Polarisation camera movies of T cells on a cover glass

The plasma membrane of live T cells (J8 LFA-1) was labelled using the Nile red-derivative NR4A, and 3D movies were recorded using a polarisation camera. Two unprocessed datasets are included here:

• Tcell_NR4A_EPI_stack: A 3D stack of a single T cell, imaged with the excitation in EPI configuration.
• Tcell_NR4A_HILO_stack_movie: A 3D movie of live T cells interacting with an OKT3 antibody-coated cover glass, imaged with the excitation in HILO configuration.

For each .tif file, the corresponding Micro-Manager metadata file is included.

Optical setup:

Imaging was performed on a widefield microscope equipped with a polarisation camera (CS505MUP, Thorlabs). The sample was excited using a 515 nm laser, coupled into a square-core multi-mode fiber (M97L02, Thorlabs) with a custom vibration-motor-based mode scrambler. The measured power density at the image plane was on the order of 0.004 kW/cm^2. A dichroic (Di03-R514-t1, Semrock) was used to seperate fluorescence from the excitation. The emission was filtered using a long-pass filter (FF01-515/LP, Semrock) and bandpass filter (FF01-650/200, Semrock) before detection.

Sample preparation:

J8 LFA-1 cells were incubated overnight (~18 h) in complete-RPMI (StableCell RPMI-1640 media (Sigma) supplemented with 10 % (v/v) fetal calf serum (FCS), 1 % (v/v) HEPES buffer, and 1 % (v/v) pen/strep antibiotics. 1 mL of cells were collected by centrifugation and resuspended in phenol-red free RPMI supplemented with 1 % HEPES.

Round coverslips were rinsed with IPA, MilliQ, dried, and Ar-plasma cleaned for 20 minutes. Grace Bio-Labs CultureWells were attached, and the slide was incubated with OKT3 antibody (provided by the Human Immunology Unit, WIMM, Oxford) for 30 minutes. The slide was washed 5 times with phenol-red free RPMI supplemented with 1 % HEPES and a final wash with phenol-red free RPMI supplemented with 1 % HEPES and 200 nM NR4A before imaging.