Materials and Reagents

1. Plant materials
   1. Arabidopsis thaliana transgenic plants expressing GFP-TALIN in wild-type or mutant plants (Kong et al., 2013). Actin probe lines such as LIFEACT-YFP and GFP-fABD2, THRUMIN1-GFP transgenic lines are also useful.
      
   2. Young and fully expanded rosette leaves of 3-4-week-old plants grown under 100 μmol/m²/s white light (16 h)/dark (8 h) cycles at 23 °C.
      Note: Plants grown on soil are better than those in the plate of MS medium. The reason is that the leaves of the plants grown in the plate are more susceptible to wither during sample handling than those grown on soil.
      
2. Red cellophane film (TokyoButaiShowmei, catalog number: No. 20 )
   
3. (Optional) 2,3-butanedione monoxime (BDM) (Sigma-Aldrich, catalog number: B0753 ) as an inhibitor of myosin ATPase that inhibits actin dynamics
   
4. Evacuation solution containing Silwet L-77 (Bristol-Myers Squibb Company, catalog number: BMS-SL7755 ) (see Recipes)

Equipment

1. Syringe (10 ml)
   
2. Confocal microscope (Leica Microsystems, model: SP5 equipped with a 63x/1.20 W objective lens and multi-line 100 mW argon laser)
   
3. Plant growth room or chamber
   
4. Red safe-light (red LED or fluorescent lamp filtered with red films)
   
5. Forceps
   
6. Razor blade
   
7. Scissors
   
8. A custom-made cuvette system and ring holder: this system is composed of two steel rings mating each other with complementary threads, two round cover glasses (22 mm in diameter, No. 1 and No. 5), and a silicon ring (100 μm in thickness) (Wada and Kong, 2011; see Video 1)
   

Software

1. Open source software ImageJ (http://rsbweb.nih.gov/ij/)

Procedure