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Measuring Cyanobacterial Metabolism in Biofilms with NanoSIMS Isotope Imaging and Scanning Electron Microscopy (SEM)
利用纳米SIMS同位素成像和扫描电子显微镜(SEM)测量蓝藻生物膜中的代谢   

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mBio
May/Jun 2016

Abstract

To advance the understanding of microbial interactions, it is becoming increasingly important to resolve the individual metabolic contributions of microorganisms in complex communities. Organisms from biofilms can be especially difficult to separate, image and analyze, and methods to address these limitations are needed. High resolution imaging secondary ion mass spectrometry (NanoSIMS) generates single cell isotopic composition measurements, and can be used to quantify incorporation and exchange of an isotopically labeled substrate among individual organisms. Here, incorporation of cyanobacterial extracellular organic matter (EOM) by members of a cyanobacterial mixed species biofilm is used as a model to illustrate this method. Incorporation of stable isotope labeled (15N and 13C) EOM by two groups, cyanobacteria and associated heterotrophic microbes, are quantified. Methods for generating, preparing, and analyzing samples for quantifying uptake of stable isotope-labeled EOM in the biofilm are described.

Keywords: Stable isotopes (稳定同位素), Extracellular matrix (细胞外基质), Extracellular organic matter (细胞外有机物), Microbial ecology (微生物生态学), Substrate incorporation (底物掺入), NanoSIP (NanoSIP)

Background

Stable isotope labeling combined with NanoSIMS (‘NanoSIP’) is an established method to quantify incorporation of stable isotope labeled substrates into individual microbial cells, which can then be extrapolated to estimate incorporation for a population of cells (for example, Lechene et al., 2006 and Woebken et al., 2012). Tracing multiple stable isotope labels (e.g., 13C and 15N) into individual cells can be used to examine differential incorporation between treatments over time (for example, Popa et al., 2007 and Stuart et al., 2016a). Biofilms present specific challenges to quantifying incorporation of label. Since individual organisms are embedded in an extracellular matrix and have a diverse range of cell sizes and shapes, cell counts and biomass calculations are difficult. Additionally, polymeric labeled substrates, such as EOM, can adhere to the matrix and cell surfaces, so unincorporated label needs to be accounted for. Imaging-based methods such as NanoSIMS, paired with SEM and fluorescence microscopy, are well-suited to address these challenges because cell sizes and unincorporated label can be identified. Here, we describe methods to address these challenges in order to quantify the incorporation of labels (13C and 15N) from a polymeric substrate (EOM) into a photosynthetic biofilm. EOM is extracellular material that is loosely associated with cells, and is separated from the cells in the biofilm. One drawback of this method is that biofilm spatial structure (the extracellular matrix) is not preserved. If the examination of spatial arrangements is desired, embedding and sectioning of the biofilm samples may be necessary (for example, Lechene et al., 2006).

Materials and Reagents

  1. Sterile cell scrapers (VWR, catalog number: 89260-224 or 89260-222 )
    Note: The product “ 89260-224 ” has been discontinued.
  2. 0.2 µm syringe filters (polyethersulfone membranes, e.g., Pall, catalog number: 4652 )
  3. Pipet tip  
  4. 1.7 ml microcentrifuge tubes
  5. Sterile syringes
  6. Silicon (Si) wafers, sized to fit NanoSIMS holder (e.g., 5 x 5 mm, Ted Pella, catalog number: 16008 )
  7. Sterile tissue grinder (Fisher Scientific, catalog number: 02-542-08 )
  8. Biofilm culture
    Note: We examined a unicyanobacterial mixed species biofilm, however, the protocol is suitable for analysis of most biofilm types, provided that an aqueous substrate can be added to the sample, with even distribution. A detailed description of our biofilms and culturing procedures can be found in Stuart et al., 2016a.
  9. Stable isotope labeled compound appropriate to support growth, or metabolism, of organism(s) of interest (e.g., 99 atom percent excess [atm%] 13C-NaHCO3, Cambridge Isotope Laboratories, catalog number: CLM-441-1 )
  10. Appropriate defined medium for growth (e.g., modified artificial seawater base [ASN] medium, described in Stuart et al., 2016a)
  11. 50% ethanol (EtOH)
  12. 37% formaldehyde solution (Sigma-Aldrich, catalog number: 252549 )
  13. 10x phosphate buffered saline (Sigma-Aldrich, catalog number: P5493 )
  14. Sodium chloride (NaCl)
  15. Sterile MilliQ water
  16. 4% PFA (see Recipes)
  17. Sterile 10% sodium chloride (NaCl) solution (see Recipes)
  18. Modified artificial seawater base (ASN) medium recipe (with nitrate) (see Recipes)

Equipment

  1. Dounce homogenizer (WHEATON, catalog number: 357546 )
  2. Microcentrifuge, capable of variable rpm generating up to 15,000 x g
  3. Isotope-ratio mass spectrometer (e.g., ANCA-IRMS, PDZE Europa Limited, Crewe, England)
  4. Chambers for biofilm cultivation (e.g., Pyrex sealable flasks, Corning, PYREX®, catalog number: 4985-1L )
  5. Incubator (as appropriate for cultivating the organism of interest)
  6. Dissecting microscope (e.g., Fisher Scientific, model: Fisher ScientificTM 420 Series , catalog number: 11-350-124)
  7. Desiccator cabinet (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5317-0070 )
  8. Epifluorescence light microscope (e.g., Leica Microsystems, model: Leica DMI600B )
  9. Diamond or carbide scribe (e.g., Ted Pella, catalog number: 54412 )
  10. Gold (or other conductive metal) sputter coater (e.g., Cressington, model: Sputter Coater 108 )
  11. NanoSIMS 50 or 50 L (AMETEK, Cameca, model: NanoSIMS 50 or NanoSIMS 50 L )
  12. Scanning electron microscope (SEM) with better than 50 nm resolution and micrograph recording capability (e.g., FEI, model: FEI Inspect F SEM )

Software

  1. Ion image data processing software (e.g., LIMAGE, L. Nittler, Carnegie Institution of Washington, Washington, DC, USA; ImageJ with MIMS plugin; Look@NanoSIMS [Polerecky et al., 2012] or similar)

