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Using Light and Electron Microscopy to Estimate Structural Variation in Thylakoid Membranes
使用光学和电子显微镜估计类囊体膜的结构变异   

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参见作者原研究论文

本实验方案简略版
Physiologia Plantarum
May 2017

Abstract

The shapes of chloroplasts and the architectures of internal thylakoid membranes are altered by growth and environmental changes (Lichtenthaler et al., 1981; Kutik, 1985; Terashima and Hikosaka, 1995). These morphological alterations proceed via transitional intermediates, during which dynamic and heterogeneous thylakoid membranes are observed in cells (Nozue et al., 2017). Light microscopy is useful for the detection of morphological differences in chloroplasts. The thylakoid architecture of such morphologically variable chloroplasts is confirmed by transmission electron microscopy (TEM). The method of monitoring structural variation by light microscopy in combination with electron microscopy is described.

Keywords: Light microscopy (光学显微镜检查), TEM (TEM), Chloroplast (叶绿体), Thylakoid membrane (类囊体膜), Arabidopsis thaliana (拟南芥)

Background

Functional coupling of ultra-structural morphology to photosynthesis and metabolic pathways in thylakoid membranes has been suggested (Oswald et al., 2001). This is supported by the fact that thylakoid membranes are heterogeneous during leaf maturation and during the transition from the vegetative to the flowering growth phase. There is a certain time lag associated with morphological alteration (Nozue et al., 2017). Rearrangement of thylakoid membranes occurs in parallel with changes in the shapes of chloroplasts, which go from having a typically elongated lenticular appearance to a swollen appearance that is identifiable by light microscopy.


Part I. Light microscopy

Materials and Reagents

  1. 1.5-ml microtubes (Ina-Optika, Bio Bik, catalog number: RC-0150 )
  2. Mineral wool (Nippon Rockwool, Yasaihana-block50)
  3. Razor blades (Feather Safety Razor, catalog number: FAS-10 )
  4. Double-edged razor blades (Feather Safety Razor, catalog number: FA-10 )
  5. Cell culture dish (Violamo, AS ONE, catalog number: 2-8590-02 )
  6. Glass microscope slide (Matsunami Glass, catalog number: S1214 )
  7. Glass microscope cover slip (Trophy, Matsunami Glass, 18 m/m)
  8. Arabidopsis thaliana ecotype Columbia
  9. Hydroponic solution (OAT Agrio, OAT House Fertilizer)
  10. 25% glutaraldehyde (NISSHIN EM, catalog number: 3052 )
  11. Agar, powder (Wako Pure Chemical Industries, catalog number: 016-11875 )
  12. Fixing solution (see Recipes)
  13. 5% agar (see Recipes)

Equipment

  1. Extra-high-pressure mercury lamp (Nikon Instruments, model: INTENSILIGHT C-HGFI )
  2. Fluorescence microscope (Nikon Instruments, model: ECLIPSE 80i )
  3. Stainless-steel pincette (Top well) (AS ONE, catalog number: 5-1076-01 )
  4. Laboratory glass bottle, screw cap, 100 ml (SCHOTT, DWK Life Sciences, Duran, catalog number: 21 805 24 04 )
  5. High-pressure steam sterilizer (TOMY SEIKO, model: LSX-500 )
  6. Microwave (Nisshin EM, model: MWF-2 )
  7. Refrigerator (Panasonic, model: NR-B52T2-H )
  8. Micro-slicer (DOSAKA EM, model: DTK-Zero1 )
  9. Objective lens 20x (Nikon Instruments, model: Plan Fluor 20x/0.50, catalog number: MRH00201 )
  10. Objective lens 40x (Nikon Instruments, model: Plan Fluor 40x/0.75, catalog number: MRH00401 )
  11. Objective lens 100x (Nikon Instruments, model: Plan Fluor 100x/1.30 Oil, catalog number: MRH01902 )
  12. CCD camera (Nikon Instruments, model: DS-Ri1 )
  13. Condenser (Nikon Instruments, models: D-CUD , D-CUO DIC )
  14. Autoclave

Software

1. Imaging software (Nikon Instruments, model: NIS-Elements)

Procedure

  1. Growth conditions and sampling of leaves
    1. Arabidopsis thaliana seeds (ecotype Columbia) are germinated and cultured on mineral wool using a hydroponic solution (EC: electrical conductivity, 1.2 ds/m, pH 7.0).
    2. Fluorescent lighting tubes and white LEDs consisting of blue (450 nm), green (545 nm) and red (660 nm) lights are used as light sources for growth.
    3. The growth condition is controlled at a temperature of 22-24 °C and 50-70% humidity, and the PPFD on the growth area is adjusted to 70-80 μmol m-2 sec-1 with a 12-h light photoperiod.
    4. Leaves are harvested from the plants at 1-3 h of the light period after a 12-h dark period.

  2. Preparation of leaf discs
    1. A fresh leaf is placed on a plastic mat of semisoft material. Different sizes of leaf blocks–3 x 5 mm for light microscopy and 1-2 x 3-5 mm for electron microscopy–are cut out with a razor-sharp blade. The razor blade, in vertical contact with the leaf, is carefully drawn so as not to damage cells by excessive compression. Major veins should not be included in the leaf blocks selected (Figure 1).
      Note: A slightly larger size makes light-microscopic sectioning easier, and a size of 2 mm or less makes electron-microscopic sectioning easier.
    2. The excised leaf tissues are fixed in fixing solution (see Recipes) (a 2% glutaraldehyde-phosphate buffer solution [pH 7.0]) for a minimum of 3 h at room temperature. For convenience, the leaf tissues in fixing solution can be stored in a refrigerator overnight.
      Note: To avoid tissue damage by grasping with tweezers, the leaf discs are transferred into the fixing solution by attachment to wet tweezers (Figure 1).


