必要,采用 突出结构用于通过目视检查进行微调 "调整"处理工具组的"通道移位"。

笔记

  1. 在3D中PALM和SIM图像的对准需要仔细调整用于数据采集的仪器。您必须确保图像大小相同。如果您使用Andor iXon 885相机,其8米像素尺寸的芯片用于SIM和安道尔iXon 897相机,其16米像素用于PALM,PALM的帧尺寸必须是一半(8μm/16μm) SIM。因此,如果以512×512的帧大小记录SIM图像,则PALM图像必须以256×256的帧大小拍摄。这意味着SIM的帧大小在我们的情况下必须可以除以2,获得PALM的帧大小的整数。
  2. 在3D PALM实验中,SIM图像堆栈的中心切片必须是焦平面。必须补偿在SIM捕获和以下PALM记录之间发生的z中的任何漂移。在长PALM采集的过程中,主动漂移控制是必要的。当在z中匹配两个图像时,PALM切片厚度必须通过ZEN软件的"PALM-Rendering"视图选项卡精确地适应SIM切片厚度。即使如此,应该意识到在SIM中,通过去卷积(DCV)的光贡献的分配将比分子的定位更精确。因此,存在不匹配结构的机会。对齐一个平面的SIM和PALM图像更可靠。在这种情况下,SIM图像平面用于PALM记录,所有本地化事件可以分配到一个平面
  3. SIM原始数据采集应从较长的波长开始,以尽量减少漂白和交叉激发。
  4. 如果在3D-SIM原始数据采集期间需要裁剪,则仅集中裁剪,不使用平移,否则PALM和SIM卡图像以后不匹配。
  5. 在3D-SIM原始数据采集期间,切片数目必须设置为不均匀的数目,以使焦平面稍后作为中心切片。
  6. 如果用于校正模板的珠在盖玻片上,则不出现折射率不匹配。如果样品也固定在盖玻片上,获得的校正是可靠的。然而,如果样品固定在载玻片上,折射率不匹配可能具有影响。因此,校正可能不太准确,并且珠应该嵌入在与样品相同的介质中
  7. 要执行PALM的通道对准和分子的共定位,请注意,仅当存在基准标记时,"通道对准"处理功能才能在PALM矢量图上起作用。

食谱

  1. 磷酸盐缓冲盐溶液10x浓缩物(10x PBS)
    1x溶液通过在ddH 2 O中10倍稀释制备

致谢

我们感谢Andrea Kunze和Joachim Bruder的优秀技术援助。该协议是基于以前发表的Schubert(2014),Schubert和Weisshart(2015)的工作开发的。

参考文献

  1. Dole?el,J.,Binarová,P。和Lucretti,S。(1989)。 通过流式细胞术分析植物细胞中的核DNA含量。 em> 31:113-120。
  2. Jackson,D.A.,Hassan,A.B.,Errington,R.J.and Cook,P.R。(1993)。 人类核内转录的聚焦位点的可视化 EMEM (3):1059-1065。
  3. Papantonis,A.和Cook,P.R。(2013)。 转录工厂:基因组组织和基因调控 Chem Rev 113(11):8683-8705。
  4. Rieder,D.,Trajanoski,Z.和McNally,J.G。(2012)。 转录工厂 前基因 3:221. < br />
  5. Schubert,V。(2014)。 RNA聚合酶II在黑麦和拟南芥中形成转录网络 Cytogenet Genome Res 143(1-3):69-77。
  6. Schubert,V。和Weisshart,K。(2015)。 RNA聚合酶II在拟南芥间期细胞核中的丰度和分布。 a> J Exp Bot 66(6):1687-1698。
  7. Zhao,Z.W.,Roy,R.,Gebhardt,J.C.,Suter,D.M.,Chapman,A.R。和Xie,X.S。(2014)。 RNA聚合酶II在哺乳动物细胞核内的空间组织通过反射光片超分辨率显微镜显示。/a> Proc Natl Acad Sci USA 111(2):681-686。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Weisshart, K., Fuchs, J. and Schubert, V. (2016). Structured Illumination Microscopy (SIM) and Photoactivated Localization Microscopy (PALM) to Analyze the Abundance and Distribution of RNA Polymerase II Molecules on Flow-sorted Arabidopsis Nuclei. Bio-protocol 6(3): e1725. DOI: 10.21769/BioProtoc.1725.
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