Understanding cells as integrated systems is central to modern biology. Although fluorescence microscopy can resolve subcellular structure in living cells, it is expensive, is slow, and can damage cells. We present a label-free method for predicting three-dimensional fluorescence directly from transmitted-light images and demonstrate that it can be used to generate multi-structure, integrated images. The method can also predict immunofluorescence (IF) from electron micrograph (EM) inputs, extending the potential applications.

The axial deformation of a pipette-pressurized fluid membrane bag produces minuscule yet well-defined, reproducible forces. The stiffness of this ultrasensitive force transducer is tunable and largely independent of the constitutive membrane behavior. Based on a rigorous variational treatment, we present both numerical as well as approximate analytical solutions for the force-deflection relation of this unique biophysical force probe. Our numerical results predict a measurably nonlinear force-deflection behavior at moderate-to-large deformations, which we confirm experimentally using red blood cells. Furthermore, considering nearly spherical membrane shapes and enforcing proper boundary conditions, we derive an analytical solution valid at small deformations. In this linear regime the pressurized membrane bag behaves like a Hookean spring, with a spring constant that is significantly larger than previously published for the biomembrane force probe.

We assess the cross-reactivity of both cellular as well as recombinant E- and N-cadherins using functionalized bead arrays assembled on atomic-force-microscope cantilevers. This new approach builds upon and enhances the utility of a recently developed force probe that integrates a custom-built, horizontal atomic force microscope with micropipette manipulation. It enables us to test multiple biomolecular interactions of the same cell in a swift sequential or cyclic manner and thus to resolve subtle differences between individual interactions that otherwise would be obscured by cell-cell baseline variability. For each cell, we contrast heterophilic E:N-cadherin binding with the respective homophilic bonds and with a suitable control. Clarifying previous literature reports, we establish that specific bonds between E- and N-cadherins form readily, albeit less frequently than homophilic bonds of either cadherin. We support this assessment with a rough estimate of the ratio of on-rate constants of E/N-cadherin binding.

We present a multipurpose nanomechanical force probe that combines a sideways-mounted elastic cantilever and an optical-lever detection module with automated micropipette manipulation. It allows us to apply and measure compression, stretching, adhesion, and dissociation forces in the horizontal direction while providing a "side view" of ongoing experiments. The integrated micropipette setup facilitates the easy manipulation and mechanical interrogation of individual cells, functionalized particles, and synthetic membrane capsules. Pipette-held test objects are translated perpendicularly to and from the stationary cantilever, eliminating the need to attach them to a carrier surface and substantially reducing unwanted hydrodynamic coupling effects. Moreover, the test objects can be brought into contact with the cantilever anywhere along its length, which considerably enlarges the range of forces that can be applied with a single cantilever. Advantages of this instrument are demonstrated in example measurements of single-cell compression, membrane-tether extrusion, oligonucleotide stretching, and extraction of individual lipids from surfactant-monolayer surfaces of microbubbles.

We report on the refinement and validation of one of the earliest ideas of "reverse" biophysics: the use of individual red blood cells as reliable, ultrasensitive mechanotransducers. Our analysis is based on the numerical prediction of the force exerted by a micropipette-held red cell as it is pushed against a test object. Examining this red-cell transducer, in conjunction with a custom-built "horizontal" force microscope, we were able to soundly corroborate its utility, while at the same time, accurately calibrating the spring constants of atomic-force microscope cantilevers.