Orca flash4.0 v3 digital cmos camera

Live-cell microscopy methods have been previously described [26 (link)]. Cells were counted by hemocytometer and plated at known density in 96 well imaging plates, then subsequently incubated at 37˚C under 5% CO₂ for ~24 hours to facilitate cell adhesion and generate conditions of steady state cell polarization. In the bottom of each well, cells attach to a flat glass surface and many spontaneously polarize in the absence of an attractant gradient. Two microscopes were employed. (i) Images for Figs 1A and 1B and 4C and 4D and S1 and S3 were captured using a Nikon TiE microscope equipped with a 40x, 0.95 N.A. objective and a Hamamatsu ORCA-Flash 4.0 V3 Digital CMOS camera. (ii) Images for Figs 1C and 1D and 4E–4L and S2 were acquired with a Nikon TiE spinning-disc confocal microscope equipped with a Yokogawa CSU-X1 scanning unit, an Andor iXon 888 EMCCD camera, and a 60x, 1.3 N.A. water-immersion objective (Fig 1C and 1D) or a 40x, 0.95 N.A. objective (Figs 4E–4L and S2). For both microscopes, the imaging stage was enclosed in an environmental chamber maintaining humidity, 5% CO₂, and 37°C.

PDMS surface replicas mimicking various textures, placed in the bottom of a polystyrene 24-well chamber, were first sterilized under UV light. PBMCs, pre-stained with CellTracker Fluorescent Probes (Thermo Fisher Scientific Inc.), were seeded onto these surfaces. In experiments involving bacterial interaction, a suspension of S. epidermidis (strain ATCC 12228, OD = 0.1) in Tryptone Broth was added to the wells and incubated for 24 h at 37°C, after which the bacterial medium was replaced with the PBMC solution. Imaging was performed on a DMI8 Leica microscope, utilizing a 20× air objective and maintained under climate control at 37°C. For each experimental condition, 10 images across different vertical planes were captured using an ORCA-Flash 4.0 V3 Digital CMOS camera (Hamamatsu). Time-lapse imaging was conducted at 10-min intervals for up to 5 h, employing Metamorph (v7.10.1.161) for image acquisition.

The double-barrel nanopipette was mounted on a custom-designed holder. An Ag/AgCl and an Ag electrode were inserted in the aqueous and organic barrels, respectively. Each electrode was connected to a headstage amplifier (Axopatch CV-7B) mounted on the SICM frame. The headstage amplifiers were connected to a patch-clamp amplifier (MultiClamp 700B) and to an analog-to-digital converter (Digidata 1550B). The ion current was sampled using a sampling frequency of 10 kHz. The SICM setup consisted of an Axon MultiClamp 700B amplifier, an MM-6 micropositioner (Sutter Instrument), and a P-753 Linear actuator (Physik Instrumente) to allow precise three-dimensional movement of the nanopipette. The SICM software was used to control the positioning and topographical scanning capabilities of the SICM (ICAPPIC, London, UK). The z-piezo actuator had a travel range of 38 μm, while the travel range for x and y piezo was 96 μm. An Eclipse Ti2 inverted microscope (Nikon Instruments) and LED illumination system (pE-4000 CoolLED) with filter sets for DAPI, FITC, and TxRed were used for bright-field and epifluorescence imaging. An ORCA-Flash4.0 V3 Digital CMOS camera (C13440-20CU, Hamamatsu) was used to acquire optical and fluorescent micrographs.

Flow cells were constructed using a microscope slide, double-sided tape, and coverslips coated in 0.2% nitrocellulose (LADD Research Industries; catalog no.: 53152) diluted in amyl acetate. SNAP-PDZ (plasmid provided by the Spudich Lab (58 (link)), purified from E. coli) at 3 μM diluted in 1× assay buffer (20 mM Mops [pH 7.0], 25 mM KCl, 5 mM MgCl₂, and 10 mM DTT) was flowed into each chamber of the flow cell and incubated at room temperature for 2 min. 1 mg/ml bovine serum albumin (BSA) diluted in 1× assay buffer was flowed through each chamber followed by myosin S1 diluted to 0.4 μM (∼54 μg/ml) in 1× assay buffer and incubated at room temperature for 3 min. Each chamber was blocked again with 1 mg/ml BSA followed by labeled actin and incubated at room temperature for 1 min. Finally, motility buffer consisting of 1× assay buffer, 3 mM ATP, 1 mg/ml BSA, 1 mM EGTA, and 0.5% methylcellulose and an oxygen scavenging solution of 4 mg/ml glucose, 0.135 mg/ml glucose oxidase (Sigma; catalog no.: G2133), 0.0215 mg/ml catalase (Sigma; catalog no.: C30). Motility videos were obtained at 25 °C with a frame rate of 1 frame per second for 30 s using a 100× oil objective on a Nikon Ti-E Eclipse Inverted Fluorescence Microscope equipped with a Hamamatsu ORCA-Flash 4.0 V3 Digital CMOS Camera.

The PWS microscopy optical instrument is built into a Nikon Ti-E inverted microscope with an automated filter turret, a Ti-S-ER sample stage, and a 100x 1.49NA oil objective. Broadband illumination is provided by a white X-Cite 120LED lamp (Excelitas Technologies). To acquire spectral information, a VariSpec liquid crystal tunable filter (LCTF; Cambridge Research & Instrumentation) has been added to the microscope. Spectral data cubes for captured light intensity I(λ, x, y), where λ is the wavelength and (x, y) correspond to pixel positions in the field of view, are collected with a high-speed ORCA-Flash4.0 V3 digital CMOS camera (Hamamatsu) over the wavelength range 500–700 nm at 2 nm intervals [32 (link)]. Microscope control and image acquisition are performed using a custom PWS microscopy acquisition plugin (https://github.com/BackmanLab/PWSMicroManager) for μManager [63 (link),64 (link)]. For live-cell measurements, cells were imaged under physiological conditions (5% CO₂ and 37°C) via a stage top incubator (Tokai Hit) with type 37 immersion oil (Electron Microscopy Sciences, #16914–01). Fixed cells were imaged at room temperature (22°C) in trace CO₂ (open air) conditions using type N immersion oil (Nikon, #MXA22203).

Fluorescent flatmount images were acquired with Nikon Ti inverted widefield microscope with a Prior ProScanIII motorized stage. The objective used was Plan Apo Lambda 10 x/0.45 Air DIC N1 objective, and the camera used was Hamamatsu ORCA-Flash 4.0 V3 Digital CMOS camera. Fluorescent retina section images were acquired with W1 Yokogawa Spinning disk confocal microscope with 50 µm pinhole disk and 488, 561, and 640 laser lines. The objectives used were either Plan Apo 20 x/0.75 air or Plan Apo 60 x/1.4 oil objectives, and the camera used was Andor Zyla 4.2 Plus sCMOS monochrome camera. Nikon Elements Acquisition Software (AR 5.02) was used for image acquisition and Fiji or Adobe Photoshop CS6 was used for image analysis.