使用Olympus双光子激光扫描显微镜分析完整的拟南芥花药中正在发育的花粉粒

Teagen D. Quilichini; A. Lacey Samuels; Carl J. Douglas

doi:10.21769/BioProtoc.1677

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Analysis of Developing Pollen Grains within Intact Arabidopsis thaliana Anthers by Olympus Two-Photon Laser Scanning Microscopy

使用Olympus双光子激光扫描显微镜分析完整的拟南芥花药中正在发育的花粉粒

Teagen D. Quilichini

AS A. Lacey Samuels

Carl J. Douglas email

发布: 2015年12月05日第5卷第23期 DOI: 10.21769/BioProtoc.1677 浏览次数: 9908

评审: Tie LiuNing LiuAnonymous reviewer(s)

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of The Plant Cell, featuring study using the protocol.

Nov 2014

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

活体植物根部细胞核的近红外自发荧光成像

Akira Yoshinari and Masayoshi Nakamura

2025年04月20日 1081 阅读

利用活细胞成像系统研究拟南芥珠被中淀粉体的复制

Makoto T. Fujiwara [...] Ryuuichi D. Itoh

2025年06月05日 1441 阅读

活体叶片切片成像观察细胞内叶绿体运动及细胞间相互作用

Yuta Kato [...] Mitsutaka Taniguchi

2025年08月05日 1014 阅读

Abstract

The method consists of imaging developing pollen grains as they form within intact, immature Arabidopsis thaliana anthers. Using two-photon excitation in the infrared wavelength range, the intrinsic fluorescence (autofluorescence) of developing pollen grains and surrounding sporophytic tissues of the anther wall, including the tapetum, middle layer, endothecium and epidermis, can be visualized in the three-dimensional space of an intact anther. In contrast to conventional confocal microscopy, the application of red-shifted light by two-photon microscopy improves depth penetration into specimens, while the scattering of light and subsequent phototoxicity is minimized, making this a superior method for imaging the developing pollen grains and tapetal cells enclosed within anthers. The technique described was optimized for the detection of autofluorescent components of the pollen wall, including sporopollenin and the pollen coat, and provided spatial and developmental data on the autofluorescent metabolites in anthers of wild-type and pollen wall mutant plants (Quilichini et al., 2014). The use of two-photon imaging of live, intact anthers holds potential for future studies aimed at understanding the spatial relationship between gametophytic and sporophytic tissues during pollen development and the distribution of metabolites or fluorescently-tagged proteins within developing anthers.

Materials and Reagents

Ruler with millimetre divisions
Glass bottom Petri dishes (MatTek Corporation, catalog number: P35G-1.5-14C )
Coverslips (No.1.5, 0.17 mm thick, 22 x 22) (Thermo Fisher Scientific, catalog number 12-541B )
Transfer pipettes 1.5 ml
Arabidopsis thaliana plants in the flowering stage
Non-toxic food-grade paraffin wax maintained in a liquid state at 40 ºC in a paraffin bath (Thera-Band, catalog number: 24050 )
Distilled water

Equipment

Spectra-Physics MaiTai HP Titanium:Sapphire mode-locked pulsed laser, with tuneable wavelengths spanning 690 to 1,040 nm.
Olympus Fluoview 1000 scan head (Olympus America Inc., model: FV1000MPE ) modified by Olympus for two-photon imaging by the inclusion of a light path for the pulsed laser input and the housing for the non-descanned, multialkali side window photomultiplier tubes (PMTs).
Olympus BX61WI upright microscope
Olympus multiphoton microscope filter cube (440/40 bandpass filter) (OLYMPUS, model: FV10-MRVGR/XR 4CH NDD FILTER )
Olympus multiphoton microscope dedicated objective lens optimized for transmitting IR light (25x, 1.05 numerical aperture, 2.0 mm working distance) (OLYMPUS, model: FV10-SNPXLU )
Light tight, custom made multiphoton microscope enclosure. To build an enclosure, aluminum extruded components (Item North America) and flat-black painted fibre board can be used
Forceps for fine dissections (Electron Microscopy Sciences, catalog number: 72700-D )
Olympus stereomicroscope (magnification range of 6.3-63x) (OLYMPUS, model: SZX10 )

Software

Olympus FluoView FV10-ASW 3.01
Spectra-Physics MaiTai Control laser software
Volocity version 6.1.1 software package
Note: ImageJ 1.47v can also be used to process images.

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Quilichini, T. D., Samuels, A. L. and Douglas, C. J. (2015). Analysis of Developing Pollen Grains within Intact Arabidopsis thaliana Anthers by Olympus Two-Photon Laser Scanning Microscopy . Bio-protocol 5(23): e1677. DOI: 10.21769/BioProtoc.1677.