Lsm 710 nlo confocal microscope

For in vivo photoactivation of individual LNs, 5-day-old female flies (see Supplementary Table 1) were dissected as in the calcium imaging experiments and scanned with a ZEISS LSM 710 NLO confocal microscope (Carl Zeiss, Jena, Germany) equipped with an infrared Chameleon Ultra diode-pumped laser (Coherent, Santa Clara, USA) using a ×63 water immersion objective (W Plan-Apochromat 63×/1.0 VIS-IR, Carl Zeiss, Jena, Germany). A precise region of interest was placed on a single LN soma of the right AL and continuously illuminated for 25–30 min at a wavelength of 760 nm. Subsequently, flies were kept in a dark humidified chamber for approximately 25 min to allow photoconverted GFP molecules to diffuse within the LN. Flies were then killed by removing the body to eliminate movements in the final scan. A z-stack scan of the whole right antennal lobe was acquired at a laser wavelength of 925 nm at an interval of 0.77 µm and with a pixel resolution of 624 × 624. For 3D reconstructions the acquired scans were processed with AMIRA 5.6 software (Fei Visualization Sciences Group) using the labelfield module and the semi-automated reconstruction module “hxskeletonize”⁷⁰ to reconstruct glomeruli and single LNs, respectively.

Confocal imaging of seedling was set up as previously described^{65 (link)}. Imaging was performed with 5× and 40× oil-immersion lenses in multitrack mode using a Zeiss LSM710 NLO confocal microscope (Carl Zeiss Microscopy). Plants expressing iNAP1, iNAP4, and iNAPc in various subcellular compartments were excited sequentially at 405 nm and 488 nm, and emission was detected at 520 ± 16 nm. R_iNAP, R_iNAPc, and R_SoNar represent the raw ratios of emission excited at 405 nm and 488 nm for iNAP1/4, iNAPc, and SoNar. Autofluorescence was recorded at 431–469 nm and chlorophyll fluorescence was collected at 629–700 nm.
Confocal images were processed with a custom MATLAB-based analysis suite^{66 (link)}. The ratiometric images were analyzed on a pixel-by-pixel basis using x, y noise filtering, and fluorescence background subtraction was conducted based on the intensity of the cell samples not expressing sensors from the dark side of the images. Visually, all the ratio profiles in the mesophyll were displayed in pseudocolors. iNAPc is the pH control sensor of iNAP and SoNar^{3 (link)}. The pH-corrected ratio (normalized R_405/488) was calculated with the formula below, $\mathrm{Normalized}{R}_{\frac{405}{488}}=R_{\mathrm{iNAP}\mathrm{or}\mathrm{SoNar}}/R_{\mathrm{iNAPc}}$

Nuclear localization of Nrf2 was detected by immunofluorescence confocal microscopy. Cells were exposed to DON at 1 μM for 3 hours for Nrf2. The duration of DON treatment for Nrf2 was determined based on our preliminary experiments (data not shown). For the Sch A pre-treated samples, 10 μM Sch A exposure was carried out for 24 hours, followed by DON exposure at 1 μM for 3 hours. Cells were also exposed to 10 μM Sch A alone for 24 hours. After treatment, cells were fixed with 10% (v/v) formalin and ice cold absolute methanol, followed by permeabilization with 0.1% Triton X-100. They were then incubated with Nrf2 primary antibody (1:500 dilution) (#12721; Cell Signalling) and goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077, Abcam) as secondary antibody (1:200), and subsequently Hoechst staining and viewed and analyzed in a Zeiss LSM 710 NLO confocal microscope (Carl Zeiss, Thornwood, NY, USA).

Mouse whole pancreas tissue was fixed in 4% formalin and processed for paraffin embedding. Antigen retrieval was performed by microwave heating in antigen retrieval solution-citrate buffered (Prosan, Merelbeke, Belgium). Detection was done using fluorochrome-conjugated secondary antibodies (Jackson Laboratory, Westgrove, Pennsylvania, USA) according to manufacturer’s instructions.
The following primary antibodies were used: anti-insulin (Guinea pig Polyclonal—C. Van Schravendijk, Diabetes Research Center, Brussels, Belgium); anti-PP (Rabbit Polyclonal—R.E. Chance, Lilly Research, Indianapolis, USA); anti-Glut2 (Rabbit Polyclonal—Alpha Diagnostic—San Antonio, Texas, USA), anti-KDEL (Mouse monoclonal – Enzo Life Sciences – Farmingdale, NY).
Pictures were acquired with ZEISS LSM7 10 NLO confocal microscope using ZEN 2009 software (Carl Zeiss, Oberkochen, Germany). Quantification of insulin and PP-positive areas and pancreas tissue area (85,29±9,58 mm² spread across 3 non-consecutive sections) was performed with IPlab 4.0 software (Becton Dickinson, San Jose, CA, USA).

Stable STYK1-eGFP overexpressing HCC827 and empty vector control cells were seeded in a black Microclear 96-well plate (655090, Greiner, Frickenhausen, Germany). At 90% confluency, the cells were fixed using 4% paraformaldehyde for 15 min at 4 °C and subsequently permeabilized with 0.25% Triton X-100 in PBS for 15 min. Subsequent washes were performed with 0.05% Tween-20 in PBS. Primary antibody for EGFR (GRO1, Sigma-Aldrich) was diluted in 0.05% Tween-20, 1% BSA and 22.5 mg/mL Glycine, and was incubated for one hour, followed by one hour staining with an AlexaFluor 647-labeled secondary antibody. Nuclei were stained with Hoechst3342. Images were acquired using a ZEISS LSM710 NLO Confocal microscope using the ZEN 2009 software (Carl Zeiss, Oberkochen, Germany).

A LSM-710-NLO confocal microscope (Zeiss) with a Chameleon Vision II titanium sapphire tunable laser (680–1080 nm, 140 fs, Coherent) was used for imaging (Fig. 1 A). Light was focused with a LCI Plan-Neofluar 25X/0.8 immersion objective (Zeiss) and the SHG was collected in the transmission direction (forward path) with a 0.5 N.A. condenser. The excitation wavelength used was 810 nm and non-descanned detection was performed after a low pass filter (LP 490) with photomultiplier tubes (Hamamatsu), using Zeiss NDD units (Fig. 1A). The average power before the objective was 13 mW or 20 mW. The calculated intensities 〈I〉 = 〈P〉/S, considering ${\omega }_{\mathit{xy}}=\frac{0.325\lambda }{\sqrt{2}{\text{NA}}^{0.91}}$ ^{49 (link),50 (link)} are 7.96 MW/cm² and 12.24 MW/cm².

CHO cells were maintained in growth media (F-12K + 10% FBS and Penicillin/ Streptomycin) on glass chamber slides for 24 h (eight chamber, Nunc). The cells were then washed with DPBS once and incubated with Ag-488 or QD-488, with or without T-Ag in DMEM medium for 1 h at 37 °C. Here, 2x concentration of NPs was used compare to flow cytometry study. After incubation, cells were etched for 1 min, aspirated, then washed with DPBS twice and fixed with 4% PFA. The chamber slides were then mounted in DAPI-containing mounting medium (Vector Laboratories, Burlingame, CA) with a coverslip and examined under a Zeiss LSM 710 NLO confocal microscope. Cells were also incubated in AA-free and Cys medium mixed with Ag-488 and T-Ag and treated similarly to image.