Back-focal-plane imaging and linear density measurements of sub-diffraction sized biological filaments and particles.

Biological filaments and their networks are studied to gain a deeper understanding of cell and tissue properties. Imaging of filaments and networks frequently relies on fluorescence microscopy to achieve high-contrast, high-specificity images. However, fluorescence microscopy studies of filament mechanical properties are hindered by phototoxicity, fluorophore induced changes in mechanical properties, and the difficulty of precise local filament thickness measurements. High-contrast label-free methods are needed to visualize filaments under physiological conditions without fluorescence. Higher contrast can be achieved by measuring the transmitted light intensity distribution. This method, known as differential phase contrast, has been implemented in electron and optical microscopy. Similarly, optical tweezers frequently use back-focal-plane detection to track the position of single, trapped, sub-diffraction sized particles with sub-nanometer precision and MHz bandwidth. Here, this method of single particle tracking is extended to visualizing more complex objects such as filaments. We demonstrate that back-focal-plane-detection-equipped optical tweezers can be used for high contrast microscopy by imaging single collagen fibrils and microtubules. By modeling filaments as a line of individual scattering particles, local linear density and thickness is quantified. The sample-orientation-dependent detector response for filaments can be used for a unique background subtraction method. This is demonstrated by removing the protein aggregate background from surface-bound microtubule images. We show that measurements can be made far from the coverslip, making this an excellent tool for studying the link between the structure and mechanics of filaments and filament networks. Optical tweezer setups with back-focal-plane detection can implement this method without significant optical modifications.