Rapid and Inexpensive Method of Loading Fluorescent Dye into Pollen Tubes and Root Hairs

Abstract

The most direct technique for studying calcium, which is an essential element for pollen tube growth, is Ca2+ imaging. Because membranes are relatively impermeable, the loading of fluorescent Ca2+ probes into plant cells is a challenging task. Thus, we have developed a new method of loading fluo-4 acetoxymethyl ester into cells that uses a cell lysis solution to improve the introduction of this fluorescent dye into pollen tubes. Using this method, the loading times were reduced to 15 min. Furthermore, loading did not have to be performed at low (4°C) temperatures and was successful at room temperature, and pluronic F-127 was not required, which would theoretically allow for the loading of an unlimited number of cells. Moreover, the method can also be used to fluorescently stain root hairs.

3D Manipulation and Imaging of Plant Cells using Acoustically Activated Microbubbles

“…The closed microchannel design enforces a limitation to optical feedback and, thereby complicates the thorough investigation of the specimen. Here we demonstrate the rotation of pollen grains in open channels, which additionally provides access for micromechanical interrogations of the sample by integration with microforce sensing systems, such as the cellular force microscope, or for microinjection of high molecular weight dyes or DNA, which are cell wall or membrane impermeable . The open‐microchannel arrangement is developed by bonding the PDMS microchannel upside‐down onto a glass slide, as shown in Figure and Figure S2 in the Supporting Information.…”

“…Here we demonstrate the rotation of pollen grains in open channels, which additionally provides access for micromechanical interrogations of the sample by integration with microforce sensing systems, [51,52] such as the cellular force microscope, or for microinjection of high molecular weight dyes or DNA, which are cell wall or membrane impermeable. [53,54] The open-microchannel arrangement is developed by bonding the PDMS microchannel upside-down onto a glass slide, as shown in Figure 6 and Figure S2 in the Supporting Information. Submerged lily pollen grains are placed dropwise onto the microchannel, simultaneously trapping air bubbles in the microcavities due to the hydrophobic surface properties of the PDMS.…”

Loading calcium fluorescent probes into protoplasts to detect calcium in the flesh tissue cells of Malus domestica

“…Calcium imaging is a useful technique for studying the roles of Ca 2+ in living cells 18 . In plants for which stable transgenic systems are difficult to establish, small- 20 . We used enzymatic hydrolysis to obtain viable apple flesh protoplasts and then loaded Ca 2+ fluorescent probes into the protoplasts for cytoplasmic calcium imaging.…”

“…Therefore, the indicator should be added with an acetoxymethyl (AM) ester, and the dye should be made neutral so that it can cross the cell membrane 41 . However, esterases on the cell membrane can cleave AM groups and prevent the indicator from entering the cell 20 wall is a pool of Ca 2+ that interferes with the fluorescent intensity of Ca 2+ in the cytoplasm when loaded with a fluorescent probe 31 . To avoid this interference, the microinjection method is also used to avoid the cell wall 44 .…”

“…However, this method is very complicated and slow 31,33 . In a previous study, we used cell lysates to slightly degrade the cell membrane without reducing cell viability and allowed the calcium fluorescent probe to enter the pollen tube 20 . In the present study, we successfully removed the cell wall of flesh cells by enzymatic hydrolysis and loaded fluorescent probes into protoplasts at a high temperature (37°C).…”