Microfluidic cell unroofing for the in situ molecular analysis of organelles without membrane permeabilization

Molecular networks of organelle membranes are involved in many cell processes. However, the nature of plasma membrane as a barrier to various analytical tools, including antibodies, makes it challenging to examine intact organelle membranes without affecting their structure and functions via membrane permeabilization. Therefore, in this study, we aimed to develop a microfluidic method to unroof cells and observe the intrinsic membrane molecules in organelles. In our method, single cells were precisely arrayed on the bottom surface of microchannels in a light-guided manner using a photoactivatable cell-anchoring material. At sufficiently short cell intervals, horizontal stresses generated by the laminar flow instantly fractured the upper cell membranes, without significantly affecting some organelles inside the fractured cells. Subsequently, nucleus and other organelles in unroofed cells were observed via confocal fluorescence and scanning electron microscopy. Furthermore, distribution of the mitochondrial membrane protein, translocase of outer mitochondrial membrane 20, on the mitochondrial membrane was successfully observed via immunostaining without permeabilization. Overall, the established cell unroofing method shows great potential to examine the localization, functions, and affinities of proteins on intact organelle membranes.