Procedure

  1. Rare isotope substrate generation, biofilm incubations, fixation and preparation on Si wafers
    1. Generate rare isotope enriched extracellular organic matter (EOM)
      Note: Substrate for isotope labeling will depend on hypotheses being tested that are specific to the experiments conducted. Here, we describe generation of a polymeric rare isotope labeled substrate, EOM, but substrates may also be simple defined compounds (e.g., 13C-amino acids, glucose, bicarbonate, or 15N nitrate, ammonium). Substrate used will be dependent on hypotheses being tested.
      1. Inoculate and grow a biofilm (or pure culture) in sealed flasks (with no headspace) to late exponential phase, with fresh media of similar composition to experimental conditions but where the growth substrate of interest is replaced by a rare isotope analog (e.g., ASN with 3 mM 13C sodium bicarbonate and 1.76 mM K15NO3).
        Notes:
        1. Biofilms are cultured without headspace in order to avoid CO2 (without 13C label) to equilibrate from the air into the medium, however, the 13C sodium bicarbonate will become depleted and will need to be refreshed over time. Frequency of addition of 13C sodium bicarbonate will depend on C-fixation rates of the biofilm. Our additions were made every 48 h.
        2. Maintaining biofilms in conditions to which they have been previously acclimated is recommended. In this case, biofilms were cultured with a 12:12 light:dark cycle at 23 °C and 20 μmol m-2 sec-1 light.
      2. Transfer the biofilm(s) an additional three times into the rare-isotope media at 1:50 dilution.
        Note: The target biomass of late exponential biofilm for these cultures is 50-100 mg dry weight (in this case, 50 ml cultures, 3 weeks of growth).
      3. Inoculate this rare-isotope-labeled culture into a larger volume fresh rare isotope media and grow to late exponential phase, at the volume(s) needed to generate approximately 1-5 g dry weight. Inoculum can be split into multiple flasks at this stage, ensuring equal inoculation of each culture. For our biofilms, this was achieved with four 500 ml flasks of cultures grown for 3 weeks.
      4. Harvest biofilm(s) and separate EOM (adapted from Jiao et al., 2010; Stuart et al., 2016b).
        Note: Methods described here are specific to hypersaline filamentous cyanobacterium, ESFC-1, and will need to be tested and modified for different species.
        1. Decant and discard media overlaying biofilm and transfer biofilm to sterile Dounce homogenizer. If biofilm is adherent, use sterile cell scraper.
        2. Add 1-2 ml sterile solution.
          Note: For our hypersaline biofilms, 10% NaCl was used, and salt concentration chosen based on that of defined medium, (e.g., ASN).
        3. Homogenize biofilm.
          Note: This will require microscopic examination initially to ensure minimal cell lysis during homogenization.
        4. Incubate homogenate for 15 min at 40 °C.
          Note: This is to loosen the exopolysaccharides from the cells. This will require initial tests, such as microscopic examination, to ensure minimal cell lysis is occurring during this step.
        5. Centrifuge homogenate at 15,000 x g at 4 °C for 20 min.
        6. Separate supernatant from cell pellet and filter through 0.2 µm syringe filter. Filtered supernatant is EOM. Store at -80 °C until use.
          Note: Polyethersulfone membranes are recommended since they have low protein and polysaccharide binding.
      5. Determine the bulk isotope ratio of cell pellet and EOM by isotope-ratio mass spectrometry (e.g., ANCA-IRMS, PDZE Europa Limited, Crewe, England).
    2. Inoculate cells into replicate preferred cultivation chambers, cultivate for biofilm development, measuring growth over time, a detailed description of growth conditions for our cyanobacterial biofilms can be found in Stuart et al., 2016b.
      Notes:
      1. Minimize volume of biofilm cultures as much as possible, while maintaining relevant conditions, to conserve use of stable isotope substrate (EOM, in this case), generated in step A1.
      2. To obtain quantitative measurements of elemental uptake (µg C or N fixed), biomass measurements for all biofilm community members are necessary. Microscopy of a subportion of the fixed biofilm may be used to generate biovolume measurements (Stuart et al., 2016b).
      3. A fully defined medium (e.g., ASN [Stuart et al., 2016a]) is recommended, in which all C and N sources are known, so that biofilm C and N budgets can be estimated.
    3. Add stable isotope labeled substrate(s) to naïve biofilm culture
      1. Add isotopically rare growth substrate (step A1) to unlabeled biofilm cultures (step A2).
      2. Control treatments should include (at minimum):
        1. Killed controls–fix biofilms with 4% PFA (see Recipes below) for 1 h prior to experiment start, rinse off fixative, then treat similarly to other live samples.
        2. Controls with no stable isotope added.
        3. Optional: other controls to address hypotheses regarding specific metabolisms (for example, in presence of specific inhibitors).
      3. At chosen time points, remove spent media from biofilm cultures.
        Note: Time points are dependent on metabolic rates being targeted, and growth rates of test organisms. Our time points ranged from 1 h to 24 h, based on the growth rate of our organism (approximately 0.1 day-1) and bulk rates of C-fixation based on 13C-bicarbonate incorporation (IRMS measurements).
      4. Optionally: Save spent media for further analysis–filter through 0.2 µm polyethersulfone syringe filters and freeze the filtrate at -80 °C.
    4. Fix tissues to preserve ultrastructure
      1. Add 4% PFA to biofilm tissues and incubate either 1 h at room temperature or overnight at 4 °C.
        Note: Glutaraldehyde or ethanol are acceptable alternative fixatives.
      2. Remove 4% PFA and rinse biofilms carefully a minimum of 3 times with sterile H2O.
        Note: It is important to thoroughly remove salt and fixatives, as these interfere with NanoSIMS measurements.
      3. Store biofilms in 50% EtOH, in enough volume to cover the biofilms, at -20 °C.
        Note: Fixed biofilms can be stored under these conditions for up to a few years.
    5. Prepare fixed biofilm samples for microanalysis and imaging
      1. Using a pipet tip, transfer a piece of fixed biofilm into a sterile tissue grinder containing 1 ml of sterile H2O.
      2. Grind gently to break up biofilm and transfer to a 1.7 ml microcentrifuge tube.
      3. Centrifuge at 10,000 x g for 1 min, remove supernatant and add 1 ml of sterile H2O. Repeat rinse process, remove supernatant and then resuspend in 10 µl of 50% EtOH. These steps help to remove excess extracellular organic matter that adheres to cell surfaces. This organic matter can interfere with NanoSIMS analysis of cellular uptake.
      4. For mixed species biofilms (i.e., non-axenic mixtures of bacteria and cyanobacteria), the centrifugation speed must be high enough or long enough to collect bacterial cells in addition to filaments (e.g., 15,000 x g, and/or more than 20 min).
      5. Under a dissecting microscope, gently score silicon wafers with a diamond scribe to create a ~2 x 2 mm spaced grid (see Figure 1A). Pipet 1-2 µl of sample onto one square of the grid, let dry and examine under the microscope–aim for approximately 30 filaments per spot, if there are too many or too few, wash off sample and concentrate/dilute sample in tube as necessary. In the ideal preparation, cells should form a monolayer, with minimal overlap or clumping.
      6. Replicate samples and treatments may be pipetted into additional grid squares; they should be close together, without risking overlap.
      7. Dry samples overnight or longer at room temperature, protected from dust and humidity.
      8. Optional: image with an epifluorescence microscope to assess cell density and configuration. Be aware that fluorescence imaging may not be performed after metal coating.


        Figure 1. Representative image of samples and analysis. A. Silicon wafer with 5 samples spotted on grid. Scale bar is 1 mm. Red box and arrow indicate location of SEM image. B. SEM image. Red box indicates a representative NanoSIMS analysis area, NanoSIMS output of this area is shown in C-E. C. NanoSIMS 12C14N ion image, showing cumulative counts in a 30 x 30 µm area, with a 2562 pixels beam spot size, over a duration of 30 cycles. D. 13C/12C ratio image; E. 15N/14N ratio image. Numbered white circles in (C-E) indicate representative regions of interest (ROIs) for analysis. ROIs 1-3 values are in Table 1. Scale bars for (B-E) are 10 µm.

    6. Use sputter coater to coat samples with a highly conductive metal surface
      1. A gold or iridium coat is typical; aim for a 5 to 10 nm coating; thicker for larger cells or preparations with increased topographic relief. Carbon coating may be used, but will interfere with carbon isotope measurements.
      2. Metal coating is not necessary for low-relief samples (< 1 μm), but it improves SEM imaging and initial imaging of the sample in the NanoSIMS.
    7. Place Si wafers in a holder
      1. If the SEM chamber is large enough, samples may be placed in a NanoSIMS holder; otherwise, SEM image samples prior to loading in a NanoSIMS holder.

  2. SEM imaging
    1. SEM imaging is very useful for selecting analysis targets and ensuring they are clear of interfering debris or non-target cells. Analysis locations selected in the SEM may be relocated in the NanoSIMS if SEM images of the target location are collected at three scales (100x, 500x, and 3,000x). SEM stage coordinates can also be used to relocate targets that are too small or indistinct to identify in the NanoSIMS light imaging system (CCD camera); this is not necessary for most cell dispersions on Si wafers.
    2. Place holder with samples into the SEM.
    3. Adjust spot size and kV as needed for the instrument; low voltage imaging can reduce charging if the sample has not been metal coated.
    4. Select analysis targets and image at low, medium and high magnification (100x, 500x, 3,000x).
      1. Choose locations where multiple filaments are adjacent or overlap, but are still clearly distinguishable (see Figure 1B).
        Note: Overlapping filaments will allow for analysis of 2-3 filaments in one analysis window, maximizing NanoSIMS analysis time, however, if they are not clearly distinguishable this will compromise the analysis.
      2. If heterotropic bacterial cells are of interest, collect high magnification images (> 3,000x) in order to clearly visualize them (Figure 1B).
      3. Collect enough SEM images for analysis of at least 20 filaments per sample.

