Plan apo vc 100

Live cell fluorescence microscopy was performed using Hoechst 33342 (Molecular Probes; 15 µg/ml) and mCherry protein fusions to localize IVA. Samples were prepared on agarose pads as previously described (39 (link)) except that the samples were not imaged until 30 min after harvesting, since this time frame allowed for reconstitution of mCherry fluorescence signal in the anaerobically growing bacteria. Sporulating cells were exposed to ambient oxygen for a maximum of 80 min to minimize DNA fragmentation; no cell lysis was observed under these conditions. Differential interference contrast (DIC) and fluorescence microscopy were performed using a Nikon PlanApo Vc 100× oil immersion objective (1.4 numerical aperture [NA]) on a Nikon Eclipse Ti2000 epifluorescence microscope. An EXi Blue Mono camera (QImaging) with a hardware gain setting of 2.0 was used to acquire multiple fields for each sample in 14-bit format with 2-by-2 binning using NIS-Elements software (Nikon). The Texas Red channel was used to acquire images after a 300- to 400-ms exposure, 75- to 90-ms exposures were used to visualize the Hoechst stain, and ~10- to 20-ms exposures were used for DIC microscopy. Twenty-megahertz images were subsequently imported into Adobe Photoshop CS6 for minimal adjustments in brightness/contrast levels and pseudocoloring.

An optical microscope (Zeiss Axiovert 135) is used to image fluorescent samples of QDs. A two-axis piezo stage (P-542.2SL, Physik Instrumente) is used to position the sample. For illumination, a 473 nm pulsed picosecond laser diode (Edinburgh Instruments) is used, coupled to a single-mode fibre. The repetition rate of this laser is set to 20 MHz. A 1.4 numerical aperture objective lens (Plan Apo Vc 100 × , Nikon) is used to tightly focus the illuminating laser. The fluorescence is collected by the same objective lens and filtered by dichroic mirrors and filters (FF509-FDi01, SP01-785RS, BLP01-532R, Semrock). A Galilean beam expander (BE05-10-A, Thorlabs) is placed following the relay lens to magnify the imaged fluorescence spot on to a fibre bundle (A.R.T. Photonics GmbH, Germany). This fibre bundle consists of multimode 100/110 μm core/clad fibres, fused on one side and fan-out to individual multimode fibres on the other side, and is used to guide photon from an imaged spot to 15 fibre coupled single-photon avalanche photodiodes (SPCM-AQ4C, Perkin-Elemer). For a detailed characterization of the fibre bundle setup see Supplementary Note 4. The overall detection efficiency of our setup is 12%, and further details about the efficiency are found in the Supplementary Note 1 and Supplementary Table 1.

Cells were washed, blocked with 2% BSA and incubated with 1:1000 of VesB antiserum that was pre-incubated with ΔvesABC cells to remove cross-reactive antibodies. Following incubation with 1:1000 of Alexa Fluor 488 F(ab′)₂ goat anti-rabbit immunoglobulin G (Invitrogen) and washing, fluorescence was measured (Ex 495 nm/ Em 519 nm). The cells were also visualized by differential interference contrast and fluorescent microscopy using a Nikon Eclipse 90i fluorescence microscope equipped with a Nikon Plan Apo VC 100× (1.4 numerical aperture) oil immersion objective and a CoolSNAP_HQ² digital camera as previously described [57 (link)].

FISH was performed on 3-µm tissue sections on positively charged slides using the probe cocktail composed of hTERC (3q26), c-Myc (8q24), and SE7 Triple color (KBI-10704, Leica Microsystem). Tissue digestion and probe hybridization was performed according to the manufacturer's recommendations using Poseidon Tissue Digestion Kit I (KBI-60007 Tissue Digestion Kit I, Leica Microsystem) with the following modifications: tissue was pretreated in solution A (LK-110B) at 96 °C to 98 °C for 10 minutes and digested using pepsin solution (LK-110B) for 5 minutes. FISH digital images were captured by using a Nikon Eclipse fluorescence inverted microscope equipped with a charge-coupled device camera (Andor Neo sCMOS), using filter sets for DAPI/YGFP/TRITC/CY GFP with an objective lens (Plan Apo VC 100×, Nikon). All images were captured with 100× magnification of the objective and a pixel size of 0.07 µm. For each selected field, 21 Z sections were taken with a step size of 0.3 µm. Large images of 7 × 7 fields were automatically captured from each tissue section, and the 5 best fields of view with adequate tumor tissue, free of optical artifact, were chosen for further analysis with the exception of JBLAB-178 where only 2 fields of view were suitable.

For ergosterol staining, C. neoformans cells at an OD₆₀₀ of 0.3 were cultivated in 25 mL YPD at 30°C for 6 h. The cell pellets were washed with PBS and fixed with 4% paraformaldehyde (PFA) solution for 20 min on an 18-rpm rotator. After washing with PBS, the cells were stained with 10 μg/mL filipin III (Cayman Chemical, Ann Arbor, MI) in the dark for 5 min on an 18-rpm rotator. After washing with PBS, cell pellets were resuspended in 500 μL PBS and observed immediately. Cells were visualized with an Eclipse Ti-E fluorescence microscope (Nikon) equipped with a Nikon DS-Qi2 camera and a Plan Apo VC 100× oil differential interference contrast (DIC) N2 (numerical aperture, 1.4) lens using an excitation of 360 nm and an emission of 480 nm. Images were processed based on the NIS-Elements microscope imaging software (Nikon).