Lattice light-sheet microscopy

Lattice light-sheet microscopy images were acquired using a 3i Lattice Light Sheet microscope. Here, lasers are individually expanded in the laser launch to 2.5 mm, collimated and aligned to be co-linear. All lines pass through an Acousto-Optic Tunable Filter (AOTF). Frequency modulation of the AOTF regulates the degree of higher order light that is transmitted, thus regulating the laser power input into the system. Once in the Lattice Light Sheet optical path, a set of cylindrical lenses expands the 2.5-mm input beam in X to 25 mm to uniformly illuminate a stripe on the spatial light modulator (SLM). The SLM is programmed to display binary images of user generated multi-Bessel patterns generating an optical lattice of Bessel beams. The Bessel beam is projected onto an annular mask, which filters the zeroth order, removes artifacts and lengthens the sheet. The mask is serially conjugate to Z and X galvo mirrors, as well as the rear pupil of the excitation objective, allowing the light sheet to be translated in y and z and to rapidly oscillate in x for the dithered mode of operation. The beam is focused through the illumination objective to create a pattern of the Bessel beams at the sample plane that is conjugate to the projection off of the SLM. This pattern is dithered by the X galvo to form the sheet of illumination that is observed by the sCMOS camera (ORCA-Flash4.0 v2, Hamamatsu), through the detection objective. The 25 × detection objective, in conjunction with the 500-mm tube lens, gives a 62.5 × magnification at the camera.

The volumetric data acquisition can be performed in two modalities: sample scan or sheet scan. In sample scan mode the stage moves while the light sheet and the objectives remain stationary. This mode allows to scan big areas but, since the objective is tilted at an angle with respect to the axis of stage movement, the scan produces a lateral offset between images from neighboring z planes. Therefore, these images have to be shifted (or deskewed) in post-processing to retrieve the original positions. In sheet scan mode, instead, the light sheet and objective are moved in tandem so that there is no offset between the volumetric slices and no deskewing operation is needed.

HaCaT keratinocytes were generously provided by Kowalczyk Lab at Emory University. They were cultured in DMEM (Corning, Tewksbury, MA) supplemented with 10% fetal bovine serum and 1% Antibiotic/ antimycotic. Cells grown on 5-mm coverslips were transfected according to manufacturer’s instruction with Viromer RED (OriGene, Rockville, MA). Briefly, plasmids were incubated with Viromer RED transfection reagent and buffer for 20 min at room temperature. This plasmid/reagent mix was then added to cells in culture dishes. Cells were then fixed 24 h after transfection with 4% PFA for 15 min. The mCherry-VAPB (human) plasmid construct was purchased from Addgene (Plasmid #108126).

Mouse embryonic fibroblasts (MEFs) were generously provided by Chan lab at Caltech. They were plated onto 5 mm matrigel-coated coverslips 24 h before experimentation, at about 60–70% confluence. Cells were grown in DMEM + 10% FBS. The day of the experiment, the medium was swapped with imaging medium (phenol red-free DMEM with HEPES, Gibco, Catalog #: 21063–029) along with SiRActin dye (diluted to a 1 µM concentration; Cytoskeleton Inc, Cat. # CY-SC001) at least 1 h before experimentation. Phenol red-free medium with SiRActin dye was used throughout the LLSM imaging experiment and was not washed out.

Human lung cancer cells (NCI-H1299 NSCLC, ATCC, Manassas, VA) expressing gd2PAL-Dendra2 (Bassell lab, Emory University) were cultured on glass coverslips in Roswell Park Memorial Institute (RPMI-1640) media supplemented with 10% fetal bovine serum and 100 units/mL of penicillin/streptomycin, and maintained at 37 °C and 5% CO₂.