Circularly polarised luminescence laser scanning confocal microscopy to study live cell chiral molecular interactions.

EDCC imaging

Microscope control and image acquisition and analysis were performed with Leica’s commercial microscope software LAS-X. EDCC imaging is achieved using ImageJ software (v1.49) 31 , with its built-in image calculator add-on software by subtracting one CPL channel from the other, and vice versa. The convention used herein is: left-handed enantioselective contrast = left CPL—right CPL. Right-handed enantioselective contrast = right CPL—left CPL. Images for a typical 1024 × 1024 pixel FOV with 10 line accumulation bidirectional scanning sequence were typically acquired in 90 s (corresponding to 10 full-frame accumulations). Confocality—axial (z) resolution—was governed by the applied pinhole diameter denoted in Airy disk units to preserve resolution and maximise light detection from the FOV. Images were obtained using the detector saturation mode of LAS-X where each image is assessed for maximum intensity value. Each image is only recorded if no 4 × 4 pixel cluster (Nyquist sampling covering an area determined by the systems optical resolution, 126 × 126 nm at 355 nm excitation using 1.4 NA objective) possesses average intensity values of 255 on an 8-bit greyscale. This allows the employed gain of each detector to be synchronised and kept constant, so no error associated with pixel intensity saturation is included accidentally contributing to pixel uncertainties, lowering S/N and exponentially increasing the limit of detection values. Non-live cell 8-bit average pixel chiroptical contrast value calculations were facilitated by selecting and averaging five different positions non-overlapping equal size and shape arbitrary area of the sample with respect to each enantiomer and dark background. Due to the 1024 × 1024 pixel size of each recorded image total FOV, this arbitrary area has been kept at a constant area of 100 × 100 pixels region of interest (ROI). The average maximum 8-bit grayscale pixel intensity values were determined using the LSCM’s built-in LAS-X software that is employing a maximum average value ROI histogram methodology that is based on standard Gaussian distribution profiling of the average intensity values. Due to the employed methodology and the averaging nature of image acquisition and ROI calculation the limit of detection (error associated with) 8-bit greyscale contrast value is below 1% (