Neo scmos

For the purpose of this study the microscope was equipped with a triaxial Helmholtz coilset and controller (C-SpinCoil-XYZ, Micro Magnetics Inc.), a fluorescence camera (2560×2160 pixels; NeosCMOS, Andor Technology), a high speed camera (540 fps; 1696×1710 pixels; CR3000x2, Optronis), two illumination sources for the fluorescence (pE-100, 400 nm and 470 nm, CoolLED Ltd.) and an illumination source for transmission imaging (pE-100, 635 nm, CoolLED Ltd.) (Note S1). The 3D-axis Helmholtz coils were used to generate DC magnetic fields with a precision of 5% of the Earth's magnetic field (±2.5 µT, manufacturer's specifications). Using this setup, six magnetic conditions were applied under computer control using a LabView based program (LabView, National Instruments). A 3-axis magnetic sensor (Micro Magnetics Inc.) was zeroed in a zero gauss chamber and used to generate a magnetic field that cancels out the ambient magnetic field. The magnetic field was then set to 0 µT, 50 µT or 500 µT along the long axis of the capillary, and to 50 µT at 90° and 45° to the capillary long axis.

The samples were illuminated with a 100 W mercury lamp and visualized by an epifluorescence microscope (Eclipse Ti, Nikon) using an oil-coupled Plan Apo 60× N.A.1.4 objective (Nikon). UV cut-off filter blocks (TRITC: EX 540/25, DM565, BA605/55; GFP-B: EX460-500, DM505, BA510-560; Nikon) were used in the optical path of the microscope. Images were captured using a cooled-CMOS camera (NEO sCMOS, Andor) connected to a PC. Two ND filters (ND4, 25% transmittance for TRITC and ND1, 100% transmittance for GFP-B) were inserted into the illumination light path of the fluorescence microscope to reduce photobleaching of the samples.

The droplet of tubulin solution after drying and stabilizing with taxol buffer, was illuminated with a 100 W mercury lamp and visualized by an epifluorescence microscope (Eclipse Ti, Nikon) using 2×, 20× objective lens (Nikon). UV cut-off filter block (GFP-B: EX460-500, DM505, BA510-560; Nikon) was used in the optical path of the microscope. Images were captured using a cooled-CMOS camera (NEO sCMOS, Andor) connected to a PC. ND filter (ND32, 3.1% transmittance for GFP-B) was inserted into the illumination light path of the fluorescence microscope to reduce photobleaching of the samples.

The samples were illuminated with a 100 W mercury lamp and visualized by an epifluorescence microscope (Eclipse Ti, Nikon) using an oil-coupled Plan Apo 60× N.A.1.4 objective (Nikon). UV cut-off filter blocks (TRITC: EX 540/25, DM565, BA605/55; GFP-B: EX460-500, DM505, BA510-560; Nikon) were used in the optical path of the microscope. Images were captured using a cooled-CMOS camera (NEO sCMOS, Andor) connected to a PC. Two ND filters (ND4, 25% transmittance for TRITC and ND1, 100% transmittance for GFP-B) were inserted into the illumination light path of the fluorescence microscope to reduce photobleaching of the samples. In order to isomerize the azobenzene units, the flow cell was irradiated with the light passed through a UV-1A filter block (UV-1A: EX 365-410, DM400, BA400; Nikon).

Zebrafish were imaged in 96-well plates using Nikon Ti-E with a CFI Plan Achromat UW 2X N.A. 0.06 objective lens, using Intensilight fluorescent illumination with ET/sputtered series fluorescent filters 49002 (Chroma, Bellow Falls, VT, USA). Images were captured with Neo sCMOS, (Andor, Belfast, UK) and NIS-Elements (Nikon, Richmond, UK). Images were exported as tif files and further analysis performed in ImageJ (Schneider et al., 2012). Images were individually cropped to remove the side of the 96-well or any bright debris or noise within the well. Pixels above the intensity corresponding to C. neoformans strain H99GFP were selected using a threshold. The same threshold was used for all images. Thresholded images were converted to binary images and the number of pixels counted using the ‘analyse pixel’ function.

Cells were plated prior to imaging on 10 μg/ml fibronectin-coated glass-bottomed dishes (MatTek Corporation). The time-lapse images of cells with transient transfection of CAV-1-mEGFP, mCherry-actin, and CAV-1-mCherry were acquired with 3I Marianas imaging system (3I intelligent Imaging Innovations), consisting of an inverted spinning disk confocal microscope Zeiss Axio Observer Z1 (Zeiss) and a Yokogawa CSU-X1 M1 confocal scanner. Appropriate filters, heated sample environment (+37°C), controlled CO₂, and 63×/1.2 WC-Apochromat Corr WD = 0.28 M27 objective (Zeiss) were used. The images were acquired via SlideBook 7.0 software (3I intelligent Imaging Innovations) and recorded via Neo sCMOS (Andor) camera. The recording was set as every 1 sec for 10 min and one focal plane was recorded for all live cell videos. For tracking and speed measurement of CAV-1 vesicles, the Imaris 9.2 (Bitplane) ‘Track’ module with globular-objects over time was used as in previous study (Jiu, 2018 (link)). Then, 2 μm estimated XY diameter, 5 μm max distance, and 3 max gap size were set for analyzing.