Insight ds

Two-photon microscopy was used to track the rate of amyloid deposition or clearance around implanted microelectrodes in 6 m.o. WT and APP mice or 18 m.o. WT and hAbeta/APOE4/Trem2*R47H over a 12–16 week implantation period (0, 2, 4, 7, 14, 21, 28, 56, 84, and 112 d post-implantation), as described previously [8 (link), 10 (link), 26 (link), 27 (link)]. The microscope was equipped with a scan head (Bruker, Madison, WI), a OPO laser (Insight DS+; Spectra-Physics, Menlo Park, CA), non-descanned photomultiplier tubes (Hamamatsu Photonics KK, Hamamatsy, Shizuoka, Japan), and a 16X, 0.8 numerical aperture water-immersive objective lens (Nikon Instruments, Melville, NY). To visualize plaques, mice were injected intraperitoneally with methoxy-X04 (2 mg kg⁻¹, Abcam, #ab142818) 24 h prior to each imaging session[20 (link)]. Mice were retro-orbitally injected with FITC-dextran (2 MDa, 0.03 ml at 10 mg ml⁻¹) immediately prior to imaging to visualize surrounding blood vessels. The vasculature was used as a landmark to ensure similar ROI fields were captured between subsequent imaging sessions. methoxy-X04 was excited at a 740 nm laser excitation wavelength and care was given not to exceed >20–30 mW of power during chronic imaging. Z-stacks were collected along the full depth of the implant at a step size of 2–3 µm, ∼5 s frame rate, and zoom factor of ∼1.5-2x.

Fluorescence imaging was performed using a custom-built multiphoton microscope (Bruker Fluorescence Microscopy, Middleton, WI). Illumination was provided with a pulsed Ti:Saph laser (Insight DS+, Spectra Physics, Santa Clara, CA), tuned to 750 nm for NAD(P)H excitation, and 890 nm for FAD excitation. Excitation and emission were coupled using a 40X water-immersion objective (Nikon, 1.15 NA). Fluorescence emission of NAD(P)H was isolated with a 440/80 nm bandpass filter cube, and fluorescence emission from FAD was isolated using a 550/100 nm filter cube. Images were acquired with a resolution of 256 x 256 px, with an optical zoom of 1.16 and a pixel dwell time of 4.8  $\mathrm{\mu }$ s over a 60 s integration time. A minimum of 6 fields-of-view were collected for each treatment.

The carpal nodules and control areas were excised from the mice, placed on a coverglass, and imaged by SHG, using an inverted laser scanning two-photon microscope (MPE-RS, Olympus, Center Valley, PA, USA) equipped with a tunable laser (Insight DS+, Spectra Physics, Santa Clara, CA, USA). Samples were excited at 900 nm and the SHG signal (450 nm) was collected on a GaAs detector using a dichroic mirror (SDM570) and a bandpass filter (BP/410-470). Low magnification images were acquired using a 4× air objective [UPLSAPO4X(F), Olympus] whereas high magnification images were acquired with a silicon oil immersion 30× objective (UPLSAPO30XIR, Olympus).

The H&E sections (5 μm thick) from 33 esophageal biopsies were scanned under a multimodal nonlinear microscope previously described [24 (link)]. Briefly, a femto-second laser (Insight DS+, Spectra-Physics, USA) tuned to 940 nm was used for excitation, with ~12.5 mW average power on the specimen (Fig. 1a). Images (1024 × 1024 pixels; 381 × 381 μm) were collected via galvanometric scanning and a 20X objective (Olympus UPLXAPO, 20 × 0.8 NA). The images were stitched using MosaicJ in FIJI/ImageJ [25 (link), 26 (link)] across the entire lamina propria and used for quantitative analysis. A blue filter (447/60 nm | Semrock, USA) was used to isolate SHG emission (470 nm) in the forward and backward directions to visualize collagen. A red filter (625/90 nm | Semrock, USA) was used to collect two-photon excitation fluorescence (TPEF), which allowed for visualization of the epithelium and underlying muscularis mucosa (if present). The signals were collected using photomultiplier tubes (Amplified PMT, Thorlabs, USA) with 5–10 μs pixel dwell times, with the intensities of each image being normalized to its dwell time. A digital H&E image and its corresponding SHG and TPEF stitched images from a patient biopsy section are shown in Fig. 1b and c, respectively.