      Figure 1. Preparation of Arabidopsis leaf segments for glutaraldehyde fixation. Cut out mesophyll tissues including chloroplasts (A). Wet tips of pincette (B) and carry tissue into the fixing solution (C).

  3. Embedding and sectioning for light-microscopic observation
    1. The 5% solid agar (once dissolved and autoclaved according to the recipe, the agar is stored as a stock of solid agar, see Recipes) is heated until boiling in a microwave, repeating 2-3 times until all the solid agar is completely dissolved. The agar solution is allowed to cool to 50-60 °C and then poured into plastic Petri dishes approximately 50 mm high.
      Note: Preparation of an agar block with similar hardness to leaf mesophyll cells is important to make tissue sections with natural, round, cellular shapes.
    2. Tissue blocks are embedded to keep the leaf cross-sections upright.
    3. After cooling the agar containing leaf sections in a refrigerator at 4 °C, an agar block with the embedded leaf tissue is cut out (Figure 2).


      Figure 2. Preparation of agar block with embedded leaf. Pour melted agar into a Petri dish (A) and embed tissues in agar (B). Cut out agar block (C) for slicing.

    4. The block is placed on the stage of the micro-slicer, and water is poured into the vessel. Half of a double-edged razor blade is fitted to the prescribed position to be able to precisely cut the block to a minimum thickness of 10 μm. A 100-μm-thick leaf section is scooped onto a glass slide and then subjected to light-microscopic observation (Figure 3).


      Figure 3. Slicing of leaf traverse section. Place a sample block on the stage (A) of the micro-slicer (B). Scoop a leaf section (C) onto a glass slide (D).

  4. Microscopic observation
    1. A drop of water is placed on a glass microscope slide. A floating tissue slice is scooped up with the tip of a stainless-steel pincette and allowed to float on the water droplet on a glass slide. A glass microscope cover slip is placed on the sample to eliminate air bubbles (Figure 3).
    2. Observations are first made with the 20x objective lens, which enables whole cross-sectional observation from the surface to the underside of the leaf. The chloroplasts distributed in each cell are viewed as a whole. To verify the shape of each chloroplast, the image is magnified to 40x and 100x, focusing on a chloroplast attached to the cell membrane. Differential interference contrast (DIC) imaging makes it easier to detect the differences in membrane structures (Figure 4).


      Figure 4. Light microscopic observation of Arabidopsis chloroplasts. Chloroplasts lined the inner cell membrane of a young leaf in the vegetative growth phase (A) and of a mature leaf in the floral phase (B). The Arabidopsis plants were grown under controlled conditions of a 12-h photoperiod at 22 °C. Leaves with sizes of 1 x 2 cm (A) and 2 x 5 cm (B) were subjected to microscopic observation. Scale bars represent 10 μm.

Data analysis

Starting with an overview of chloroplasts using a wide microscopic field, a variety of different shapes were observed, which is caused by the natural placement of chloroplasts at either surface of the leaf (which reflects the different lighting conditions between the two surfaces) and the direction of observation of the chloroplasts (which reflects the non-uniformity of shape). To detect morphological differences in the outer shapes of chloroplasts, the observational analysis was carried out by focusing on chloroplasts attached to inner cell membrane like a ring (Figure 4). Chloroplasts released from membranes or attached to the membrane on the reverse side of the sample are often seen as round shapes lacking thylakoid structures.

Notes

The shapes of chloroplasts, whether lens-like or not, can be indicators of thylakoid structure. Sampling of leaves within several hours of growth in light is important in order to observe lens-like outlines of chloroplasts. Large starch granules accumulate in chloroplasts during long lighting periods due to deformation.

Recipes

  1. Fixing solution
    10 ml of 25% glutaraldehyde
    115 ml of 50 mM K-phosphate buffer, pH 7.0
  2. 5% agar
    3.5 g of agar powder
    Add 70 ml of distilled water
    Dissolve agar completely by autoclaving at 105 °C for 5 min
    Agitate gently to achieve a homogeneous liquid state
    Cool down to 50-60 °C for use
    Store at room temperature

Part II. Transmission electron microscopy

Materials and Reagents

  1. 1.5-ml microtubes (Ina-Optika, Bio Bik, catalog number: RC-0150 )
  2. Disposable Pasteur pipets (Fisher Scientific, catalog numbers: 13-678-30 , 13-678-20C )
  3. Filter paper (ADVANTEC No.1) (AS ONE, catalog number: 00011090 )
  4. Bottle for collecting waste solution
  5. Reagent bottle, narrow mouth, amber glass (Spectrum Chemical, Wheaton, catalog number: 989-11698 )
  6. Disposable plastic syringe (Terumo, catalog numbers: SS-01T , SS-20ESZ , SS-50ESZ )
  7. Disposable plastic beaker (Nisshin EM, catalog number: 405 )
  8. Silicon embedding plate (Nisshin EM, catalog number: 420-1 )
  9. Glass sheet for glass knife (Nisshin EM, catalog number: 5402 , 8-mm thick)
  10. 150 Cu Grid (Nisshin EM, Veco grids)
  11. Parafilm M (Pechiney Plastic Packaging, AS ONE, catalog number: 6-711-01 )
  12. Arabidopsis thaliana ecotype Columbia
  13. 99.5% ethanol (NACALAI TESQUE, catalog number: 14713-53 )
  14. Propylene oxide (Nisshin EM, catalog number: 311 )
  15. Toluidine blue (Sigma-Aldrich, catalog number: 198161 )
  16. Lead stain solution (Sigma-Aldrich, catalog number: 203580 )
  17. Glutaraldehyde, 25% aqueous ampule (Nisshin EM, catalog number: 3020-2 )
  18. Osmic acid solution (Nisshin EM, catalog number: 3028 )
  19. Potassium dihydrogen phosphate (KH2PO4) (KANTO KAGAKU, catalog number: 32379-23 )
  20. Dipotassium hydrogen phosphate (K2HPO4) (KANTO KAGAKU, catalog number: 32378-23 )
  21. Epon 812 resin embedding kit (TAAB Laboratories 500 g) (Nisshin EM, catalog number: 342-2 )
  22. Uranyl acetate (Merck KGaA, Darmstadt (Discontinued), Replacement: Polysciences, 400 Valley Road Warrington, PA 18976) (Polysciences, catalog number: 21447-25 )
  23. Molecular sieves 3A (NACALAI TESQUE, catalog number: 04176-55 )
  24. 2% glutaraldehyde (see Recipes)
  25. 2% osmium solution (see Recipes)
  26. 0.1 M potassium phosphate buffer (pH 7.0) (see Recipes)
  27. Washing buffer (see Recipes)
  28. 100% ethanol (see Recipes)

