Silicone Gels

To prepare the gel layers, the components of the HRI gel pre-polymers, parts A and B of QGel 920 and parts A and B of QGel 903 (both by Quantum Silicones LLC, Richmond VA; refractive index of 1.49 when cured), were mixed in various proportions (Table 1) and coated onto 25 mm no. 1 round cover glasses using a home-built spin-coater rotating at 1920 rpm. Each cover glass was baked at 100°C for 2 hr to create a layer of cured gel on it with a thickness, µm. After baking, the gels on the cover glasses were treated with 3-aminopropyl trimethoxysilane for 5 minutes and incubated for 10 minutes at room temperature under a suspension of 40 nm carboxylated far-red fluorescent beads (excitation/emission 690/720 nm, by Invitrogen, Carlsbad, CA) in a 100 µg/ml solution of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in water to covalently link beads to the gel surface. This technique made it possible to have all beads in one plane corresponding to the surface of the gel. Therefore, the beads could be imaged under wide-field (epi-fluorescence illumination) with minimal background and their displacements reflected the deformation of the very top of the substrate. To promote cell adhesion, fibronectin (FN) was covalently linked to the gel surface by incubation in 50 µg/ml of FN with 100 µg/ml EDC in PBS, pH 7.4 for 30 min at room temperature.
The elastic modulus (Young's modulus), E, of the gels was evaluated by applying a known hydrodynamic shear stress, τ, to the gel surface using a custom-built microfluidic device, measuring the resulting bead displacement, , calculating the shear of the gel, , and applying the equation , where ν is the Poisson ratio, as explained in detail elsewhere [26] . Because the Poisson ratio of silicone gels is nearly equal to 0.5 [27] , the equation was reduced to . To measure the gel thickness, , a small amount of the 40 nm far-red fluorescent beads was deposited on the cover glass surface before it was coated with the gel pre-polymer. The fluorescence microscope was first focused on beads on the glass surface and then on those on the gel surface and the difference in the readings of the nosepiece (z-axis) knob was recorded (with a correction for the mismatch between the refractive indices of the gel and immersion liquid), resulting in ∼1 µm accuracy. The shear, , was found to be a zero-crossing linear function of τ for of up to at least 3 µm (greater than produced by HUVECs; see below) for all gels, with no sign of plastic deformations (see also [26] ), thus validating the use of the equation , which applies to linear materials. Furthermore, measurements of vs. at different constant values of τ resulted in linear dependencies, indicating homogeneity of mechanical properties of the gel layers.

TIRF-SIM of low illumination NA, so called GI-SIM, and astigmatic microscopy was performed on a custom-built system controlled by acquisition software written in LabView^{25 (link),27 (link)}. A single 488 nm excitation laser was used (MPB Communications Inc., 2RU-VFL-P-500-488-B1R) to excite both the Lifeact-citrine channel and the fluorescent beads to facilitate rapid acquisition. The excitation beam was first collimated and passed through the acousto-optic tuneable filter (AOTF, AA Quanta Tech, AOTFnC-400.650-TN). The beam was then expanded and sent to a phase-only modulator, consisting of a polarising beam splitter, an achromatic half-wave plate (HWP, Bolder Vision Optik, BVO AHWP3) and a ferroelectric spatial light modulator (SLM, Forth Dimension Displays, QXGA-3DM). The SLM displays a grating pattern with parameters matching the excitation wavelength, which was used to generate diffraction patterns. The diffraction orders of +1 and −1 were focused on opposite sides of back focal aperture of objective lens (Olympus UAPON 100XOTIRF 1.49NA), and then total internal reflected at the cell–gel interface. The evanescent waves generated by +1 and −1 order light interfered to form the illumination pattern. To maximise the pattern contrast, the diffracted light was maintained with s-polarisation using a polarization rotator. The fluorescent images of Lifeact-citrine and fluorescent beads generated by the applied illumination pattern were collected by the same objective, separated by a dichroic beam splitter (Chroma, ZT405/488/560/647tpc), and focused by respective tube lens onto corresponding sCMOS camera (Hamamatsu, Orca Flash 4.0 v3). A cylindrical lens (Optosigma, CLB-3030-1000PM) was introduced in front of the camera to generate astigmatic imaging of the marker beads. For each frame, the acquisition time was 10–100 ms, leading to a super-resolution image (three angles and three phase, nine frames total) every 90–900 ms calculated using a reconstruction method described previously^{27 (link),43 (link)}. For live-cell imaging, a micro-incubator (H301, Okolabs, Naples, Italy) at 37 °C and 5% CO₂ was used.
During the aTFM acquisition, the TIRF interface at the top of the silicone gel is displaced in the axial direction due to cell generated normal stresses. This will lead to a change in the angle of incidence relative to the gel–sample interface. For the acquired data, the dynamic range of axial displacement was of the order of 500 nm. If this is assumed to act over the contact area of the cell (15 µm), this equates to an angular distortion of the gel of ~1.5°. The excitation light has an incident NA ranging from 1.38 to 1.41; therefore, the incident angle necessary to achieve TIRF illumination was sin⁻¹(1.41/1.515) = 68.5°. If the surface deviates from being flat, the incident angle is 68.5° ± 1.5°, giving a corresponding range in NA from 1.395 to 1.42. TIRF illumination for the described setup is typically achieved for NA > 1.38, indicating that TIRF illumination is maintained during deviations from a flat surface inherent to the aTFM acquisition.