  3. NanoSIMS analysis
    1. Load sample into NanoSIMS 
      1. Samples may require 1-12 h to come to the necessary vacuum, depending on the sample type, dryness and density of cell material.
    2. Analysis for C and N isotopes is performed using a Cs+ analysis beam. Negative secondary ions are extracted in this mode.
    3. Tune the instrument and set up masses for the desired analysis. To measure 13C or 15N enrichment in cells, ion masses of 24, 25, 26, 27 (12C2-, 13C12C-, 12C14N-, and 12C15N- respectively) should be collected. Previous work indicates the C dimer (e.g., 12C2-) has superior yield to the 12C monomer and better coincidence with the secondary mass spectrometer settings for CN-(Pett-Ridge and Weber, 2012). 
      1. Mass resolving power (ΔM/M) > 6,800 is necessary to resolve isobaric interferences.
      2. Reference materials should be analyzed to ensure correct instrument performance. This can simply be unlabeled controls, though a consistently run sample can provide higher confidence in the results (Pett-Ridge and Weber, 2012).
    4. Choose analysis beam current
      1. Typically, analysis speed is the primary factor considered in the choice of beam current, because CN- is a high yield species, and the maximum sustainable count rate for the NanoSIMS detectors is ~250,000 counts per second, which is easily achieved from cells with 1 to 2 pA Cs+. These settings will typically yield ~150 nm diameter analysis beam. This diameter sets the ultimate spatial resolution of the analysis.
      2. If higher spatial resolution is desired, lens and aperture settings that reduce the diameter of the analysis beam and reduce the beam current should be used.
    5. Choose and save locations to analyze
      1. General locations are first recorded based on points located in the CCD camera (light imaging). If desired targets cannot be visualized in the CCD camera, other land marks must be located, and measurements determined from an SEM image or a coordinate transformation can be used to locate the targets in SIMS mode.
      2. SIMS real-time imaging is necessary to ensure that targets selected in CCD imaging are centered in the imaged field.
    6. Set up analysis parameters to use on each mapped location and analyze. This includes:
      1. Size of analysis raster, which will depend on the size of the cells being analyzed. Usually between 10 x 10 µm and 25 x 25 µm.
      2. Pixel number, which is typically based on the analysis beam spot size (linear pixel number) ~2 x (raster size)/(spot size). For a typical 150 nm diameter analysis beam, 1282 pixels would be reasonable for a 10 x 10 µm raster, and 2562 pixels for a 25 x 25 µm raster.
      3. Pre-analysis sputtering: typically, a high-current Cs+ beam is rastered over the sample before data collection to enhance and stabilize the secondary ion count rate from the sample. The sample is sputtered to approximately twice the depth of Cs+ implantation, which is on the order of 60 nm. One can estimate the necessary duration of sputtering based on the sputter rate (ξ), primary ion beam current (I), sputtering time (t), raster area (A) and sputtered depth (d):



        For biological materials, the sputter rate may be estimated as 2.5 nm µm2 pA-1 sec-1 (Ghosal et al., 2008). Longer pre-analysis sputtering may be necessary to remove residual materials from cell surfaces. The optimal duration of sputtering can be determined empirically by monitoring count rates and isotopic ratios with depth.
      4. Analysis duration, which should be timed according to the number of counts needed from the smallest target to achieve sufficient analysis precision; 10,000 counts of the minor isotope will result in ~1% analysis precision.
        Notes:
        1. Determining consistent data collection parameters will likely be an iterative process, especially for a new sample, where enrichment levels, residual material on the cell surfaces, and within-sample variability, is unknown. Samples may need to be analyzed and examined, after which conditions may be altered slightly to ensure appropriate data collection.
        2. It is critical that unlabeled and killed controls be run. These results are used for the enrichment calculations below.

Data analysis

  1. Analysis of NanoSIMS ion images requires special software that can quantitatively process the images on a pixel by pixel basis. Several software packages are available (e.g., LIMAGE [L. Nittler, Carnegie Institution of Washington, DC]), including free versions.
  2. Results can be visualized based on quantitative ion images (such as 12C14N- ion Figure 1C) or ion ratio images (13C/12C, Figures 1D-1E) for each analyzed area. Ratios are calculated on a pixel by pixel basis.
  3. For stable isotope labeling experiments such as these, data are typically presented as ratios, in delta notation, atom percent of the minor isotope, or atom percent excess (APE) of the minor isotope. APE is an ideal presentation since it is simple and a direct representation of the uptake of the label (see equation below). While common in the literature, delta notation may be less intuitive for highly enriched samples. The choice of units, however, does not affect data visualization, either for images or graphs.
  4. Regions of interest (ROIs) surrounding target cells must be determined for quantification of isotopic ratios (Figures 1C-1E). ROIs are typically drawn manually, but automated routines can also be successful.
    1. For biofilm bacterial cells or cyanobacterial filaments, ROIs can be hand drawn based on secondary electron and 12C14N- ion images (Figure 1C), both of which yield a good indication of cell boundaries. It is also useful to examine 13C/12C and 15N/14N images, as these images may allow hotspots of residual labeled substrate to be excluded (Figures 1D-1E).
    2. Isotopic ratios, uncertainties, and pixel size are extracted for each ROIs.
    3. Atom percent enrichment (APE) of the ROI can be calculated according to (Table 1):
      1. APE = [Rf/(Rf/(Rf + 1) - Ri/(Ri + 1)] x 100%
      2. Rf is the final ratio, use the measured ratio of the isotope in the ROI (i.e., 13C/12C). Ri is the initial ratio, the measured ratio in killed controls or no-isotope added controls.
  5. Net fixation of stable isotope (the percentage of stable isotope incorporated relative to initial stable isotope content) can then be calculated (Popa et al., 2007) for each ROI and averaged (Table 1):
    1. Fxnet = {Rf[1 - Ri/(Ri + 1)] - Ri/(Ri + 1}/{Rs/(Rs + 1) - Rf[Rs/(Rs + 1]} x 100%
    2. Rf is the isotopic ratio of the treated biofilm, from NanoSIMS measurement (i.e., 13C 12C counts/12C2 counts in an ROI).
    3. Ri is the isotopic ratio in the initial biofilm, from NanoSIMS measurement (i.e., a time zero sample or killed control ratio in an ROI).
    4. Rs is the isotopic ratio in the substrate added. This can be obtained by IRMS, or if using a purchased substrate, from the manufacturer.
  6. Net fixation can be extrapolated to calculate the average net μg C or N assimilated per biofilm using biovolume measurements on the biofilms.
    1. C content can be estimated using biovolume-to-carbon conversion factors from the literature (e.g., 2.2 x 10-13 g C/μm3 for bacteria and 1.8 x 10-13 g C/μm3 for filamentous diazotrophic cyanobacteria) (Bratbak, 1985; Goebel et al., 2008), and then used to calculate average µg C in the biofilm.
    2. N content can be estimated based on C/N ratios determined via elemental analysis of biomass coupled with IRMS analysis, and literature for marine bacteria (C/N of 4.0 for cyanobacteria and 4.4 for heterotrophs), (Fukuda et al., 1998) then used to calculate average µg N in the biofilm.
    3. Multiply the average µg C or N in the cyanobacteria or heterotroph biomass in the biofilm by the average net fixation rate for that organism.
  7. If examining substrates with more than one stable isotope label (i.e., 13C as well as 15N), relative use efficiency can be calculated (Mayali et al., 2013), and mean use efficiencies compared to a theoretical mean of 1.0 using a t-test, to test for elemental preferences (Table 1).
    1. C/N relative use efficiency is calculated using this equation: [FCnet x (C:N)b]/[FNnet x (C:N)s] where (C:N)b is the C/N ratio for the biomass and (C:N)s is the C/N ratio for the substrate, which can be estimated based on the literature or determined via elemental analysis.