Equipment

  1. Storage bottle (sample tube bottle No.3, 10 ml) (AS one, catalog number: 9-851-05 )
  2. Program-controlled incubator (AS ONE, model: ICV-450P )
  3. Ultra-microtome (Leica, model: Leica EM UC7i )
  4. Tweezers (PEER-VIGOR Type 7) (Nisshin EM, catalog number: 2246 )
  5. Hotplate (AS ONE, model: HP-A1914B )
  6. Transmission electron microscope (JEOL, model: JME-1400 )
  7. Diamond knife (Biel Switzerland, DiATOME 2501)
  8. Glass knife (Leica glass Knife Strip, Nisshin EM, catalog number: 542 )
  9. Vacuum evaporator (JEOL, model: JEE-420T )
  10. CCD camera (Gatan, model: ORIUS® SC 1000, Model 832 TEM )
  11. Invitro shaker (Taitec, model: wave-S1 slim )
  12. Water purification system (Sartorius, model: Arium® 611DI )
  13. Automatic lab mixer (AS ONE, model: HM-10H )
  14. Aspirator (AS ONE, model: AS-01 )
  15. Rotator (Sakura, model: VEM-16 )
  16. Grid stick kit (Micro Star Corporation)

Procedure

Note: This part is modified from Desmond, 1965.

  1. Double fixation
    1. Pre-fixation: 4-6 pieces of 1-2 x 2-4 mm fresh leaf tissues are put into a 1.5-ml microtube filled with 0.5-1.0 ml of 2% glutaraldehyde-0.1 M phosphate buffer solution (see Recipes) for 1-2 h, and then, the tubes are placed in a vacuum desiccator for 1-2 h.
    2. A visual check is performed to ensure that the leaf discs sink to the bottom.
    3. After pre-fixation, the fixing solution is removed with a Pasteur pipet and transferred to a waste storage bottle.
    4. Using a new Pasteur pipet, 1-2 ml of K-phosphate washing buffer (see Recipes) is added into the microtube with the leaf discs.
    5. The leaves are washed for 10 min while shaking the tube gently by hand. While shaking, care is taken to prevent the leaves from getting dry. This washing is repeated 6 times.
    6. Post-fixation: the tissues are fixed with 2% osmium tetroxide (OsO4) (see Recipes) at 4 °C for 1 h.

  2. Dehydration
    1. The tissues are dehydrated with a series of solutions containing increasing concentrations of ethanol–50%, 60%, 70%, 80%, 90% and 95%–with rotation for 15 to 30 min.
    2. Subsequently, the tissues are dehydrated with 100% ethanol (see Recipes) for 60 min, 3 times, with shaking.

  3. Substitution
    1. The tissues are permeated twice with 100% propylene oxide for 15 min.
    2. To increase the concentration of epoxy resin gradually, the tissue is infiltrated into 7:3, 5:5, and 3:7 mixed solutions of propylene oxide and epoxy resin, for 60 min each time.
    3. Finally, the tissue is infiltrated into the pure epoxy resin for 60 min.

  4. Resin embedding and hardening
    1. The silicon embedding plate is filled with pure epoxy resin and the tissues are placed in the resin.
    2. The blocks of epoxy resin are hardened by heat in the program-controlled incubator.
      Note: The heat program starts at 35 °C for 8 h, rises to 45 °C for 8 h, and then heats at 60 °C for 48 h.

  5. Preparation of specimen for light microscopy
    1. The epoxy resin block is attached to an ultra-microtome and cut with a glass knife to obtain 3-μm thick sections.
    2. Using tweezers, a section is placed on a glass slide where a drop of distilled water has been placed.
    3. The glass slide is dried at 60-70 °C on a hotplate.
    4. After drying, the tissue is stained a with 1% toluidine blue staining solution while warming for 60 sec.
    5. After washing with water and drying, desired portions of tissues sample are selected by optical microscopy.

  6. Trimming
    The sample is trimmed again under a stereoscopic microscope to an ultrathin section size of approximately 0.5 x 0.5 mm, which includes the site to be observed.
    The ultrathin section size of the specimen to be observed by electron microscopy is smaller than the that of the optical microscopy specimen, and the section size depends on the blade width of the diamond knife. The minimum width size of optical microscopy specimens is 25 mm, while the maximum size of electron microscopy specimens is 3 mm. Therefore, it is necessary to select and re-trim the site for observation from the optical microscope specimen to use it for electron microscopy.

