Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

Two-photon Photoactivation to Measure Histone Exchange Dynamics in Plant Root Cells

双光子光活化法测定植物根细胞中的组蛋白交换动力学

SR Stefanie Rosa
PS Peter Shaw

发布: 2015年10月20日第5卷第20期 DOI: 10.21769/BioProtoc.1628 浏览次数: 8783

评审: Tie LiuVinay PanwarAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
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.

Dec 2014

Advertisement

Special Issue on Protocols for Organoid Research
High-throughput T-cell antigen discovery technology platform and its applications
Technologies for RNA Detection
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

利用延时共聚焦显微成像分析拟南芥茎生叶发育过程

利用延时共聚焦显微成像分析拟南芥茎生叶发育过程

Constance Le Gloanec

2026年05月20日 540 阅读

ROOT-ExM：利用膨胀显微技术实现拟南芥根部蛋白质的超分辨率成像

ROOT-ExM：利用膨胀显微技术实现拟南芥根部蛋白质的超分辨率成像

Yutaro Shimizu [...] Magali S. Grison

2026年06月05日 610 阅读

拟南芥根尖的双色tau-STED超分辨显微成像

拟南芥根尖的双色tau-STED超分辨显微成像

Louise Fougère [...] Magali Grison

2026年08月05日 321 阅读

Abstract

Chromatin-binding proteins play a crucial role in chromatin structure and gene expression. Direct binding of chromatin proteins both maintains and regulates transcriptional states. It is therefore important to study the binding properties of these proteins in vivo within the natural environment of the nucleus. Photobleaching, photoactivation and photoconversion (photoswitching) can provide a non-invasive experimental approach to study dynamic properties of living cells and organisms. We used photoactivation to determine exchange dynamics of histone H2B in plant stem cells of the root (Rosa et al., 2014). The stem cells of the root are located in the middle of the tissue, which made it impossible to carry out photoactivation of sufficiently small and well-defined sub-cellular regions with conventional laser illumination in the confocal microscope, mainly because scattering and refraction effects within the root tissue dispersed the focal spot and caused photoactivation of too large a region. We therefore used 2-photon activation, which has much better inherent resolution of the illuminated region. This is because the activation depends on simultaneous absorption of two or more photons, which in turns depends on the square (or higher power) of the intensity-a much sharper peak. In this protocol we will describe the experimental procedure to perform two-photon photoactivation experiments and the corresponding image analysis. This protocol can be used for nuclear proteins tagged with photoactivable GFP (PA-GFP) expressed in root tissues.

Keywords: Histone exchange (Histone交换) search Two-Photon Microscopy (双光子显微镜) search FRAP (FRAP) search Plant stem cells (植物干细胞) search

Materials and Reagents

  1. Sterile 9 cm square Petri dishes for plant growth media
  2. Cover slips No 1.5 (VWR International, catalog number: 631-0125 )
  3. Microscope slides with frosted end (VWR International, Superfrost®, catalog number: 631-0114 )
  4. Secure Seal Adhesive Sheets (0.12 mm thick) (Grace Bio-Labs, Inc., catalog number: 620001 )
  5. Arabidopsis lines expressing a protein of interest tagged with PA-GFP (Patterson and Lippincott-Schwartz, 2002)
    Note: In our case, for measuring histone exchange dynamics in the root stem cells we placed the H2B-PAGFP construct under the control of a root cell-specific promoter (pSCR) (Rosa et al., 2014), which derives expression simultaneously in quiescent centre cells, endodermis/cortex initials, and root endodermis. This setup allowed us to compare the dynamics of a histone protein in pluripotent stem cells as they progress into more differentiated states. In this protocol we will not mention particular cells or tissues as the procedure can be applied to any cell type in the root.
  6. Murashige & Skoog Basal Medium with Vitamins (any equivalent source would be suitable) (PhytoTechnology Laboratories®, catalog number: M519 )
  7. 10% bleach (contains 5-10% sodium hypochlorite) in dH2O (Vortex, Procter & Gamble)
  8. NH4NO3 (pH 5.8) (ForMediumTM)
  9. Sucrose (Sigma-Aldrich)
  10. Phytagel (Sigma-Aldrich, catalog number: P8169 )
  11. Murashige and Skoog medium (see Recipes)

Equipment

  1. Plant growth chamber
  2. Two-photon photoactivation experiments were performed on an Ultima two-photon laser scanning microscope (Buker Coopration, Prairie Technologies, model: Ultima 2-Photon Microscope )
    Note: Live images were acquired using Olympus 60x 0.9 NA water immersion objectives at 512 x 512 resolution (0.203 μm/pixel) and 1 μm focal steps.
  3. Laminar flow cabinet

Software

  1. ImageJ software
  2. MS Excel
  3. Graphpad prism software

Procedure

English
中文翻译

文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Rosa, S. and Shaw, P. (2015). Two-photon Photoactivation to Measure Histone Exchange Dynamics in Plant Root Cells. Bio-protocol 5(20): e1628. DOI: 10.21769/BioProtoc.1628.

ris ris Download Citation in RIS Format

分类

植物科学 > 植物细胞生物学 > 细胞成像

细胞生物学 > 细胞成像 > 双光子显微镜

您对这篇实验方案有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件