Application suite program

MCF-7 cells (1 × 10⁴ cells/cm²) were grown in supplemented RPMI medium in glass bottom dishes (35-mm dishes with a 0.17-mm thick cover glass on the bottom; Greiner Bio-One, Germany) for 24 h. After washing, cells were incubated in RPMI medium supplemented with 2% FBS with 0.62 µM ZnPc3, 0.3 mg/mL ZnPc3/FF-MNSs or 0.3 mg/mL FF-MNSs for 2 h. Cells were washed with calcium- and magnesium-free saline solution, and images were acquired on Leica AF6000 microscopy system (Leica Microsystems, Germany) using a fluorescence buffer (1.5 mM CaCl₂, 130 mM NaCl, 5.6 mM KCl, 0.8 mM MgSO₄, 1 mM Na₂HPO₄, 25 mM glucose, 2 mM HEPES and 2.5 mM NaHCO₃–pH 7.3). Fluorescence emission was recorded using the filter cube Y5 (Leica Microsystems), with 620 nm excitation and 60 nm bandpass, 660 nm dichroic mirror and 700 nm emission with 75 nm bandpass. Differential interference contrast microscopy images were acquired by using an HCPL Apo 20× /0.7 and HCX PL Apo CS. 63x/1.40-0.60 OIL numerical aperture plan apochromatic objective coupled to an ultrafast Leica DFC365 FX digital camera (Leica Microsystems, Germany). The fluorescence intensity, relative to the amount of photosensitizer cell uptake, was quantified using the Leica Application Suite program (LAS, v. 3, Leica Microsystems).

A tube formation assay was performed to evaluate the effect of CHI3L1 on the angiogenesis ability of HUVECs. In brief, the cells were cultured in a medium containing 10% of FCS pre-cultured with MEK inhibitor, PI3K inhibitor for 1 h, and corresponding shRNAs for 48 h, followed with or without 400 ng/ml of CHI3L1 for 24 h. Finally, the cells seeded into a 96-well plate at a density of 5 × 10³ cells/well were overlaid with Matrigel substrate (BD Biosciences, CA), and were incubated at 37°C for 6 h. Capillary tube structures were observed and quantified (Hou et al., 2017 (link)). Representative images were captured using Leica Application Suite Program (magnification ×100). The value of tubular branches representing the capacity of angiogenesis was determined using Image J software (National Institutes of Health, Bethesda, MD, USA).

At the end of the study, the animals were humanely euthanized with an intravenous overdose of pentobarbital sodium and phenytoin sodium (Euthasol; Virbac, Westlake, TX, USA). Immediately following death confirmation, the eyes were enucleated, fixed, and processed as previously described (Beltran et al., 2017 (link)). The sections were stained with H&E and different IHC markers (Table S4) following protocols previously established (Beltran et al., 2017 (link)). Immunolabeled sections were examined by confocal microscopy (Leica TCS SP5; Leica Microsystems, Buffalo Grove, IL, USA), and digital images were acquired and processed using the Leica Application suite program. Additional details can be found in supplemental experimental procedures S4.
Immunohistochemical quantification of the integration events was performed in a subset of normal and mutant animals (see supplemental experimental procedures S5).

Insect batches were sorted with respect to size into subjectively defined groups for comparison with lidar data and identified according to Chinery34 . One of the batches (Table 1, batch number 2, caught between 22:18–23:18 local summer time) contained a large number of insects and therefore identification was based on a ¼ subsample. After sorting, the body and wing width and length of one wing was measured on three (where available) randomly chosen specimens from each category and batch, using a Leica MZ5 microscope and the Leica application suite program. The measurements of the widths and lengths were made on wings that were laid flat down (to obtain the widest and longest cross-sectional distances of the wing) and from the lateral side of the body. This was decided because the lidar algorithm estimates the peak sizes of the body and wing OCS. The body and wing measurement were used to estimate the cross-sectional area of the body and wings for each grouping. For insects with two sets of wings, both the fore- and hindwings were included. We used an elliptic approximation to estimate the body and wing cross section area for comparison with the lidar data. We then calculated the body:wing proportions as: body OCS/(body OCS + wing OCS).

Five taxa of Amaranthus (A. blitoides var. blitoides, A. blitum subsp. blitum var. blitum, A. dubius, A. graecizans subsp. graecizans, and A. viridis) were collected from Saudi Arabia (Fig. 1) during the spring of 2021. They were identified and deposited in the herbaria PNUH and RO (Thiers, 2022 ). High resolution images of the synflorescences were obtained using the Leica IC80 HD photo camera and Leica Application Suite program, version 4.5.0. (Leica, Wetzlar, Germany). The images were later processed using Helicon Focus, version 6.6.1 Pro where different focus of the same sample were merged together.
The map was prepared using ArcGIS programme (Esri, Redlands, California, USA).