  7. Ultrathin section preparation (Williams and Carter, 2009)
    1. The re-trimmed epoxy resin block is attached to the ultra-microtome, and the knife is changed from a glass knife to a diamond one.
    2. The parallax is readjusted because of the change in parallelism between the block and the knife.
    3. After adjusting the parallelism, the re-trimmed epoxy resin block is sliced with the diamond knife. The feed volume and cutting speed of the ultra-microtome are used to adjust the thickness.
    4. Ultrathin sections made by the diamond knife are collected on water. The thickness of the ultrathin section is set to 100 nm.
    5. The thin sections floating on the water in the diamond knife boat are transferred to the grid.
      Extra water is wiped off with filter paper, and the grids are placed into a Petri dish.

  8. Staining for electron microscopy
    1. The ultrathin section grid is attached to the contact tape of a stick stain holder. The stick stain holder is immersed for 10 min in a centrifuge tube filled with 1% uranyl acetate solution.
    2. The stick stain holder is placed in a beaker filled with distilled water.
    3. The grids on the stick stain holder are washed in 3 changes of water.
    4. After washing, the stick stain holder is wiped with filter paper.
    5. The stick stain holder is immersed into a lead citrate solution for 5 min.
    6. The stick stain holder is placed in a beaker filled with distilled water.
    7. After washing, the stick stain holder is wiped with filter paper.

  9. Carbon coating
    1. Carbon coating has the effect of preventing damage caused by electron beam irradiation to ultrathin sections during electron microscopic observation.
    2. The sample is placed in the carbon vacuum evaporator.
    3. The carbon layer thickness is set to 20 nm.

Note: Procedure A-I is summarized in Figure 5.


Figure 5. Ultrathin section preparation and staining. Fresh 1-2 x 2-4 mm leaf tissues are cut to a size of approximately 1 mm with a razor and placed in a 1.5-ml microtube (A), epoxy resin blocks are trimmed in preparation for ultrathin sectioning and then sectioned on an ultra-microtome (B). Ultrathin sections from the diamond knife are collected on water. The thin, sliced sections floating on the water in the diamond knife boat are picked up and transferred to the grid (C). Electron microscopic staining: The grid with the ultrathin sections grid is attached to the contact tape of a stick stain holder, which is then immersed for 10 min into a centrifuge tube filled with 1% uranyl acetate solution and for 5 min into lead citrate solution (D).



  1. Transmission electron microscopic observations
    1. For electron microscopy, low- and high-magnification observations are carried out using JEM 1400.
    2. Observation data are recorded and stored by the Gatan CCD camera and Digital Microscopy software (Figure 6).


      Figure 6. TEM observations distinguishing the fine structures of thylakoid membranes. Stroma-grana thylakoid membrane of a young leaf (A) and isolated grana thylakoid membrane of a mature leaf (B). Scale bars represent 10 μm.

Data analysis

The TEM imaging data were in agreement with the light microscopic images. The collection of data from more than 5 repetitions of independent experiments showed similar changes in chloroplasts shape and thylakoid structure. Architectural differences in thylakoid membranes are shown as measurements of thicknesses of grana layers, lengths of stroma lamella, and angles of curvature (Nozue et al., 2017).

Recipes

  1. 2% glutaraldehyde
    1 ml of 25% glutaraldehyde
    11.5 ml of 0.1 M K-phosphate buffer
  2. 2% osmium solution
    2 ml of 4% osmic acid solution
    2 ml of 0.1 M K-phosphate buffer
    Note: The osmium solution is stored in a brown bottle for protection from light, at 4 °C.The stopper is sealed with Parafilm.
  3. 0.1 M potassium phosphate buffer (pH 7.0)
    A: 0.2 M KH2PO4 (27.2 g/1,000 ml H2O)
    B: 0.2 M K2HPO4 (45.6 g/1,000 ml H2O)
    Mix 39 ml A and 61 ml B
    Adjust pH to 7.0 by adding solution A or B
  4. Washing buffer
    0.05 M K-phosphate buffer, pH 7.0, is prepared from refrigerated 0.1 M K-phosphate buffer stocks
  5. 100% ethanol
    Put an appropriate amount of molecular sieve in a 500-ml reagent bottle and then add 95% ethanol. The initially turbid ethanol solution becomes clear over time. Use the supernatant liquid
  6. Epon 812 resin embedding kit
    For 50 ml of Epon 812 resin:
    23.2 ml of Epon 812
    12.6 ml of DDSA (dodecenylsuccinic anhydride)
    14.2 ml of MNA (Methyl nadic anhydride)
    For solidification of resin, add 0.75 ml of DMP30 (2,4,6-Tris(dimethylaminomethyl) phenol)
    Gently agitate the Epon resin for 30 min on a shaker
    Notes:
    1. Plastic syringes are used to measure the reagent volume and collected in a plastic centrifuge tube (50 ml).
    2. All Epon reagents are stored at 4 °C. The reagents warmed to room temperature (22-24 °C under temperature-controlled conditions) are used for resin preparation. Application conditions vary based on temperature, humidity and storage conditions.

Acknowledgments

Prof. Y Kaneko provided instruction on the fundamentals of the resin embedding method for plant samples. We also thank Ms. M. Kondo for technical advice on TEM analysis. We thank Dr. K. Fukamoto for her assistance in preparing the manuscripts.