      Table 1. Representative NanoSIMS ROI extracted data and analysis
      ROIa
      12C2b
      13C 12Cb 
      14N 12Cb
      15N 12Cb
      13C/12Cb
      15N/14N
      15N
      APEc
      13C
      APEc
      FCnetc
      FNnetc
      C/N rel
      usec
      1
      279691.90
      8454.86
      374425.10
      2154.07
      0.015
      0.006
      0.206
      0.378
      0.540
      0.188
      1.996
      2
      210659.20
      7840.00
      303345.00
      2481.86
      0.019
      0.008
      0.445
      0.715
      1.062
      0.471
      1.568
      3
      493302.90
      14908.48
      657695.20
      4458.77
      0.015
      0.007
      0.307
      0.377
      0.539
      0.308
      1.219
      Initial
      --
      --
      --
      --
      0.011
      0.004
      --
      --
      --
      --
      3.34d
      EOM
      --
      --
      --
      --
      2.09
      6.27
      --
      --
      --
      --
      4.80e
      aRepresentative ROIs from Figures 1D-1F.
      bcounts and ratios extracted from NanoSIMS software (LIMAGE)
      ccalculated based on equations in Data analysis section
      d(C:N)b
      e(C:N)s

Recipes

  1. 4% PFA (100 ml)
    9.25 ml of 37% formaldehyde solution
    90.75 ml of 1x phosphate buffered saline (diluted with MilliQ H2O from 10x stock, pH 7.4)
    Mix together and use immediately
  2. Sterile 10% sodium chloride (NaCl) solution (100 ml)
    10 g NaCl
    100 ml H2O
    Mix to dissolve, autoclave at 121 °C for 25 min to sterilize
  3. Modified artificial seawater base (ASN) medium recipe (with nitrate)

    Chemical
    g/L
    Final conc.
    1
    NaCl 
    25
    428 mM
    2
    MgCl2·6H2O
    2
    9.84 mM
    3
    KCl
    0.5
    6.71 mM
    4
    MgSO4·H2O
    3.5
    14.2 mM
    5
    CaCl2·2H2O
    0.5
    3.4 mM
    6
    NaHCO3
    0.2
    2.38 mM
    7
    Tricine
    0.025
    0.14 mM
    8
    KH2PO4
    0.0494
    0.0363 mM
    9
    NaNO3
    0.0748
    1.76 mM
    10
    FeCl3
    0.001898
    0.0117 mM
    11
    EDTA
    0.004355
    0.0149 mM
    12
    H3BO3
    0.00286
    0.0463 mM
    13
    MnCl2·4H2O
    0.00181
    9.15 μM
    14
    ZnSO4·7H2O
    0.000222
    0.772 μM
    15
    NaMoO4·2H2O
    0.00039
    1.61 μM
    16
    Co(NO3)2·6H2O
    0.0000494
    0.17 μM
    17
    Cyanocobalamin
    0.000005
    0.00369 μM
    18
    Biotin
    0.000005
    0.0205 μM
    19
    Thiamin HCl
    0.0002
    0.593 μM

    1. Stock a: Prepare 1,000x stock solution of 8 in distilled water
    2. Stock b: Prepare 500x stock solution of 9 in distilled water
    3. Stock c: Prepare 1,000x stock of 10-11 in distilled water
    4. Stock d: Prepare 1,000x stock solution of 12-16 in distilled water
    5. Stock e: Prepare 1,000x stock solution of 17-19, filter sterilize and freeze in 1 ml aliquots
    6. Add 1-7 to 1 L distilled water
    7. Add 1 ml of stock solutions a, c and d and 2 ml of stock solution b
    8. Autoclave at 121 °C for 20 min and once cooled add stock e
    9. Stock solutions a-d can be stored at 4 °C for up to 6 months

Acknowledgments

Funding was provided by the DOE Genomic Science Program under contract SCW1039. Work at Lawrence Livermore National Laboratory was performed under the auspices of DOE contract DE-AC52-07NA27344. This protocol is based on previous work described in Stuart et al. (2016a and 2016b).

References

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  2. Fukuda, R., Ogawa, H., Nagata, T. and Koike, I. I. (1998). Direct determination of carbon and nitrogen contents of natural bacterial assemblages in marine environments. Appl Environ Microbiol 64(9): 3352-3358.
  3. Ghosal, S., Fallon, S. J., Leighton, T. J., Wheeler, K. E., Kristo, M. J., Hutcheon, I. D. and Weber, P. K. (2008). Imaging and 3D elemental characterization of intact bacterial spores by high-resolution secondary ion mass spectrometry. Anal Chem 80(15): 5986-5992.
  4. Goebel, N. L., Edwards, C. A., Carter, B. J., Achilles, K. M. and Zehr, J. P. (2008). Growth and carbon content of three different-sized diazotrophic cyanobacteria observed in the subtropical North Pacific(1). J Phycol 44(5): 1212-1220.
  5. Jiao, Y., Cody, G. D., Harding, A. K., Wilmes, P., Schrenk, M., Wheeler, K. E., Banfield, J. F. and Thelen, M. P. (2010). Characterization of extracellular polymeric substances from acidophilic microbial biofilms. Appl Environ Microbiol 76(9): 2916-2922.
  6. Lechene, C., Hillion, F., McMahon, G., Benson, D., Kleinfeld, A. M., Kampf, J. P., Distel, D., Luyten, Y., Bonventre, J., Hentschel, D., Park, K. M., Ito, S., Schwartz, G. B., Slodzian, G (2006). High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry. J Biol 5:20.
  7. Mayali, X., Weber, P. K., Pett-Ridge, J. (2013). Taxon-specific C/N relative use efficiency for amino acids in an estuarine community. FEMS Microbiol Ecol 83: 402-412.
  8. Pett-Ridge, J. and Weber, P. K. (2012). NanoSIP: NanoSIMS applications for microbial biology. Methods Mol Biol 881: 375-408.
  9. Polerecky, L., Adam, B., Milucka, J., Musat, N., Vagner, T. and Kuypers, M. M. (2012). Look@NanoSIMS--a tool for the analysis of nanoSIMS data in environmental microbiology. Environ Microbiol 14(4): 1009-1023.
  10. Popa, R., Weber, P. K., Pett-Ridge, J., Finzi, J. A., Fallon, S. J., Hutcheon, I. D., Nealson, K. H. and Capone, D. G. (2007). Carbon and nitrogen fixation and metabolite exchange in and between individual cells of Anabaena oscillarioides. ISME J 1(4): 354-360.
  11. Stuart, R. K., Mayali, X., Boaro, A. A., Zemla, A., Everroad, R. C., Nilson, D., Weber, P. K., Lipton, M., Bebout, B. M., Pett-Ridge, J. and Thelen, M. P. (2016a). Light regimes shape utilization of extracellular organic C and N in a cyanobacterial biofilm. MBio 7(3).
  12. Stuart, R. K., Mayali, X., Lee, J. Z., Craig Everroad, R., Hwang, M., Bebout, B. M., Weber, P. K., Pett-Ridge, J. and Thelen, M. P. (2016b). Cyanobacterial reuse of extracellular organic carbon in microbial mats. ISME J 10(5): 1240-1251.
  13. Woebken, D., Burow, L. C., Prufert-Bebout, L., Bebout, B. M., Hoehler, T. M., Pett-Ridge, J., Spormann, A. M., Weber, P. K. and Singer, S. W. (2012). Identification of a novel cyanobacterial group as active diazotrophs in a coastal microbial mat using NanoSIMS analysis. ISME J 6(7): 1427-1439.

