Zen 2.3 sp1 operatingsoftware

The FCS
measurement was performed on the solution of RhB-labeled RNase or
citrine in the absence and presence of PICsome using a confocal laser
scanning microscope LSM880 (Carl Zeiss, Oberkochen, Germany) with
a laser of 488 nm. The fluctuation of the fluorescent intensity from
RhB or citrine was measured to obtain the autocorrelation function G(t). The experimental autocorrelation
curve was fitted using a following function: where N is number of molecules, t is correlation time, τ is the diffusion time of
component, k is structural constant, F is the fraction of particles that have entered the triplet state,
and τ_trip is the relaxation time of the corresponding
triplet state. The fitting was performed by ZEN 2.3 SP1 operating
software (Carl Zeiss, Oberkochen, Germany). The diffusion coefficient
and hydrodynamic diameter of RhB-labeled RNase in the absence and
presence of PICsome were estimated from the fitting curves.

Prior to infiltration into A. thaliana seedlings, citrine encapsulated in CPP-modified
PICsome (citrine@CPP-PICsome)
was prepared according to the procedure described above. Ten 4-day-old
seedlings of A. thaliana were added
to a solution of citirine@CPP-PICsome in a 2 mL microtube. The microtube
was subjected to vacuum at −0.08 MPa for 1 min and pressurized
at 0.08 MPa for 1 min. The seedlings were then transferred on agarose
gel containing half MS media and grown for 1 day. The seedlings were
stained with calcofluor white (0.1 g L^–1) for 10
min and subjected to CLSM observation by LSM880 (Carl Zeiss, Oberkochen,
Germany). Images were acquired at excitation wavelength of 405 nm
(for calcofluor white) and 514 nm (for Citrine) and visualized under
a 63× oil-immersion objective. Colocalization analysis of micrographs
was performed using the Zen 2.3 SP1 operating software (Carl Zeiss).