Reference

  1. Desmond, H. K. (1965). Techniques for electron Microscopy. 2nd edition. Blackwell Scientific Publications.
  2. Kutik, J. (1985). Photosynthesis during leaf development. In: Sestak, Z. (Ed.). Chloroplast Development. Dr W. Junk Publishers pp: 51-75.
  3. Lichtenthaler, H. K., Buschmann, C., Doll, M., Fietz, H. J., Bach, T., Kozel, U., Meier, D. and Rahmsdorf, U. (1981). Photosynthetic activity, chloroplast ultrastructure, and leaf characteristics of high-light and low-light plants and of sun and shade leaves. Photosynth Res 2(2): 115-141.
  4. Nozue, H., Oono, K., Ichikawa, Y., Tanimura, S., Shirai, K., Sonoike, K., Nozue, M. and Hayashida, N. (2017). Significance of structural variation in thylakoid membranes in maintaining functional photosystems during reproductive growth. Physiol Plant 160(1): 111-123.
  5. Oswald, O., Martin, T., Dominy, P. J. and Graham, I. A. (2001). Plastid redox state and sugars: interactive regulators of nuclear-encoded photosynthetic gene expression. Proc Natl Acad Sci U S A 98(4): 2047-2052.
  6. Terashima, I. and Hikosaka, K. (1995). Comparative ecophysiology of leaf and canopy photosynthesis. Plant Cell Environ 18: 1111-1128.
  7. Williams, D. B. and Carter, C. B. (2009). Transmission electron microscopy: A textbook for materials science. Springer.

简介

叶绿体的形状和内部类囊体膜的结构被生长和环境变化所改变(Lichtenthaler等,1981; Kutik,1985; Terashima和Hikosaka,1995)。 这些形态改变经由过渡中间体进行,在此期间在细胞中观察到动态和非均匀的类囊体膜(Nozue等人,2017)。 光学显微镜可用于检测叶绿体中的形态差异。 透射电子显微镜(TEM)证实了这种形态变化的叶绿体的类囊体结构。 描述了用光学显微镜结合电子显微镜监测结构变化的方法。
【背景】已经提出了超结构形态学与类囊体膜中的光合作用和代谢途径的功能性偶联(Oswald等人,2001)。 这是由类囊体膜在叶成熟期间和从营养期向开花生长期转变期间的异质性支持的。 形态改变有一定的时间滞后(Nozue等人,2017)。 类囊体膜的重排与叶绿体形状的变化同时发生,叶绿体的形状从具有典型的细长透镜状外观变为通过光学显微镜可识别的肿胀外观。

关键字:光学显微镜检查, TEM, 叶绿体, 类囊体膜, 拟南芥


第一部分。光学显微镜

材料和试剂

  1. 1.5-ml微型管(Ina-Optika,Bio Bik,目录号:RC-0150)
  2. 矿棉(日本岩棉,Yasaihana-block50)
  3. 剃刀刀片(羽毛安全剃刀,目录号:FAS-10)
  4. 双刃剃须刀(羽毛安全剃刀,产品目录号:FA-10)
  5. 细胞培养皿(Violamo,AS ONE,目录号:2-8590-02)
  6. 玻璃显微镜幻灯片(Matsunami玻璃,目录编号:S1214)
  7. 玻璃显微镜盖滑(奖杯,松浪玻璃,18米/平方米)
  8. 哥伦比亚生态型拟南芥
  9. 水培溶液(OAT Agrio,OAT家用肥料)
  10. 25%戊二醛(NISSHIN EM,目录号:3052)
  11. 琼脂粉(和光纯药工业,目录号:016-11875)
  12. 解决方案(请参阅食谱)
  13. 5%琼脂(见食谱)

设备

  1. 超高压汞灯(尼康公司,型号:INTENSILIGHT C-HGFI)
  2. 荧光显微镜(尼康仪器,型号:ECLIPSE 80i)
  3. 不锈钢镊子(顶部)(AS ONE,目录号:5-1076-01)
  4. 实验室玻璃瓶,螺旋盖,100毫升(SCHOTT,DWK生命科学,杜兰,目录号:21 805 24 04)
  5. 高压蒸汽灭菌器(TOMY SEIKO,型号:LSX-500)
  6. 微波炉(Nisshin EM,型号:MWF-2)
  7. 冰箱(松下,型号:NR-B52T2-H)
  8. 微切片机(DOSAKA EM,型号:DTK-Zero1)
  9. 物镜20倍(尼康仪器,型号:Plan Fluor 20x / 0.50,目录号:MRH00201)
  10. 物镜40倍(Nikon Instruments,型号:Plan Fluor 40x / 0.75,目录号:MRH00401)
  11. 物镜100倍(Nikon Instruments,型号:Plan Fluor 100x / 1.30油,目录号:MRH01902)
  12. CCD相机(Nikon Instruments,型号:DS-Ri1)
  13. 聚光镜(尼康仪器,型号:D-CUD,D-CUO DIC)
  14. 高压灭菌器

软件

1.成像软件(Nikon Instruments,型号:NIS-Elements)

程序

  1. 叶子的生长条件和采样
    1. 使用水培溶液(EC:电导率,1.2ds / m,pH7.0)使拟南芥种子(哥伦比亚生态型)萌发并在矿棉上培养。
    2. 荧光灯管和由蓝色(450纳米),绿色(545纳米)和红色(660纳米)组成的白色LED用作生长的光源。
    3. 将生长条件控制在22-24℃和50-70%湿度的温度下,并将生长区域上的PPFD调节至70-80μmolm -2•s -1 1/12小时光照。

    4. 在12小时黑暗期之后,在光照期的1-3小时从植物收获叶
  2. 叶盘的制备
    1. 将一片新鲜的叶子放在半软质材料的塑料垫上。用一把锋利的刀片切出不同尺寸的叶片块,3×5毫米的光学显微镜和1-2×3-5毫米的电子显微镜。与叶片垂直接触的剃刀刀片被小心地拉制,以免由于过度压缩而损伤细胞。
      主叶脉不应该包括在选定的叶块中(图1) 注:稍大的尺寸使得光学切片更容易,而2mm或更小的尺寸使电子显微切片更容易。
    2. 将切下的叶组织在室温下固定在固定溶液(参见配方)(2%戊二醛 - 磷酸盐缓冲液[pH 7.0])至少3小时。为了方便起见,定影液中的叶子组织可以放在冰箱里过夜。
      注意:为了避免用镊子抓住组织而造成的损伤,通过附着湿镊子将叶片转移到固定溶液中(图1)。