简介

为了提高对微生物相互作用的理解,解决复杂群落中微生物代谢贡献的重要性越来越重要。生物膜的生物体特别难以分离,形成和分析,需要解决这些局限性的方法。高分辨率成像二次离子质谱(NanoSIMS)产生单细胞同位素组成测量,可用于量化在各生物体内同位素标记的底物的掺入和交换。在这里,使用由蓝藻混合物种生物膜的成员掺入的蓝细菌胞外有机物(EOM)作为模型来说明该方法。通过两组(蓝细菌和相关的异养微生物)掺入稳定同位素( 15 N和 13 C)EOM的量化。描述了用于定量生物膜中稳定同位素标记的EOM摄取的样品的生成,制备和分析方法。

背景 与NanoSIMS(“NanoSIP”)结合的稳定同位素标记是一种确定的稳定同位素标记底物掺入到单个微生物细胞中的方法,然后可将其推断以估计细胞群体的掺入(例如,Lechene et al。 ,2006和Woebken等人,2012)。可以将多个稳定同位素标记(例如, 13 C和 15 N追踪到单个细胞中可用于检查治疗之间随时间的差异并入(例如,Popa等人,2007和Stuart等人,2016a)。生物膜对量化掺入标签提出了具体的挑战。由于个体生物被包埋在细胞外基质中并具有不同范围的细胞大小和形状,所以细胞计数和生物量计算是困难的。另外,诸如EOM的聚合物标记的底物可以粘附到基质和细胞表面,因此需要考虑非公司标签。基于成像的方法,如NanoSIMS,与SEM和荧光显微镜配对,非常适合解决这些挑战,因为可以识别细胞大小和非组合标签。在这里,我们描述了解决这些挑战的方法,以量化从聚合物底物(EOM)到标签(光子生物膜)中的标记( 13 C和 15 N)的掺入。 EOM是与细胞松散相关的细胞外物质,并与生物膜中的细胞分离。该方法的一个缺点是生物膜空间结构(细胞外基质)不被保留。如果需要对空间布置进行检查,则生物膜样品的嵌入和切片可能是必要的(例如,Lechene等人,2006)。

关键字:稳定同位素, 细胞外基质, 细胞外有机物, 微生物生态学, 底物掺入, NanoSIP

材料和试剂

  1. 无菌细胞刮刀(VWR,目录号:89260-224或89260-222)
    注意:产品"89260-224"已经停产。
  2. 0.2μm注射器过滤器(聚醚砜膜,例如,Pall,目录号:4652)
  3. 吸管尖端
  4. 1.7ml微量离心管
  5. 无菌注射器
  6. 尺寸适合NanoSIMS夹持器(例如,5×5mm,Ted Pella,目录号:16008)的硅(Si)晶片
  7. 无菌组织研磨机(Fisher Scientific,目录号:02-542-08)
  8. 生物膜文化
    注意:我们检查了一种单体混合物种生物膜,但是,该方案适用于大多数生物膜类型的分析,条件是水性底物可以添加到样品中,具有均匀的分布。我们的生物膜和培养程序的详细描述可以在Stuart et al。,2016a找到。
  9. 稳定的同位素标记化合物,适合于支持感兴趣的生物体的生长或代谢(例如,99原子百分比过量[atm%] 13 C/sub> 3 ,Cambridge Isotope Laboratories,目录号:CLM-441-1)
  10. 用于生长的合适的限定培养基(例如,在Stuart等人,2016a)中描述的修饰的人造海水基础[ASN]培养基
  11. 50%乙醇(EtOH)
  12. 37%甲醛溶液(Sigma-Aldrich,目录号:252549)
  13. 10x磷酸缓冲盐水(Sigma-Aldrich,目录号:P5493)
  14. 氯化钠(NaCl)
  15. 无菌MilliQ水
  16. 4%PFA(见食谱)
  17. 无菌10%氯化钠(NaCl)溶液(参见食谱)
  18. 改性人造海水基(ASN)介质配方(含硝酸盐)(见配方)

设备

  1. Dounce均质机(WHEATON,目录号:357546)
  2. 微量离心机,能够产生高达15,000 x g的可变转速
  3. 同位素比质谱仪(例如,ANCA-IRMS,PDZE Europa Limited,Crewe,England)
  4. 用于生物膜培养的室(例如,Pyrex密封烧瓶,Corning,PYREX,目录号:4985-1L)
  5. 孵化器(适合培育感兴趣的生物体)
  6. 解剖显微镜(例如,Fisher Scientific,型号:Fisher Scientific TM 420系列,目录号:11-350-124)
  7. 干燥器柜(Thermo Fisher Scientific,Thermo Scientific,Supest TM),目录号:5317-0070)
  8. 荧光光学显微镜(例如,Leica Microsystems,型号:Leica DMI600B)
  9. 金刚石或硬质合金划片(例如,Ted Pella,目录号:54412)
  10. 金(或其他导电金属)溅射涂布机(例如,Cressington,型号:溅射涂布机108)
  11. NanoSIMS 50或50 L(AMETEK,Cameca,型号:NanoSIMS 50或NanoSIMS 50 L)
  12. 扫描电子显微镜(SEM)具有超过50nm分辨率和显微照片记录能力(例如FEI,型号:FEI Inspect F SEM)

软件

  1. 离子图像数据处理软件( e ,LIMAGE,L. Nittler,Carnegie Institution of Washington,华盛顿特区,美国; ImageJ与MIMS插件; Look @ NanoSIMS [Polerecky et al。,2012]或类似的)