Four male and three female Long-Evans rats (300–500 grams) were used in this study. All surgeries were performed on isoflurane anesthetized animals head fixed on a stereotaxic frame. After removing hair from the head, the incision area was cleaned using alcohol and betadine. Next, an incision was made to expose the skull underneath. The skull was cleaned of tissues and blood, after which hydrogen peroxide was applied. Coordinates for probe implantation were marked above the dorsal hippocampus (AP: −3.36, ML: ±2.2) following measurement of bregma and lambda. Craniotomies were drilled at the marked location. Using a blunt needle, the dura was removed carefully to expose the brain surface. After cessation of bleeding, animals were implanted with 64 channel (8 shank “Buzsaki” probe; Neuronexus, MI; X animals) or 128 channel (8 shanks, Diagnostic Biochips, MD, 7-X animals) silicon probes. Ground and reference screws were placed over the cerebellum. Craniotomy was covered with DOWSIL silicone gel (3–4680, Dow Corning, Midland, MI) and wax. A copper mesh was built around the implant for protection and electrical shielding. All procedures involving animals were approved by the Animal Care and Use Committee at the University of Michigan.

For pharmacological inactivation, ≈500-μm-diameter craniotomies were drilled over cool (32–22 °C)-responsive areas in S1 and pIC in mice anaesthetized with isoflurane (1.5–2% in O₂). Both regions were identified functionally using the widefield calcium imaging response to thermal stimuli. Following the craniotomy, the dura was covered with transparent silicone gel (3-4680, Dow Corning). A 300 nl volume of muscimol or Ringer’s solution was injected at a rate of 100 nl min⁻¹ 300–500 μm below the pial surface using a pulled glass pipette and a hydraulic injection system (MO-10, Narashige). muscimol (Abcam, ab120094) was dissolved in Ringer’s solution to a concentration of 5 mM. Imaging sessions were carried out >10 min following the end of Ringer’s solution or muscimol injection (Fig. 1f,g).

Cell cultures prepared as described in “Preparation ofC. albicanscell culture with Venetin-1 and fluconazole” section of Materials and methods were centrifuged at room temperature at 2500×g for 10 min. The supernatant was discarded and the pellet was suspended in 70 µL of a fixing solution (10 ml phosphate buffer pH = 7, 10 ml glutaraldehyde 10%, 200 mg saccharose) and incubated for 2 h at room temperature. Then, the fixing solution was discarded and 0.1 M phosphate buffer pH = 7 was added. After centrifugation (2500×g, 30 min, room temperature), a 1.5% OsO₄ solution was added and incubated with the cells for 30 min at room temperature. After that, the fungal cells were centrifuged and the supernatant was removed. Then, the cultures were washed with phosphate buffer and centrifuged. The cells prepared in this way were dehydrated in a series of acetone dilutions (15%, 30%, 50%, 70%, 100%), transferred onto SEM stages with carbon discs, and dried in a desiccator for 24 h in the presence of roasted silicone gel (for 2 h at 180 °C). Afterwards, the stages with the probes were coated with a gold layer using a K550X sputtering machine (Quorum Technologies, United Kingdom) and observed with the use of a scanning electron microscope Tescan Vega 3 (Tescan Orsay Holding, Czech Republic)^{39 (link)}.