      图1.用于戊二醛固定的拟南芥叶片段的制备。切除包括叶绿体的叶肉组织(A)。 (B)的湿尖,并将组织带入定影液(C)。

  3. 嵌入和切片进行光镜观察
    1. 将5%固体琼脂(一旦按照配方溶解和高压灭菌,将琼脂储存成固体琼脂原料,参见食谱)加热至微波沸腾,重复2-3次直至所有固体琼脂完全溶解。使琼脂溶液冷却至50-60℃,然后倒入约50mm高的塑料培养皿中。
      注意:制备与叶肉细胞具有相似硬度的琼脂块对于制作具有自然,圆形,多孔形状的组织切片是非常重要的。
    2. 嵌入组织块以保持叶片横截面直立。
    3. 将含有叶片的琼脂在4℃的冰箱中冷却后,切下具有嵌入的叶组织的琼脂块(图2)。


      图2.嵌有叶的琼脂块的制备。将融化的琼脂倒入培养皿(A)并将组织嵌入琼脂(B)中。切出琼脂块(C)切片。

    4. 将块放置在微切片机的台上,并将水倒入容器中。将一半的双刃刀片安装到规定的位置,以便能够精确地将块切割成10μm的最小厚度。将一个100-μm厚的叶片挖出到载玻片上,然后进行光镜观察(图3)。


      图3.切片叶横断面。在微切片机(B)的载物台(A)上放置一个样品块。把一个叶子部分(C)舀到载玻片(D)上。

  4. 显微观察
    1. 一滴水放在玻璃载玻片上。用不锈钢镊子的尖端舀起漂浮的组织切片并使其漂浮在载玻片上的水滴上。在样品上放置玻璃显微镜盖玻片以消除气泡(图3)。
    2. 首先用20倍的物镜进行观察,从而可以从表面到叶片下侧进行整个横截面的观察。分布在每个细胞的叶绿体被视为一个整体。为了验证每个叶绿体的形状,图像被放大到40倍和100倍,聚焦于附着在细胞膜上的叶绿体。差分干涉对比(DIC)成像使得更容易检测膜结构的差异(图4)。


      图4.拟南芥叶绿体的光学显微镜观察叶绿体内衬植物生长期幼叶的内细胞膜(A)和花的成熟叶片(B)。拟南芥属植物在22℃的12小时光周期的受控条件下生长。对尺寸为1×2cm(A)和2×5cm(B)的叶片进行显微镜观察。比例尺代表10微米。

数据分析

从广泛的微观领域的叶绿体概述开始,观察到了各种不同的形状,这是由叶片的任一表面(反映两个表面之间的不同光照条件)的叶绿体的自然位置和方向观察叶绿体(这反映了形状的不均匀性)。为了检测叶绿体外形上的形态差异,通过集中于像内环细胞膜上附着的叶绿体(图4)进行观察分析。从膜上释放或附着在样品反面的膜上的叶绿体经常被看作缺乏类囊体结构的圆形。

笔记

叶绿体的形状,无论是否透镜,可以是类囊体结构的指标。为了观察叶绿体的透镜状轮廓,在光生长几个小时内叶子的取样是重要的。由于变形,在长时间照明期间,大的淀粉颗粒积累在叶绿体中。

食谱

  1. 修复解决方案
    10毫升的25%戊二醛
    115ml 50mM K-磷酸盐缓冲液,pH7.0
  2. 5%琼脂
    3.5克琼脂粉
    加70毫升蒸馏水

    在105°C高压灭菌5分钟完全溶解琼脂 轻轻搅拌,以达到均匀的液态
    冷却到50-60°C使用
    在室温下储存

第二部分。透射电子显微镜

材料和试剂

  1. 1.5-ml微型管(Ina-Optika,Bio Bik,目录号:RC-0150)
  2. 一次性巴斯德吸管(Fisher Scientific,目录号:13-678-30,13-678-20C)
  3. 滤纸(ADVANTEC No.1)(AS ONE,目录号:00011090)
  4. 收集废液的瓶子
  5. 试剂瓶,窄嘴,琥珀色玻璃(Spectrum Chemical,Wheaton,目录号:989-11698)
  6. 一次性塑料注射器(Terumo,产品目录号:SS-01T,SS-20ESZ,SS-50ESZ)
  7. 一次性塑料烧杯(Nisshin EM,目录号:405)
  8. 硅嵌入板(Nisshin EM,目录号:420-1)
  9. 玻璃刀用玻璃板(Nisshin EM,目录号:5402,8mm厚)
  10. 150铜电网(日新电机,维科电网)
  11. Parafilm M(Pechiney塑料包装,AS ONE,目录号:6-711-01)
  12. 哥伦比亚生态型拟南芥
  13. 99.5%乙醇(NACALAI TESQUE,目录号:14713-53)
  14. 环氧丙烷(Nisshin EM,目录号:311)
  15. 甲苯胺蓝(Sigma-Aldrich,目录号:198161)
  16. 铅染色液(Sigma-Aldrich,目录号:203580)
  17. 戊二醛,25%安瓿瓶(Nisshin EM,目录号:3020-2)
  18. Osmic酸溶液(Nisshin EM,目录号:3028)
  19. 磷酸二氢钾(KH 2 PO 4)(KANTO KAGAKU,目录号:32379-23)
  20. 磷酸氢二钾(KH 2 HPO 4)(KANTO KAGAKU,目录号:32378-23)
  21. Epon 812树脂包埋试剂盒(TAAB Laboratories 500g)(Nisshin EM,目录号:342-2)
  22. 醋酸铀酰(Merck KGaA,Darmstadt(Discontinued),替代品:Polysciences,400 Valley Road Warrington,PA 18976)(Polysciences,目录号:21447-25)
  23. 分子筛3A(NACALAI TESQUE,目录号:04176-55)
  24. 2%戊二醛(见食谱)
  25. 2%锇溶液(见食谱)
  26. 0.1M磷酸钾缓冲液(pH 7.0)(见食谱)
  27. 洗涤缓冲液(见食谱)
  28. 100%乙醇(见食谱)

