Lsm 510 meta nlo system

The coding regions of Sb06g023980 (Clade I), Sb06g020670 (Clade II), Sb04g022190 (Clade III), Sb04g004960 (Clade IV), and Sb06g024110 (Clade V) were amplified and cloned into the pBIN35S:EGFP vector, which were transformed into Agrobacterium of GV3101. For transient expression of the fusion proteins in Nicotiana benthamiana, the resultant Agrobacterium culture was resuspended in infiltration medium (10 mM 4-morpholineethanesulfonic acid hydrate, pH 5.6, 10 mM MgCl₂, and 200 mM acetosyringone) and then injected into 4-week-old N. benthamiana leaves with an optical density of 0.5 OD at 600 nm. The addition of transformed 35S:EGFP Agrobacterium was used as a control. Confocal microscopy was used to assess the results at 3 days post-N. benthamiana leaf infection. Fluorescent images were obtained using an LSM 510 META NLO system (Carl Zeiss, Oberkochen, Germany).

As anticipated, the use of AuNPs was explored as multiphoton luminescence emitters^[50, ^51] to infer information on their cellular uptake, as a confirmation of the results obtained with R6G‐marked AuNPs.
The multiphoton laser scanning confocal microscope was based on a Zeiss LSM 510 Meta NLO system equipped with a Coherent Chameleon Ti:Sapphire laser coupled to an upright Zeiss Axioskop 2 microscope (Carl Zeiss Microscopy, Thornwood, NY). The ECFCs labeled with AuNPs (incubation condition: 100 × 10⁻⁶ or 150 × 10⁻⁶m Au equivalent for 24 h) were seeded onto sterile cover glasses, fixed using 3.7 vol% formaldehyde in PBS, and subjected to multiphoton imaging at an excitation wavelength of 800 nm. The photoluminescence intensity of the cells at each time point was obtained by averaging over at least 10 cells from multiple images. The laser power at different time points did not show variations larger than 1%.

LGs were fixed, sectioned, and prepared for immunofluorescence as described previously^{54 (link)}. Sections were incubated with primary rabbit anti-Cys C antibody (ab109508, abcam) diluted 1:100, followed by anti-rabbit AlexaFluor488-conjugated secondary antibody (A21206, Molecular Probes, Eugene, OR) diluted 1:200. Samples were mounted with ProLong anti-fade mounting medium (Molecular Probes) and imaged by confocal fluorescence microscopy using an LSM 510 Meta NLO system (Carl Zeiss, Oberkochen, Germany).