程序

  1. 稀土同位素底物生成,生物膜孵育,硅晶片固定和制备
    1. 产生稀有的同位素富集的细胞外有机物质(EOM)
      注意:用于同位素标记的底物将取决于被测试的具体实验的假设。在这里,我们描述了聚合稀有同位素标记底物EOM的生成,但底物也可以是简单定义的化合物(例如,13 C-氨基酸,葡萄糖,碳酸氢盐或硝酸铵,铵)。使用的基材将依赖于正在测试的假设。
      1. 接种并在封闭的烧瓶(没有顶部空间)中将生物膜(或纯培养物)生长至晚期指数期,用与实验条件相似组成的新鲜培养基,但其中感兴趣的生长底物被罕见的同位素类似物(<例如,具有3mM 13 C碳酸氢钠和1.76mM K 15 SO 3的ASN。
        注意:
        1. 培养生物膜无顶空以避免CO 2(不含13 C标签)从空气平衡到培养基中,然而,碳酸氢钠将会耗尽,并且随着时间的推移需要更新。添加 13碳酸氢钠的频率将取决于生物膜的C固定速率。我们每48小时补充一次。
        2. 推荐在以前适应环境条件下维护生物膜。在这种情况下,生物膜用12:12的光:23℃和20μmol/平方厘米的暗周期进行培养。 br />
      2. 将稀释的同位素培养基以1:50的稀释度将生物膜再次转移三次。
        注意:这些培养物的晚期指数生物膜的目标生物量为50-100mg干重(在这种情况下为50ml培养物,3周生长)。
      3. 将这种稀有同位素标记的培养物接种到更大体积的新鲜稀有同位素培养基中,并以产生约1-5g干重所需的体积生长至晚期指数期。在这个阶段,接种物可以分成多个烧瓶,确保每种培养物的接种量相等。对于我们的生物膜,这是通过四个500毫升烧瓶培养3周实现的。
      4. 收获生物膜和单独的EOM(改编自Jiao等人,2010; Stuart等人,2016b)。
        注意:此处描述的方法是针对超级丝状蓝细菌,ESFC-1,并且需要对不同物种进行测试和修饰。
        1. 倾倒并丢弃覆盖生物膜并将生物膜转移到无菌Dounce匀浆器的培养基。如果生物膜粘附,请使用无菌细胞刮刀。
        2. 加入1-2毫升无菌溶液。
          注意:对于我们的超生物生物膜,使用10%NaCl,并且基于定义的培养基(例如ASN)选择盐浓度。
        3. 均质生物膜。
          注意:这将需要初步的显微镜检查,以确保均匀化过程中细胞裂解的最小程度。
        4. 在40℃孵育匀浆15分钟。
          注意:这是从细胞中松开胞外多糖。这将需要初步测试,如显微镜检查,以确保在此步骤期间发生细胞裂解。
        5. 离心机在15,000 x g下在4℃匀浆20分钟。
        6. 分离细胞沉淀的上清液,并通过0.2μm注射器过滤器过滤。过滤的上清液为EOM。储存于-80°C直至使用。
          注意:推荐使用聚醚砜膜,因为它们具有低蛋白质和多糖结合。
      5. 通过同位素比质谱法(例如,ANCA-IRMS,PDZE Europa Limited,Crewe,England)确定细胞沉淀和EOM的体同位素比。
    2. 将细胞接种到重复的优选培养室中,培养用于生物膜发育,测量随时间的生长,对于我们的蓝藻生物膜的生长条件的详细描述可以在Stuart等人,2016b。
      注意:
      1. 尽可能地减少生物膜培养体积,同时保持相关条件,以节省在步骤A1中生成的稳定同位素底物(在这种情况下为EOM)的使用。
      2. 为了获得元素摄取(μgC或N固定)的定量测量,所有生物膜社区成员的生物量测量是必要的。可以使用固定生物膜的子部分的显微镜来产生生物体积测量(Stuart等,2016b)。
      3. 推荐使用完全限定的培养基(例如,ASN [Stuart等,2016a]),其中所有C和N源都是已知的,从而可以估计生物膜C和N预算。
    3. 将稳定的同位素标记的底物添加到天真的生物膜培养物中
      1. 向未标记的生物膜培养物中加入同位素稀有生长底物(步骤A1)(步骤A2)。
      2. 控制治疗应包括(至少):
        1. 杀死对照 - 在实验开始前,将4%PFA(见下文配方)修复生物膜1小时,冲洗固定剂,然后与其他活体样品一样处理。
        2. 不添加稳定同位素的对照。
        3. 可选:其他控制措施来解决有关特定代谢的假设(例如,在特定抑制剂的存在下)
      3. 在选定的时间点,从生物膜文化中清除废弃的媒体 注意:时间点取决于目标的代谢率和测试生物体的生长速率。我们的时间点从1小时到24小时,基于我们的生物体的生长速度(约0.1天 -1 )和基于 13的C固定体积率碳酸氢盐掺入(IRMS测量)。
      4. 可选择:保存用于进一步分析的用过的介质 - 过滤0.2μm聚醚砜注射器过滤器,并将滤液冷冻至-80°C。
    4. 修复组织以保持超微结构
      1. 向生物膜组织中加入4%PFA,并在室温下孵育1小时或在4℃下孵育过夜。
        注意:戊二醛或乙醇是可接受的替代品。
      2. 去除4%的PFA并用无菌H 2 O 2小心地冲洗生物膜至少3次。
        注意:彻底清除盐和固定剂很重要,因为这些干扰了NanoSIMS测量。
      3. 在-20℃下将生物膜储存在50%EtOH中,体积足够覆盖生物膜。
        注意:固定生物膜可以在这些条件下储存长达几年。
    5. 准备固定生物膜样品用于微量分析和成像
      1. 使用吸头,将一块固定的生物膜转移到含有1ml无菌H 2 O的无菌组织研磨机中。
      2. 轻轻研磨以分解生物膜并转移至1.7ml微量离心管
      3. 以10,000×g离心1分钟,除去上清液并加入1ml无菌H 2 O。重复冲洗过程,除去上清液,然后重悬于10μl50%EtOH中。这些步骤有助于去除附着于细胞表面的过量细胞外有机物质。这种有机物会干扰细胞摄取的NanoSIMS分析
      4. 对于混合物种生物膜(,细菌和蓝细菌的非无菌混合物),离心速度必须足够高或足够长以收集除细丝之外的细菌细胞(例如< ,> 15,000 ,和/或超过20分钟)
      5. 在解剖显微镜下,用金刚石刻字轻轻刻划硅晶片,创建一个〜2 x 2 mm间隔格(见图1A)。将1-2μl样品吸取到一个方格的网格上,让其干燥并在显微镜下检查,每个斑点约30根细丝,如果有太多或太少的样品,将样品冲洗并浓缩/稀释样品在管中,必要。在理想的制备中,细胞应形成单层,最小重叠或聚集。
      6. 复制样品和处理可以移液到另外的网格方格中;他们应该在一起,没有重叠的风险。
      7. 干燥样品在室温下过夜或更长时间,防止灰尘和潮湿。
      8. 可选:使用落射荧光显微镜的图像来评估细胞密度和构型。请注意,金属涂层后可能不会进行荧光成像

        图1.样品和分析的代表性图像。A.具有5个样品的硅片在网格上点样。刻度棒为1 mm。红色方框和箭头表示SEM图像的位置。 B. SEM图像。红色框表示代表性的NanoSIMS分析区域,该区域的NanoSIMS输出显示在C-E中。 C. NanoSIMS 12 C 14 N离子图像,显示在30×30μm区域中的累积计数,具有256 2像素束斑尺寸,持续30个周期。 C比例图像;(c) N/ 14 N比图像。 (C-E)中的编号的白色圆圈表示用于分析的代表性感兴趣区域(ROI)。投资回报率1-3的值在表1中。(B-E)的比例尺为10微米。

    6. 使用溅射涂层机涂覆高导电金属表面的样品
      1. 金或铱衣是典型的;目标为5至10nm涂层;较大的细胞或具有增加的地形缓解的制剂更厚。可以使用碳涂层,但会干扰碳同位素测量。
      2. 金属涂层对于低浮雕样品(<1μm)不是必需的,但它改善了纳米SIMS中样品的SEM成像和初始成像。
    7. 将硅晶片放在支架上
      1. 如果SEM室足够大,样品可以放置在NanoSIMS夹具中;否则,在加载到NanoSIMS持有者之前的SEM图像样本
  2. SEM成像
    1. SEM成像对于选择分析目标和确保清除干扰碎片或非靶细胞非常有用。如果以三个尺度(100x,500x和3,000x)收集目标位置的SEM图像,则在SEM中选择的分析位置可以重新定位在NanoSIMS中。还可以使用SEM舞台坐标来重新定位在NanoSIMS光成像系统(CCD摄像机)中太小或不明确的目标;这对于Si晶圆上的大多数细胞分散体是不必要的
    2. 将样品放在扫描电镜中。
    3. 根据仪器的需要调整光斑尺寸和电压kV;如果样品没有金属涂层,低电压成像可以减少充电。
    4. 在低,中,高放大倍数(100x,500x,3,000x)下选择分析目标和图像。
      1. 选择多个细丝相邻或重叠的位置,但仍然可以清楚地区分(参见图1B)。
        注意:重叠的灯丝将允许在一个分析窗口中分析2-3根灯丝,从而最大限度地提高NanoSIMS分析时间,但是如果它们不能清楚地区分,这将损害分析。
      2. 如果感兴趣的是异源细菌细胞,则可以收集高倍放大图像(> 3,000x),以便清楚地显现它们(图1B)。
      3. 收集足够的SEM图像以分析每个样品至少20个细丝。

