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
发布: 2017年12月05日第7卷第23期 DOI: 10.21769/BioProtoc.2639 浏览次数: 9959
评审: Scott A M McAdamAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2017
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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
- 1.5-ml microtubes (Ina-Optika, Bio Bik, catalog number: RC-0150 )
- Mineral wool (Nippon Rockwool, Yasaihana-block50)
- Razor blades (Feather Safety Razor, catalog number: FAS-10 )
- Double-edged razor blades (Feather Safety Razor, catalog number: FA-10 )
- Cell culture dish (Violamo, AS ONE, catalog number: 2-8590-02 )
- Glass microscope slide (Matsunami Glass, catalog number: S1214 )
- Glass microscope cover slip (Trophy, Matsunami Glass, 18 m/m)
- Arabidopsis thaliana ecotype Columbia
- Hydroponic solution (OAT Agrio, OAT House Fertilizer)
- 25% glutaraldehyde (NISSHIN EM, catalog number: 3052 )
- Agar, powder (Wako Pure Chemical Industries, catalog number: 016-11875 )
- Fixing solution (see Recipes)
- 5% agar (see Recipes)
Equipment
- Extra-high-pressure mercury lamp (Nikon Instruments, model: INTENSILIGHT C-HGFI )
- Fluorescence microscope (Nikon Instruments, model: ECLIPSE 80i )
- Stainless-steel pincette (Top well) (AS ONE, catalog number: 5-1076-01 )
- Laboratory glass bottle, screw cap, 100 ml (SCHOTT, DWK Life Sciences, Duran, catalog number: 21 805 24 04 )
- High-pressure steam sterilizer (TOMY SEIKO, model: LSX-500 )
- Microwave (Nisshin EM, model: MWF-2 )
- Refrigerator (Panasonic, model: NR-B52T2-H )
- Micro-slicer (DOSAKA EM, model: DTK-Zero1 )
- Objective lens 20x (Nikon Instruments, model: Plan Fluor 20x/0.50, catalog number: MRH00201 )
- Objective lens 40x (Nikon Instruments, model: Plan Fluor 40x/0.75, catalog number: MRH00401 )
- Objective lens 100x (Nikon Instruments, model: Plan Fluor 100x/1.30 Oil, catalog number: MRH01902 )
- CCD camera (Nikon Instruments, model: DS-Ri1 )
- Condenser (Nikon Instruments, models: D-CUD , D-CUO DIC )
- Autoclave
Software
1.Imaging software (Nikon Instruments, model: NIS-Elements)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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.
Download Citation in RIS Format
分类
植物科学 > 植物细胞生物学 > 细胞结构
细胞生物学 > 细胞成像 > 电子显微镜
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