Lsm 780 nlo microscope

Mouse neurons, and HEK293 cells were grown in 35-mm glass-bottom dishes, and maintained at 37 °C in 5% CO₂/95% air on the stage of the Zeiss LSM-780 NLO microscope. All cultures were serum- starved in Hank’s balanced salt solution (HBSS) for 2 h before addition of ATN 224 to 2 μM, and imaged before and 60 min after treatment. The laser was tuned to 740 nm with an average power of 7 mW at the specimen plane, and NAD(P)H fluorescence was collected using a 450–500 nm emission filter (Objective lens, 20×). For each experiment, 5–10 field of views were recorded in the descanned mode, and then each field of view was subjected to a 40-s acquisition in the non-descanned mode. The laser power and acquisition time were selected to ensure enough photons per pixel while avoiding photodamage to cells. Next, ROIs corresponding to mitochondria were selected from the NAD(P)H photon image for lifetime analysis. Both the lifetimes and fractions of free and enzyme-bound NAD(P)H were calculated on a per pixel basis, from which pseudo-color images of the bound NAD(P)H and histograms of the frequency distribution of a₂% (fraction of bound NAD(P)H) were generated.

Mice were prepared for intravital microscopy as previously described (Dudeck et al., 2011a (link)). In brief, animals were subjected to intubation narcosis with a mixture of Isofluran (1.0%) and oxygen (99%) with a mechanical ventilator (Mini-Vent, Hugo-Sachs Elektronik). Two-photon intravital microscopy was performed with a Zeiss LSM780 NLO microscope with simultaneous detection via four external non-descanned detectors. Illumination was performed at 920 nm with a Chameleon Vision II (Coherent, Inc.) laser (10–12% laser power, at 1,400 mW maximum power) via a 20× water-dipping lens with 1.0 NA. GFP-expressing DCs were detected with a 525/50 bandpass filter (BP), tdRFP-expressing MCs with a 600/20 BP, and blood vessels by i.v. injection of 12.5 µl Qtracker 705 nontargeted Qdots (Invitrogen) in 83 µl isotonic NaCl (BP 710/40). Collagen I structures were visualized by its SHG (<485 nm, BP 450/70). For visualization, raw data were processed and reconstructed using Imaris (Bitplane) and represented as maximum intensity projection (MIP) or 3D-rendered z-stacks. Time-lapse series were recorded with a rate of 1 z-stack per minute, a pixel size of 0.24 µm, and a z-spacing of 4 µm encompassing 84 µm in z and an area of 250 × 250 µm in xy.

For high-temporal resolution experiment, cell monolayers in capsules were embedded in 0.4% agarose for imaging in a 35 mm glass-bottom dish. The MatTek dish was priced on the side with a 19G hot needle to introduce teflon tubing into the dish sterilely for alginate lyase (Sigma-Aldrich, ref. A1603) injection. The MatTek dish was mounted onto inverted LSM780 NLO microscope (Carl Zeiss) (section Imaging). The 1mL of PBS alginate lyase solution (20 units per 1mL of PBS) was added both after 1min of imaging and after 20 min of imaging, resulting in total amount of alginate lyase of 40 units in MatTek petri dish. The imaging was performed with microscope parameters mentioned in section Imaging. For each capsule, confocal equatorial planes were acquired with time interval of 15 s for 2 hours with definite focus (autofocus). For low-temporal resolution, the experiment was conducted as a high-temporal resolution with addition of medium alginate lyase solution giving final alginate lyase amount of 75 units in MatTek dish. The imaging of capsule equatorial planes was performed with time interval of 10 min for 19 hours and with definite focus (autofocus).