Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

Abstract

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.We developed the RootChip, a polydimethylsiloxane (PDMS)-based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging 1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers 2 . This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors 3 . Video LinkThe video component of this article can be found at https://www.jove.com/video/4290/ ProtocolNote: Perform all steps preparatory steps under sterile conditions. Preparation of Plastic Cones for Seed Germination1. Fill a 10 cm Petri dish with growth medium containing 1% agar to a thickness of 5 mm. We use a half strength modified Hoagland medium 4 , but medium composition should be chosen to suit individual experimental requirements. 2. While the medium is still liquid, use a multi-channel pipette to fill 10 μl pipette tips with 5 μl of medium from the Petri dish. 3. Store the filled tips in the pipette tip box until the medium is solid, then cut to 4 mm long plastic cones and place upright into a Petri dish containing solid growth medium. Seed Germination and Seedling Growth1. Surface sterilize seeds in 5% NaOCl for 5 min, wash three times with sterile water, then place a single seed on top of each of the mediumfilled cones. 2. Seal the dish with Micropore tape (3M) and store at 4 °C to synchronize germination. 3. After three days, transfer the plates to a growth cabinet to start germination. Our growth conditions are 23 °C at a 16h high light/8h dark cycle (Light intensity: 100 μE m -2 s -1).

Live Cell Imaging with R-GECO1 Sheds Light on flg22- and Chitin-Induced Transient [Ca 2+ ] cyt Patterns in Arabidopsis

“…To study PAMP-induced Ca 2+ dynamics in roots in more detail, we performed Ca 2+ imaging of 6- to 7-day-old seedlings that had been challenged with flg22 or chitin. Seedlings were grown and imaged in RootChip16, a microfluidic platform that allows reversible and non-invasive application of elicitors via micro-perfusion (Grossmann et al, 2011, 2012; Jones et al, 2014). Since roots have been reported to be less sensitive to flg22 (Ranf et al, 2011), we used 1 μM flg22 for elicitation.…”

“…Fluorescence emission was detected between 570 and 640 nm. Images were recorded with a time interval of 1.5 s. RootChip16 sample preparation was done as described previously (Grossmann et al, 2011, 2012), using the advanced RootChip16 (Jones et al, 2014). In brief, seeds were surface sterilized and germinated on cut pipette tips, prefilled with solidified Hoagland's media (Sigma-Aldrich), and plugged into the chip.…”

In vivo biochemistry: applications for small molecule biosensors in plant biology

“…Upon perfusion of a root with ligand solution (blue background) the cytosolic sensor reports intracellular increase in ligand concentration by a changed ratio of donor and acceptor intensities (here: increase in ratio I(Acc)/I(Don); for details see [10, 46**]. …”

Fluorescent protein marker lines in maize: generation and applications

“…Such studies trace back historically to 3D reconstructions of fixed images at sequential time points, which were used to understand basic subcellular architecture in plants (Donohoe et al, 2013, Staehelin, 1997. Live imaging of FP lines now permits experimental studies of whole organ growth, interactions among neighboring cells, behavior of individual cytoskeletal elements and the ability to trace movement of proteins between cells (Grossmann et al, 2012, Gutierrez et al, 2009, Krebs et al, 2012, Lindeboom et al, 2013, Mathur et al, 2012, Sampathkumar et al, 2011, Teh et al, 2013. With the advent of microfluidic devices, FP markers can be used as biosensors for hormone induction studies during whole root or shoot growth.…”

“…Several quantitative fluorescence spectro-microscopy approaches such as fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and Fluorescence correlation spectroscopy (FCS) have been used for protein dynamic studies in plants , Grossmann et al, 2012, Harter et al, 2012. Other techniques such as dual-color fluorescence cross-correlation spectroscopy (FCCS), and fluorescence resonance energy transfer (FRET) have been used for protein-protein interaction studies.…”