设备

  1. 储存瓶(样品瓶3号,10毫升)(AS一,目录号:9-851-05)
  2. 程序控制的孵化器(AS ONE,型号:ICV-450P)
  3. 超薄切片机(Leica,型号:Leica EM UC7i)
  4. 镊子(PEER-VIGOR 7型)(Nisshin EM,目录号:2246)
  5. 电炉(AS ONE,型号:HP-A1914B)
  6. 透射电子显微镜(JEOL,型号:JME-1400)
  7. 钻石刀(比尔瑞士,Diatome 2501)
  8. 玻璃刀(徕卡玻璃刀条,日清EM,目录号:542)
  9. 真空蒸发器(JEOL,型号:JEE-420T)
  10. CCD照相机(Gatan,型号:ORIUS SC 1000,型号832TEM)
  11. Invitro摇床(Taitec,型号:wave-S1苗条)
  12. 水净化系统(Sartorius,型号:Arium 611DI)
  13. 自动实验室搅拌机(AS ONE,型号:HM-10H)
  14. 吸气器(AS ONE,型号:AS-01)
  15. 转子(樱花,型号:VEM-16)
  16. 网格棒套件(Micro Star Corporation)

程序

注:这部分是从1965年的Desmond修改过来的。

  1. 双重固定
    1. 预固定:将4-6片1-2×2-4毫米鲜叶组织放入装有0.5-1.0毫升2%戊二醛-0.1M磷酸盐缓冲溶液(参见食谱)的1.5毫升微管中1 -2小时,然后将试管置于真空干燥器中1-2小时。
    2. 进行目视检查以确保叶片下沉。
    3. 预固定后,用巴斯德移液管移除固定溶液并转移到废物储存瓶中。
    4. 使用一个新的巴斯德吸管,1-2毫升的K - 磷酸盐清洗缓冲液(见食谱)被添加到叶片的微管。
    5. 将叶子洗净10分钟,同时用手轻轻摇动管子。在摇动时,要小心防止叶子变干。该洗涤重复6次。
    6. 固定后:将组织用2%四氧化锇(OsO 4)(参见食谱)在4℃下固定1小时。

  2. 脱水
    1. 使用一系列含有增加浓度的乙醇(50%,60%,70%,80%,90%和95%)的溶液使组织脱水15至30分钟。
    2. 随后,使用100%乙醇将组织脱水(参见食谱)60分钟,3次,同时摇动。

  3. 代换

    1. 用100%环氧丙烷渗透组织两次
    2. 为了逐渐增加环氧树脂的浓度,将组织浸入环氧丙烷和环氧树脂的7:3,5:5和3:7混合溶液中,每次60分钟。
    3. 最后,将组织渗入纯环氧树脂60分钟。

  4. 树脂嵌入和硬化
    1. 硅嵌入板填充纯环氧树脂,组织放置在树脂。

    2. 在程序控制的培养箱中加热环氧树脂块 注意:加热程序从35°C开始8小时,升至45°C 8小时,然后在60°C加热48小时。

  5. 光学显微镜样品的制备
    1. 将环氧树脂块连接到超薄切片机上并用玻璃刀切割,以获得3μm厚的切片。
    2. 使用镊子,将切片放置在放有一滴蒸馏水的载玻片上。
    3. 玻片在电热板上60-70℃干燥。
    4. 干燥后,将组织用1%甲苯胺蓝染色溶液染色,同时加热60秒。
    5. 用水洗涤并干燥后,通过光学显微镜选择所需部分的组织样品。

  6. 修剪
    将样品在立体显微镜下再次修剪至约0.5×0.5mm的超薄切片尺寸,其包括要观察的部位。
    电子显微镜观察的样品的超薄切片尺寸小于光学显微镜样品的切片尺寸,切片尺寸取决于金刚石刀片的刀片宽度。光学显微镜样品的最小宽度尺寸是25mm,而电子显微镜样品的最大尺寸是3mm。因此,需要从光学显微镜样品中选择和重新修剪观察部位以用于电子显微镜。

  7. 超薄切片制备(Williams和Carter,2009)
    1. 重新修剪的环氧树脂块附着在超薄切片机上,刀由玻璃刀切换成菱形。

    2. 视差由于块和刀之间的平行度变化而重新调整
    3. 调整好平行度之后,用金刚石刀将重新镶边的环氧树脂块切成片。
      使用超薄切片机的进料量和切割速度来调整厚度
    4. 由钻石刀制成的超薄切片收集在水上。超薄部分的厚度设置为100纳米。

    5. 在钻石刀船上漂浮在水面上的薄片被转移到电网上。

      用滤纸擦去多余的水,并将网格放入培养皿中。

  8. 电子显微镜染色
    1. 超薄切片格栅附着在棒状污渍固定器的接触带上。将污渍固定器浸入装有1%乙酸铀酰溶液的离心管中10分钟。
    2. 将污渍固定器放在充满蒸馏水的烧杯中。