  3. NanoSIMS分析
    1. 将样品加载到NanoSIMS 
      1. 样品可能需要1-12小时才能达到必要的真空度,这取决于样品类型,干燥度和细胞材料的密度
    2. 使用Cs + 分析光束进行C和N同位素的分析。在此模式下提取负二次离子。
    3. 调整仪器并设置质量进行所需的分析。为了测量细胞中的 13 C或 15 N富集,离子质量为24,25,26,27( 12 - 13 C 12 C - 12 C 14 N - 12 C 15 N - )。以前的工作表明C二聚体(例如 12 C 2 - )具有优于 12 C单体,更好地符合CN - (Pett-Ridge和Weber,2012)的二次质谱仪设置。 
      1. 质量分辨率(ΔM/M)> 6,800是解决等压干扰的必要条件
      2. 应分析参考材料以确保仪器性能正确。这可以简单地是未标记的控件,尽管持续运行的样本可以提高对结果的信心(Pett-Ridge和Weber,2012)。
    4. 选择分析束电流
      1. 通常,分析速度是选择光束电流时考虑的主要因素,因为CN - 是高产量物种,NanoSIMS检测器的最大可持续计数率为每秒25万次,容易从具有1至2pA Cs + 的细胞实现。这些设置通常会产生约150 nm直径的分析光束。这个直径设定了分析的最终空间分辨率。
      2. 如果需要更高的空间分辨率,则应使用减小分析光束直径并降低光束电流的透镜和光圈设置。
    5. 选择并保存位置以分析
      1. 首先根据位于CCD相机(光成像)中的点记录一般位置。如果CCD摄像机中所需的目标不能被可视化,则必须定位其他地面标记,并且可以使用SEM图像或坐标变换确定的测量结果以SIMS模式定位目标。
      2. SIMS实时成像是必要的,以确保CCD成像中选择的目标居中在成像领域。
    6. 设置分析参数以在每个映射位置使用并进行分析。这包括:
      1. 分析栅格的大小,这取决于正在分析的单元格的大小。通常在10 x 10μm和25 x 25μm之间。
      2. 像素数,通常基于分析束斑大小(线性像素数)〜2×(光栅大小)/(光点大小)。对于典型的150 nm直径分析光束,对于10 x 10μm的光栅,128 <2>像素对于25 x 25μm的光栅将是合理的。
      3. 预分析溅射:通常,在数据采集之前,高电流Cs + 光束在样品上被扫描,以增强和稳定来自样品的二次离子计数率。将样品溅射到约为60nm量级的Cs + 注入深度的两倍。可以基于溅射速率(ξξ),一次离子束电流(I),溅射时间(ξ),溅射时间),栅格区域( A )和溅射深度( d ):



        对于生物材料,溅射速率可以估计为2.5nmμm2(以下)(ghosal等人,/em>。,2008)。可能需要更长的预分析溅射以从细胞表面去除残留的材料。通过监测计数率和深度的同位素比率,可以凭经验确定溅射的最佳持续时间。
      4. 分析持续时间,应根据最小目标所需的计数数量进行计时,以达到足够的分析精度;小计同位素的10,000个计数将导致〜1%的分析精度。
        注意:
        1. 确定一致的数据收集参数可能是一个迭代过程,特别是对于新样本,其中富集水平,细胞表面上的残留物质和样本内变异性是未知的。样本可能需要进行分析和检查,之后可能稍作改动,以确保适当的数据收集。
        2. 运行非标签和杀死的控件是至关重要的。这些结果用于下面的浓缩计算

数据分析

  1. 分析NanoSIMS离子图像需要特殊的软件,可以逐个像素地定量处理图像。几个软件包可用( e 。LIMAGE [L. Nittler,Carnegie Institution of Washington,DC]),包括免费版本。
  2. 结果可以基于定量离子图像(例如图1C)或离子比图像( 12)进行可视化(例如, 12 13,图1D-1E),每个分析区域。按像素计算比率。
  3. 对于诸如这些的稳定同位素标记实验,数据通常以三角洲符号,次同位素的原子百分数或次要同位素的原子百分比过剩(APE)的比率表示。 APE是一个理想的演示,因为它很简单,直接表示了标签的吸收(见下面的方程式)。虽然在文献中很常见,但是对于高度富集的样品,三角形记数法可能不那么直观。然而,单位的选择不会影响图像或图形的数据可视化。
  4. 必须确定目标细胞周围的感兴趣区域(ROI)用于同位素比率的定量(图1C-1E)。 ROI通常是手动绘制的,但自动程序也可以成功。
    1. 对于生物膜细菌细胞或蓝藻细丝,可以基于二次电子和/或超细离子图像手绘图像(图1C) ,这两者都产生了细胞边界的良好指示。检查 13 C/ 12 C和 15 N/ 14 N个图像也是有用的,因为这些图像可能会排除剩余标记底物的热点(图1D-1E)
    2. 为每个ROI提取同位素比率,不确定性和像素大小。
    3. ROI的原子百分比浓度(APE)可以根据(表1)计算:
      1. APE = [ R f /( R f /( R f + 1) - R /sub> /( R + 1)] x 100%
      2. 是最终比例,使用测量的ROI中的同位素比例( i e 。, 13 C/ 12 C)。 i 是初始比例,杀死对照或无同位素添加对照中的测定比例。
  5. 然后可以计算稳定同位素的净固定(相对于初始稳定同位素含量而引入的稳定同位素的百分比)(Popa a 1 。 ,2007)为每个ROI和平均值(表1):
    1. Fxnet = { R f [1 - R sub> /( R i + 1)] - R > /( R + 1}/{ R /( R + 1) - R >˚F [ - [R <子> 取值 /( - [R <子> + 1]} x 100%
    2. 是从NanoSIMS测量( i C 13 C 12 C计数/ 12 C 2 在投资回报率中)。
    3. 是初始生物膜中的同位素比率,来自NanoSIMS测量( i em> e ,ROI中的时间零样本或杀死的控制比率)。
    4. 是添加的底物中的同位素比。这可以通过IRMS获得,或者如果使用购买的基材,可以从制造商获得。
  6. 净固定可以外推,以使用生物膜上的生物体积测量来计算每个生物膜吸收的平均净微克C或N。
    1. 可以使用文献中的生物体积 - 碳转化因子来估计C含量。 细菌的13℃/μm3,对于丝状重氮营养蓝细菌为1.8×10 -6 -13℃/μm3 /(以上)( Bratbak,1985; Goebel ,2008),然后用于计算平均μgC在生物膜中
    2. N含量可以基于通过与IRMS分析的生物量的元素分析确定的C/N比率,以及海洋细菌的文献(蓝藻的C/N为4.0,异养菌为4.4),(Fukuda e
    3. 将生物膜中蓝细菌或异养生物量中的平均μgC或N乘以该生物体的平均净固定率。
  7. 如果检查具有多于一个稳定同位素标签的底物( i e , 13 C以及< sup> 15 N),可以计算相对使用效率(Mayali ,2013),以及使用 t -test与理论平均值1.0相比的平均使用效率,以测试元素偏好(表1)。
    1. 使用以下等式计算C/N相对使用效率:[F c C x N(C:N)b]/[FNnet x(C:N)其中(C:N)b 是生物量的C/N比,(C:N)是C/N比,底物,其可以基于文献估计或通过元素分析确定。

