Argon Ion Lasers

Live confocal time-lapse series of developing flower of A. thaliana Col-0 (Figure 2A–F and Figure 2—figure supplement 2), shoot apical meristem of tomato (Solanum lycopersicum) DR5 reporter line (Shani et al., 2010 (link)) (Figure 4—figure supplement 3) and leaf trichomes of Capsella rubella (Figure 5A) were acquired using SP8 or SP5 Leica confocal microscopes, as described previously (Kierzkowski et al., 2012 (link); Vlad et al., 2014 (link)). After dissection samples were stained with 0.1% propidium iodide (PI) and grown in vitro on medium (Bayer et al., 2009 (link)). Confocal imaging was performed with a 63× long distance water immersion objective and an argon laser emitting at the wavelength of 488 nm. PI signal was collected at 600–665 nm. In the case of tomato shoot apex, pDR5::3xVENUS-N7 signal was also collected, at 505–545 nm. Distance between stacks was 0.5 μm. Time intervals were 11 hr for tomato and 24 hr for A. thaliana and C. rubella time lapse series.
Mature A. thaliana embryos (Figure 2H) were fixed and stained as previously described (Bassel et al., 2014 (link)) and imaged using a Zeiss LSM710 confocal microscope with a 25× oil immersion lens. Confocal stacks of microtubule marker line TUA6-GFP (Ueda et al., 1999 (link)) in live Cardamine hirsuta fruits (Figure 3A) were acquired using a SP2 Leica microscope, with a 40× long working distance water immersion objective and an argon laser emitting at 488 nm. GFP signal was collected at 495–545 nm. The z step between stack slices was 0.2 μm.
The sequential replica method (Williams and Green, 1988 (link)) was used to acquire a stereopair of SEM images from an Arabidospsis leaf surface (Figure 1D) as described in (Elsner et al., 2012 (link)). Stereoscopic reconstruction (Routier-Kierzkowska and Kwiatkowska, 2008 (link)) was then performed for the stereo pair and converted into a triangular mesh using a custom MorphoGraphX module. All other data presented in this manuscript were acquired for previously published work or available through on-line catalogs.

Mice were anesthetized with a mixture of xylazine (6 mg/kg) and ketamine (100 mg/kg), and pupils were dilated with topical drops of Cyclomydril (Alcon Laboratories, Fort Worth, TX). Two minutes after pupil dilation, lubricating eye drops (Alcon Laboratories) were applied to the cornea. The fundus was viewed with an imaging camera, and laser photocoagulation was induced using the image-guided laser system (Micron IV, Phoenix Research Laboratories, Pleasanton, CA). The fundus image as well as the aiming beam can be observed on the monitor screen. Four laser burns at equal distance from the optic nerve were induced one by one in each eye by a green Argon laser pulse with a wavelength of 532 nm, a fixed diameter of 50 μm, duration of 70 ms, and varying power levels from 180 mW to 360 mW. If necessary, an orienting laser shot can also be generated approximately three times of the diameter of the optic nerve to help determine the relative positions of the lesions in an eye. After laser photocoagulation, the eyes were gently rinsed with sterile saline to remove the lubricating eye drops and treated with an antibiotic ointment, erythromycin (Fougera, Melville, NY). Mice were then placed on a pre-warmed warming plate at 35°C after the laser treatment until they awakened.

Images were acquired with a Zeiss LSM 710 inverted confocal laser scanning microscope and a Keyence BZ-X710 fluorescence microscope. To count the number of ACR2-expressing cells, a 10× objective lens was used in the Zeiss LSM 710 with 405-, 488-, and 561-nm argon lasers. To verify positions, a 4× objective lens was used in the Keyence BZ-X710 with DAPI, GFP, and Cy5 filter cubes (Keyence). The ImageJ software^{47 (link)} was used for adjustment of brightness and contrast, and quantification of ACR2-expressing cells (Cell Counter plugin). Three brain slices on the right side of 3 different mice were used for cell counting. Cells expressing TH or ACR2, as well as cells expressing both TH and ACR2 simultaneously, were counted.

The flow cytometry was performed to assess the apoptotic fractions of cancer cell line. After 24-h drug treatment, the cells were trypsinized and stained with 5 μL FITC-Annexin V stock solution (#556547; BD Biosciences Pharmingen, CA, USA) and 5 μL propidium iodide (PI) stock solution (#550825; BD Biosciences Pharmingen, CA, USA). Then, the samples were incubated in the dark for 15 min, followed by the addition of a 400 μL Annexin V Binding Buffer (#422201, BioLegend, CA, USA) for the subsequent measurement. Both fluorochromes were excited by an argon-ion laser at 488 nm. FITC-Annexin V was measured on FL1 channel (filter: 530/30 nm) and PI was measured on the FL2 channel (filter: 585/42 nm). Total 1 × 10⁴ events were evaluated in each sample via flow cytometry (CytoFLEX Flow Cytometry, Beckman Coulter, Brea, CA, USA). Further analysis of the apoptotic ratios was implemented by CytExpert 2.2 (Beckman Coulter, CA, USA).