    3. 在污渍夹持器上的栅格用3次水冲洗。
    4. 洗完后,用污渍纸擦干污渍。

    5. 沾污持有人浸入柠檬酸铅溶液5分钟。
    6. 将污渍固定器放在充满蒸馏水的烧杯中。
    7. 洗完后,用污渍纸擦干污渍。

  9. 碳涂层
    1. 碳涂层具有防止电子束照射在电子显微镜观察期间对超薄切片造成的损害的作用。
    2. 将样品放入碳素真空蒸发器中。
    3. 碳层厚度设置为20纳米。

注意:程序A-I总结在图5中。


图5.超薄切片制备和染色用剃刀将新鲜的1-2×2-4mm叶片组织切成约1mm大小,并置于1.5ml微管(A)中, ,剪下环氧树脂块准备超薄切片,然后在超薄切片机(B)上切片。钻石刀的超薄部分收集在水面上。漂浮在钻石刀船的水面上的薄切片被拾取并转移到电网(C)。电子显微镜染色:将带有超薄切片网格的格栅连接到棒状污渍固定器的接触带上,然后将其浸入装有1%乙酸双氧铀溶液的离心管中10分钟,并在柠檬酸铅溶液( d)。

  1. 透射电子显微镜观察
    1. 对于电子显微镜,使用JEM 1400进行低放大倍数观察。
    2. 观察数据由Gatan CCD相机和数字显微镜软件(图6)记录和存储。


      图6. TEM观察区分类囊体膜的精细结构。 (A)和分离的成熟叶片的类囊体膜(B)的基因组类囊体膜。比例尺代表10微米。

数据分析

TEM成像数据与光学显微镜图像一致。从超过5次重复的独立实验收集的数据显示叶绿体形状和类囊体结构相似的变化。类囊体膜的结构差异显示为基纹层厚度,基质片层长度和曲率角的测量值(Nozue等人,2017)。

食谱

  1. 2%戊二醛
    1毫升的25%戊二醛
    11.5毫升的0.1M K-磷酸盐缓冲液
  2. 2%锇溶液
    2毫升4%的锇酸溶液
    2毫升0.1M K-磷酸盐缓冲液
    注意:锇溶液在4°C储存在棕色瓶子中以防光。塞子用Parafilm密封。
  3. 0.1M磷酸钾缓冲液(pH7.0)
    A:0.2M KH 2 PO 4(27.2g / 1000ml H 2 O)
    B:0.2M K 2 HPO 4 4(45.6g / 1000ml H 2 O)
    混合39毫升A和61毫升B
    通过添加溶液A或B来调节pH至7.0
  4. 清洗缓冲液

    用冷冻的0.1M K-磷酸盐缓冲液制备0.05M K-磷酸盐缓冲液,pH7.0
  5. 100%乙醇
    将适量的分子筛放入500ml试剂瓶中,加入95%乙醇。最初混浊的乙醇溶液随时间变得清澈。使用上清液
  6. Epon 812树脂嵌入套件
    对于50毫升的Epon 812树脂:
    23.2毫升的Epon 812
    12.6毫升DDSA(十二碳烯基琥珀酸酐)
    14.2毫升的MNA(甲基纳迪克酸酐)
    为了固化树脂,加入0.75毫升DMP30(2,4,6-三(二甲基氨基甲基)苯酚)
    在振动筛上轻轻搅拌Epon树脂30分钟 注意:
    1. 使用塑料注射器测量试剂体积并收集在塑料离心管(50ml)中。
    2. 所有Epon试剂都储存在4°C。加热到室温(在温度控制条件下为22-24℃)的试剂用于树脂制备。应用条件因温度,湿度和储存条件而异。

致谢

Y Kaneko教授提供了植物样品树脂包埋方法的基础知识。我们也感谢孔多女士提供有关透射电镜分析的技术建议。我们感谢K. Fukamoto博士在准备手稿方面的协助。

参考

  1. Desmond,H.K。(1965)。电子显微镜技术。第二版。 Blackwell Scientific Publications 。
  2. Kutik,J。(1985)。 叶片发育过程中的光合作用在:Sestak,Z. (编)。叶绿体发育。 W. Junk Publishers博士 pp:51-75。
  3. Lichtenthaler,H.K.,Buschmann,C.,Doll,M.,Fietz,H.J.,Bach,T.,Kozel,U.,Meier,D。和Rahmsdorf,U。(1981)。 高光和弱光植物的光合活性,叶绿体超微结构和叶片特性以及太阳光和树荫叶子。 Photosynth Res 2(2):115-141。
  4. Nozue,H.,Oono,K.,Ichikawa,Y.,Tanimura,S.,Shirai,K.,Sonoike,K.,Nozue,M。和Hayashida,N.(2017)。 类囊体膜结构变异在维持生殖生长期间功能性光系统中的意义 Physiol植物 160(1):111-123。
  5. Oswald,O.,Martin,T.,Dominy,P.J。和Graham,I.A。(2001)。 质体氧化还原状态和糖:核编码的光合基因表达的交互式调节剂 < (Proc Natl Acad Sci USA)98 /(4):2047-2052。
  6. Terashima,I.和Hikosaka,K。(1995)。 叶和冠层光合作用的比较生态生理学。植物细胞环境 18:1111-1128。
  7. Williams,D.B。和Carter,C.B。(2009)。 透射电子显微镜:材料科学的教科书 Springer 。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nozue, H. and Kametani, K. (2017). Using Light and Electron Microscopy to Estimate Structural Variation in Thylakoid Membranes. Bio-protocol 7(23): e2639. DOI: 10.21769/BioProtoc.2639.
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