Flash 4 camera

HiPSCs (AICS-0086 cl.147) with RFP-tagged Nucleophosmin, GFP-tagged Fibrillarin and Halo-tagged upstream binding factor were plated on Matrigel coated Ibidi 35 mm μ-Dishes (Ibidi, # 81156). Before imaging, cells were treated with 200 nM of Janelia Fluor HaloTag Ligand 646 for 30 minutes. The cells were then imaged using a CSU-W1 spinning disc (Yokogawa) coupled to a Ti2 microscope (Nikon) through a 60x Plan Apochromat objective (NA 1.45). Excitation of GFP occurred at 488 nm, RFP occurred at 561 nm, and Halo JF 646 occurred at 640 nm. The emissions were collected for 20 to 200 ms per frame through a standard filter onto a Flash 4 camera (Hamamatsu). Cells were imaged every 10 minutes for 5 hours. Great care was taken to reduce illumination as much as possible to avoid phototoxicity.

Zebrafish larvae were anaesthetized in mivacurium and embedded in low-melting temperature agarose (1.2–2.0% in E3; egg water: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄) in a glass-bottom dish (Mat Tek). They were imaged on a Nikon two-photon microscope (A1RMP), attached to a fixed stage upright microscope, using a 25× water immersion objective (NA = 1.1). The femtosecond laser (Coherent Vision II) was tuned to 920 nm for GCaMP imaging. Stacks were collected in resonant-scanning mode with a 525/50 nm bandpass emission filter and with 8× pixel averaging; single-plane images were collected in galvano-scanning mode with 2× pixel averaging.
Light stimuli were generated by 5 mm blue LEDs (458 nm peak emission) powered by a 5 V TTL signal from a control computer and synchronized with image capture using a National Instruments DAQ board, controlled by the Nikon Elements software. Light intensity at the sample was 0.13 mW/cm².
For wide-field microscopy, excitation was provided by LEDs (Cairn OptoLED) at 470 nm. Images were captured on a Zeiss Axio Examiner with a 20× water immersion objective, using a Flash4 camera (Hamamatsu) controlled by MetaMorph. After background subtraction, change in fluorescence was measured using MetaMorph.

Electronic confocal slit detection requires precise timing and position control of cameras, lasers and galvanometric mirrors to ensure alignment of the illumination beam with the active area of the camera. As outlined above, we estimated the timing precision to be in the range of a few microseconds. For our optical setup, a galvanometric mirror amplitude of 1 V is sufficient to scan across the entire field of view of the camera (532 μm). A minimal slit size of 4 pixels thus yields a required precision in galvanometric mirror control voltage of 1 V*4/2048=2 mV. This can be achieved with 16-bit precision DAC. We used a custom written LabView (National Instruments) control software for synchronization of timings across all microscope devices. All trigger and analogue voltage traces are calculated by a field programmable gate array (FPGA, National Instrument NI PCIe-7842 R with a Virtex-5 FPGA) that ensures precise timing in the sub-μs range (40-MHz clock frequency). Following our collaboration with Hamamatsu Photonics, Japan, electronic confocal slit detection, also called ‘light-sheet mode', has been made available with version 2 (V2) release of the Hamamatsu Flash 4 camera. Other camera manufacturers have recently released cameras with similar features (Andor Technology, UK and PCO Imaging, Germany).

A total of 5 × 10⁶ cells of log-phase L. mexicana promastigotes were incubated in M199 medium on ice for 20 min before final concentration of 5 μg/ml FM4-64 (Invitrogen; from a 1000 μg/ml stock solution in dimethyl sulfoxide) was added for 1 min at RT. Cells were centrifuged at 800g for 3 min at RT, resuspended in 600 μl of prewarmed M199 at 28 °C, and then divided into three tubes of 200 μl each. Each tube was incubated at 28 °C and at each time point (10, 30, and 50 min), one of the tubes was centrifuged at 800g for 1 min at RT to concentrate cells for imaging with a Zeiss ImagerZ2 microscope with a 63× numerical aperture 1.4 objective and Hamamatsu Flash 4 camera. Captured cells were categorized according to the FM4-64 localization (Fig. 6A; FP; FP and endosome; and FP, endosome, and lysosome).