polarisation Differential Phase Contrast microscopy
Polarisation differential phase contrast microscopy (pDPC) is a single-shot implementation of DPC microscopy that simultateneously captures the 4 images with their appropriate semicircular aperture in the back focal plane of the condensor lens using a single polarisation-resolved camera based on the Sony ‘Polarsens™’ IMX250MZR sensor. This Polarsens™ camera is based on a CMOS sensor integrated with a polarisation filter mask that arrays polarisation filters orientated at 0, 135, 45 and 90 degrees with respect to the horizontal axis of the sensor over each 2x2 block of pixels. Thus, the 2448 x 2048 pixels of the CMOS sensor effectively record 4 polarisation resolved images of 1224x 1024 pixels at the camera frame rate.
As illustrated in the schematic below, we designed a circular mask comprising four quadrants of polarising film aligned with the four orientations of the polarisation filters on the Polarsens™ camera. As explained in our J Biophotonics paper, by considering the contributions to the recorded polarisation-resolved images (I1,I2,I3,I4) from the 4 quadrants of the polarising illumination mask, it is possible to calculate the images corresponding to illumination with semicircular apertures in the condenser back focal plane (i.e. IA,IB,IC,ID) and therefore to calculate the phase gradient images and then the quantitative phase images.
Because all the required image data is recorded in a single frame of the Polarsens™ camera, the quantitative phase images can be acquired at the maximum frame rate of the camera (i.e. up to 67 frames/second). Furthermore, the camra sensor enables QPM to be implemented using transmitted light throughout the visible and near infrared (NIR) spectrum. This makes it straightforward to combine pDPC with fluorescence microscopy (e.g. using the excitation light for QPM) or to implement QPM with NIR radiation to provide a low phototoxicity label-free imaging modality that can be used for cell sementation and/or cell tracking.
The figure below illustrates the application of combined pDPc and fluorescence imaging of a live transgenic adult C. elegans in which epidermal stem cell nuclei are labelled with GFP expressed in the lateral seam cells.