Materials and Reagents

1. Glass slide (26 x 76 No.1, thickness 0.8-1.0 mm) (Matsunami Glass Ind.)
   Note: Any types of glass slide suitable for fluorescence observation can be used. We attached black tape on the glass slide to envelop the sample with the cover slip (see Figure 4D).
2. Cover slip (24 x 60 No.1, thickness 0.12-0.17 mm) (Matsunami Glass Ind.)
   
3. 10 ml disposable syringe (Terumo Medical Corporation)
   
4. Razor blade (43 x 23)
   
5. Arabidopsis thaliana (ecotype Columbia) expressing peroxisome-targeted GFP (GFP-PTS1) (Mano et al., 2002)
   
6. Agar powder (Funakoshi, catalog number: BA-10 )
   
7. Distilled water
   
8. 1/3x Murashige and Skoog salts (MS) medium (Wako Pure Chemical Industries, catalog number: 392-00591 )

Equipment

1. Confocal microscope combined with amplified Ti:Sapphire femtosecond laser system (Figure 2)
1. Ti:Sapphire femtosecond laser system (Cyber Laser Inc., model: IFRIT-SP-01 )
   
2. Confocal microscope (Olympus Corporation of the America, model: FV300-IX71 )
   
3. Objective (OLYMPUS CORPORATION, model: PlanN100x )
   
4. Mechanical shutter (Gate time: 1/125 s) [SIGMAKOKI, model: 65GR (discontinued product); compatible product: SIGMAKOKI, model: SSH-25RA ]
   
5. Collimator lenses
   
6. λ/2 plate
   
7. Polarizer
   
   
   Figure 2. Experimental setup of confocal microscope and femtosecond laser system. Femtosecond laser pulses from an amplified Ti:Sapphire femtosecond laser system (Wavelength: 780 nm, Pulse duration: 230 fs, Pulse energy: <1 mJ/pulse, Repetition rate: 125 Hz) are introduced to a confocal microscope (Excitation wavelength: 488 nm, Detection wavelength: 515-550 nm). Mechanical shutter (Gate time: 1/125 s), collimator lenses, λ/2 plate and polarizer were placed in the beam line between the laser and the microscope.
   
   
2. Atomic force microscope (AFM) (Figure 3)
1. AFM [Pacific Nanotechnology, model: Nano-R2 (discontinued product); compatible product: JPK Instruments, model: NanoWizard4 NanoScience ]
   
2. Ti:Sapphire femtosecond laser system (see Equipment Section, 1a)
   
3. Confocal microscope (see Equipment Section, 1b)
   
4. Objective (see Equipment Section, 1c)
   
5. A tipless AFM cantilever (NanoWorld AG, NANOSENSORS^TM, model: TL-NCH )
   
6. Motorized stage (SIGMAKOKI, model: BIOS-206T )
   
7. Oscilloscope [TEKTRONIX, model: DP4104 (discontinued product); compatible product: TEKTRONIX, model: MDO4104c ]
   
   
   Figure 3. Experimental setup of AFM head on the microscope stage. The head of an AFM was mounted on the confocal microscope (Objective, PlanN100x) combined with the amplified Ti:Sapphire femtosecond laser system with the same setting as that mentioned in the former item. A tipless AFM cantilever is attached to the AFM head. The cantilever is placed between a water shield plate and cover glass, and immersed in distilled water or culture medium.
   
   Note: The distance between the top of the cantilever and the laser focal point was tuned by a motorized stage equipped on the microscope. The normal function of the AFM system [feedback system between quadrant photo diode (QPD) and piezoelectric (PZT) motor] is deactivated. The signal of the QPD in the AFM head is directly monitored with an oscilloscope. The detailed settings are described in Hosokawa et al., 2011.

Software

1. IGOR Pro 6.22J (WaveMetrics, https://www.wavemetrics.com)
   
2. Visual Basic 6.0J (Microsoft, https://msdn.microsoft.com/en-us/vstudio/ms788229.aspx)
   Note: Multivariate least-square fittings in procedures C and D were performed by a data analysis and graphing software, IGOR Pro 6.22J. For the least-square fitting in step C8, a user defined macro-program on the IGOR Pro was produced. The calculation program for step C9 was produced using a GUI programming language software, Visual Basic 6.0J.

Procedure