      表1.代表性的NanoSIMS ROI提取数据和分析
      投资回报率 a
      12 C <子> 2 b
      1 第3 C >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> b   1 4 Ñ >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> b
      1 5 Ñ >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> b
      1 第3 C / 1 2 C b
      1 5 Ñ / 1 4 N
      1 5 Ñ
      A P <强>电子 c
      1 第3 C
      A P <强>电子 c
      ˚F C <子> 名词 ë <强> < ˚F Ñ <子> <强> 名词 <强> 电子 C
      C / Ñ <强> rel
      û <强> 取值 e c
      1
      279691.90
      8454.86
      374425.10
      2154.07
      0.015
      0.006
      0.206
      0.378
      0.540
      0.188
      1.996
      2
      210659.20
      7840.00
      303345.00
      2481.86
      0.019
      0.008
      0.445
      0.715
      1.062
      0.471
      1.568
      3
      493302.90
      14908.48
      657695.20
      4458.77
      0.015
      0.007
      0.307
      0.377
      0.539
      0.308
      1.219
      Ini tial
      -
      -
      -
      -
      0.011
      0.004
      -
      -
      -
      -
      3.34 d
      EO M
      -
      -
      -
      -
      2.09
      6.27
      -
      -
      -
      -
      4.80 e
      图1D-1F的代表性ROI。
      b 从NanoSIMS软件(LIMAGE)提取的计数和比率
      根据数据分析部分
      中的等式计算的 c d (C:N) b
      e (C:N)

食谱

  1. 4%PFA(100 ml)
    9.25毫升37%甲醛溶液
    将90.75ml 1x磷酸缓冲盐水(用MilliQ H 2 O从10x储备液稀释,pH7.4)
    混合在一起并立即使用
  2. 无菌10%氯化钠(NaCl)溶液(100ml)
    10克NaCl
    100ml H 2 O O
    混合溶解,在121℃高压灭菌25分钟消毒
  3. 改性人造海水基(ASN)介质配方(含硝酸盐)

    Che m ical
    g/L
    最终浓缩
    1
    NaCl 
    25
    428 mM
    2
    MgCl 2·6H 2 O
    2
    9.84 mM
    3
    KCl
    0.5
    6.71 mM
    4
    MgSO 4·H 2 O O
    3.5
    14.2 mM
    5
    CaCl 2·2H 2 O O
    0.5
    3.4 mM
    6
    NaHCO 3
    0.2
    2.38 mM
    7
    Tricine
    0.025
    0.14 mM
    8
    KH 2 PO 4
    0.0494
    0.0363 mM
    9
    NaNO 3
    0.0748
    1.76 mM
    10
    FeCl 3
    0.001898
    0.0117 mM
    11
    EDTA
    0.004355
    0.0149 mM
    12
    3 <3> 3
    0.00286
    0.0463 mM
    13
    MnCl 2·4H 2 O
    0.00181
    9.15μM
    14
    ZnSO 4·7H 2 O
    0.000222
    0.772μM
    15
    NaMoO 4 2H 2 O O
    0.00039
    1.61μM
    16
    Co(NO 3 3)2< 2> 2< 2<> 0.0000494
    0.17μM
    17
    氰钴胺素
    0.000005
    0.00369μM
    18
    生物素
    0.000005
    0.0205μM
    19
    盐酸硫胺素
    0.0002
    0.593μM

    1. 储备a:在蒸馏水中制备1,000倍的8倍储备溶液
    2. 库存b:在蒸馏水中准备500倍的9号溶液
    3. 库存c:在蒸馏水中准备1000磅10-11的储存液
    4. 库存d:在蒸馏水中准备1000倍的12-16储备溶液
    5. 库存e:准备1,000磅17-19的储液,过滤灭菌并以1ml等分试样冷冻
    6. 加入1-7升1升蒸馏水
    7. 加入1ml储备溶液a,c和d和2ml储备溶液b
    8. 在121°C高压灭菌20分钟,一次冷却添加库存e
    9. 储存溶液a-d可以在4℃下储存长达6个月

致谢

资助由美国能源部基因组科学计划根据合同SCW1039提供。劳伦斯·利弗莫尔国家实验室的工作由能源部合同DE-AC52-07NA27344主持。该协议基于Stuart等人中描述的以前的工作。 (2016a和2016b)。

参考

  1. Bratbak,G。(1985)。  细菌生物体积和生物量估计。 ):1488-1493。
  2. Fukuda,R.,Ogawa,H.,Nagata,T.and Koike,II(1998)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/9726882"target ="_ blank">直接测定海洋环境中天然细菌组合物的碳氮含量。 环境微生物 64(9):3352-3358。
  3. Ghosal,S.,Fallon,SJ,Leighton,TJ,Wheeler,KE,Kristo,MJ,Hutcheon,ID and Weber,PK(2008)。  通过高分辨率二次离子质谱法成像和3D元素表征完整的细菌孢子。化学 80(15):5986-5992。
  4. Goebel,NL,Edwards,CA,Carter,BJ,Achilles,KM and Zehr,JP(2008)。  在亚热带北太平洋(1)观察到的三种不同大小的重氮营养蓝细菌的生长和碳含量 Phycol < em> 44(5):1212-1220。
  5. Jiao,Y.,Cody,GD,Harding,AK,Wilmes,P.,Schrenk,M.,Wheeler,KE,Banfield,JF and Thelen,MP(2010)。  来自嗜酸性微生物生物膜的细胞外聚合物质的表征 > pl Environ Microbiol 76(9):2916-2922。
  6. Lechene,C.,Hillion,F.,McMahon,G.,Benson,D.,Kleinfeld,AM,Kampf,JP,Distel,D.,Luyten,Y.,Bonventre,J.,Hentschel,D.,Park, KM,Ito,S.,Schwartz,GB,Slodzian,G(2006)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/17010211" target ="_ blank">使用稳定同位素质谱法对哺乳动物和细菌细胞进行高分辨率定量成像 生物 5:20。
  7. Mayali,X.,Weber,PK,Pett-Ridge,J.(2013)。  河口社区氨基酸的特异性C/N相对使用效率。 /em> 83:402-412。
  8. Pett-Ridge,J.和Weber,PK(2012)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22639220"target ="_ blank "> NanoSIP:用于微生物生物学的NanoSIMS应用程序。 -408。
  9. Polerecky,L.,Adam,B.,Milucka,J.,Musat,N.,Vagner,T.and Kuypers,MM(2012)。  Look @ NanoSIMS - 用于分析环境微生物学中nanoSIMS数据的工具。 Envir em> 微生物 14(4):1009-1023。
  10. Popa,R.,Weber,PK,Pett-Ridge,J.,Finzi,JA,Fallon,SJ,Hutcheon,ID,Nealson,KH和Capone,DG(2007)。< a class ="ke-insertfile" href ="http://www.ncbi.nlm.nih.gov/pubmed/18043646"target ="_ blank"> > abaena vibrarioides 。 我 1(4):354-360。
  11. Stuart,RK,Mayali,X.,Boaro,AA,Zemla,A.,Everroad,RC,Nilson,D.,Weber,PK,Lipton,M.,Bebout,BM,Pett-Ridge,J。和Thelen,MP (2016a)。细胞外有机碳的光照形状利用和蓝色细菌生物膜中的N. 7(3)。
  12. Stuart,RK,Mayali,X.,Lee,JZ,Craig Everroad,R.,Hwang,M.,Bebout,BM,Weber,PK,Pett-Ridge,J.and Thelen,MP(2016b)。 class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26495994"target ="_ blank">微生物垫胞外有机碳的蓝细菌再利用。 > ISM E J 10(5):1240-1251。
  13. Woebken,D.,Burow,LC,Prufert-Bebout,L.,Bebout,BM,Hoehler,TM,Pett-Ridge,J.,Spormann,AM,Weber,PK和Singer,SW(2012) class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22237543"target ="_ blank">使用NanoSIMS在沿海微生物垫中鉴定新型蓝细菌群作为活性重氮营养素分析。 J 6(7):1427-1439。
  • English
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Stuart, R. K., Mayali, X., Thelen, M. P., Pett-Ridge, J. and Weber, P. K. (2017). Measuring Cyanobacterial Metabolism in Biofilms with NanoSIMS Isotope Imaging and Scanning Electron Microscopy (SEM). Bio-protocol 7(9): e2263. DOI: 10.21769/BioProtoc.2263.
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