Sp5 microscope

Changes in the external morphology of A. flavus after exposure to the selected isolates, BJa3, MCal1, and DH5α, at a final concentration of 5 × 10⁸ CFU of bacterial solution were examined using CLSM. DH5α strain as well as microcultures not exposed to bacterial cultures were used as negative controls. Fungus were cultivated, with minor modifications, according to the protocol by Li et al. (2010) [37 (link)]. A. flavus fungus was collected after 2 days of growth using an inoculation loop to preserve the integrity of the mycelia and placed at the four corners of a PDA-medium rectangle. This structure was placed on a microscope slide and inserted into a 50 mL conical flask. Previously, cultures of the selected bacteria in 5 mL of LB medium were cultivated inside the flask. This system was incubated for 2 days at 28 °C. Later, the samples were washed in Phosphate-Buffered Saline (PBS) and stained with 50 μL of calcofluor white (Sigma-Aldrich, USA) for 10 min. Images were acquired in Confocal Microscopy Multiuser Laboratory (LMMC—USP) with a Leica SP5 microscope (Leica Microsystems, Wetzlar, Germany) using plan apochromatic 40× (NA 1.25, oil) and 63× (NA 1.4, oil). Calcofluor-stained samples were recorded in blue channel (395 nm) and the emission wavelength was 440 nm.

Fluorescence immunolocalization experiments were carried out following the protocol from Coimbra et al. (2007), and imaging was performed on a Leica SP5 microscope (Leica Microsystems, Germany) using laser beam lines of 405 nm (calcofluor white) and 488 nm (FITC; Alexa Fluor 488). Emitted fluorescence was captured between 415 and 455 nm for calcofluor white and between 500 and 550 nm for FITC and Alexa Fluor. Images were analyzed with Zeiss Zen software and Fiji (Schindelin et al., 2012).

To identify elastic microfibrils, elastin staining was performed using a Verhoeff van Gieson Elastic Stain Kit (Cat# HT25A, Sigma) in whole eye cross-sections according to the manufacturer’s protocol. Images were captured and analyzed (KEYENCE microscope).
For two-photon imaging, ex vivo mouse eyes were labeled intact, without dissection or sectioning, with Sulforhodamine-B (Thermo Fisher Scientific), a water soluble stain of elastic microfibrils. Microscopy was performed on a Leica SP5 microscope (Leica Microsystems, Heidelberg, Germany) coupled to a Chameleon Ultra-II multiphoton laser (Coherent, Santa Clara, CA) through inverted 20X/0.7NA or 63X/1.3NA objectives. We used 850 nm excitation, pulsed, and focused through green (525/50 nm) or red (585/40 nm) filters (Chroma, Bellows Falls, VT) onto a non-descanned photomultiplier tube detector (Hamamatsu, Bridgewater, NJ). Images were collected as multiple channel z-stacks using 512 × 512 or 1024 × 1024 pixel frames, 16-bit grayscale resolution, and 16X line averaging using 1–8 μm step sizes. Images were viewed and processed on LAS AF and AF Lite 2.2.1 (Leica), Image J (NIH; Bethesda, MD), Photoshop CS5 (Adobe, San Jose, CA) and Imaris (Oxford Instruments). Methods reported here have previously been described^{101 (link), 106 (link)–108 (link)}.

Twenty whole glomeruli in each slice were imaged using a Leica SP8 Multiphoton Microscope (Leica Microsystems, Wetzlar, Germany) fitted with a 20 × BABB immersion objective lens (numerical aperture, 0.95; working distance, 1950 μm). Serial optical sections were imaged utilising a z-step size of 1 μm with image series captured at a resolution of 1024 × 1024 pixel frames (Fig. 1). Imaging of 4-μm paraffin-embedded sections was completed on a Leica SP5 Microscope (Leica Microsystems) fitted with a 40 × oil immersion objective lens (numerical aperture 1.25). Representative images were captured at variable zoom at a resolution of 1024 × 1024 pixel frames.Fig. 1

Glomerular and podocyte imaging. a Three-dimensional image field containing three whole glomeruli showing immunofluorescence labelling and identification of podocytes. b Cropped whole glomerulus sampled for podometric analysis. c Confocal section through a whole glomerulus. Synaptopodin (green), p57 (red). Scale bars: a 60 μm; b, c 20 μm

Live cell-imaging was conducted with a Leica SP5 microscope (Leica-Microsystems, Germany). Cells were maintained at 37°C in an atmosphere containing 6% CO₂/94% air using a thermostatically controlled heated enclosure with passive humidification and a separate gas mixing unit (Life Imaging Services, Switzerland). Images were collected using a 40×, 1.1 numerical aperture, water immersion objective (Leica-Microsystems, Germany). Immersol W (Zeiss, UK) with a refractive index of 1.334 was used as a water-substitute immersion fluid for long-term imaging. Simultaneous RCM and brightfield images were acquired with separate photomultiplier tubes (PMT) using the Argon 488 nm laser line. The RCM signal was collected between wavelengths of 478–498 nm with a pinhole size of 57 μm. Images were collected with a line scanning speed of 600 Hz at 1024×1024 pixel resolution. A line and frame average of 2 was applied. A total of 34 fields of view per condition were followed by time-lapse microscopy. Representative fields of view were selected from structures formed by single cells. For the quantitative analysis of fiber organization, structures from 0% (n = 12), 5% (n = 11), and 50% (n = 10) Matrigel were analyzed at each hour between the first 3–12 hours following cell seeding. At day 5, morphology and fiber organization were quantified for 19 independent structures in 5% Matrigel.