Super-resolution Imaging of Live BY2 Cells Using 3D-structured Illumination Microscopy
采用三维结构照明显微镜对活的BY2细胞进行超分辨率成像
发布: 2016年01月05日第6卷第1期 DOI: 10.21769/BioProtoc.1697 浏览次数: 9772
评审: Fanglian HeAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2015
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Abstract
Light microscopy is the standard tool for studying sub-cellular structures however, owing to the diffractive properties of light, resolution is limited to 200 nm. Super-resolution microscopy methods circumvent this limit, offering greater resolution, particularly when studying fluorescently labeled sub-cellular structures. Super-resolution methods such as 3D-SIM (Structured Illumination Microscopy) fill a useful niche between confocal and electron microscopy. We have previously had success using fixed plant tissue samples with 3D-SIM (Bell and Oparka, 2014). However, sensitive structures can be altered by fixation and embedding procedures, so we developed a method for imaging live cells. In this protocol we used 3D-SIM to image the ER and Hechtian Strands in live, plasmolysed BY2 cells.
Materials and Reagents
- Microscope slides (Thermo Fisher Scientific)
- #1.5 coverslips (0.17 mm thick) (Thermo Fisher Scientific)
- BY2 cells expressing fluorescent marker
- 250 ml Erlenmeyer flasks (Thermo Fisher Scientific)
- Murashige and Skoog (MS) basal salt media (Sigma-Aldrich, catalog number: M5519 )
- Sucrose (Thermo Fisher Scientific)
- (2, 4-Dichlorophenoxy) acetic acid sodium salt monohydrate (Sigma-Aldrich, catalog number: D6679 )
- Calcofluor white/Fluorescent Brightener 28 (Sigma-Aldrich, catalog number: F3543 )
- 1 M D-Mannitol pure (Scientific Laboratory Supplies, catalog number: CHE1796 )
- Nail varnish
- BY2 growth media (see Recipes)
- Calcofluor White Stock solution (see Recipes)
Equipment
- Controlled temperature (28 °C) incubator or room
- Orbital shaker
- PersonalDV Deltavision Epi-fluorescence Inverted Microscope (GE Healthcare, Dharmacon)
- 3D-SIM microscope [DeltaVision OMX Blaze (GE Healthcare, Dharmacon) fitted with an Olympus PlanApo N 100x 1.42 NA oil objective]
- Edge sCMOS camera (PCO AG)
Software
- SoftWoRx 6.0 (GE)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Bell, K., Oparka, K. and Knox, K. (2016). Super-resolution Imaging of Live BY2 Cells Using 3D-structured Illumination Microscopy. Bio-protocol 6(1): e1697. DOI: 10.21769/BioProtoc.1697.
- Knox, K., Wang, P., Kriechbaumer, V., Tilsner, J., Frigerio, L., Sparkes, I., Hawes, C. and Oparka, K. (2015). Putting the squeeze on Plasmodesmata: A role for reticulons in primary plasmodesmata formation. Plant Physiol 168(4): 1563-1572.
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分类
植物科学 > 植物细胞生物学 > 细胞成像
植物科学 > 植物细胞生物学 > 细胞结构
细胞生物学 > 细胞成像 > 活细胞成像
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