Immunofluorescence staining of a human kidney (#1, peri-tumor area) obtained by MELC

19 marker MELC run in a human peri-tumor kidney sample (#1). Each image shows the same field of view, sequentially stained with the depicted fluorescence labelled antibodies. Images contain 2024 x 2024 pixels and are generated using an inverted wide field fluorescence microscope with a 20 x objective, a lateral resolution of 325 nm and an axial resolution above 5 µm. Scale bar 100 µm.

All raw images were aligned based on the reference phase contrast image taken at the beginning of measurement. Each fluorescence MELC image was processed by background subtraction and illumination correction, based on bleaching images. Then an “Extended Depth of Field” algorithm was applied to the 3D fluorescence stack in each cycle. Images were at last normalized in ImageJ, where a rolling ball algorithm was used for background estimation, edges were removed (accounting for the maximum allowed shift during the autofocus procedure) and fluorescence intensities were stretched to the full intensity range (16 bit -> 2¹⁶).