Matthias D. Koch

How to calibrate an object-adapted optical trap for force sensing and interferometric shape tracking of asymmetric structures

Optical traps have shown to be a flexible and powerful tool for 3D manipulations on the microscale. However, when it comes to sensitive measurements of particle displacements and forces thorough calibration procedures are required, which can be already demanding for trapped spheres. For asymmetric structures, with more complicated shapes, such as helical bacteria, novel calibration schemes need to be established. The paper describes different methods of how to extract various calibration parameters of a tiny helical bacterium, which is trapped and tracked in shape by scanning line optical tweezers. Tiny phase differences of the light scattered at each slope of the bacterium are measured by back focal plane interferometry, providing precise and high bandwidth information about fast deformations of the bacterium. A simplified theoretical model to estimate the optical forces on a chain like structure is presented. The methods presented here should be of interest to people that investigate optical trapping and tracking of asymmetric particles.

Force generation by groups of migrating bacteria

Significance Bacterial migration, aggregation, and even host infection depend on the generation of mechanical force. Despite their biomedical importance, forces between bacteria and surfaces have not yet been measured during migration. We present a first study of bacterial cell–substrate traction using Myxococcus xanthus as a model organism. M. xanthus exhibits two common mechanisms of motility, namely, twitching and gliding. We find that these mechanisms lead to distinct patterns of traction during motion as an individual or in groups. Twitching leads to local, uncoordinated traction, and gliding in groups allows for collective emergence of directional traction. The forces produced by twitching or gliding of individual cells are significantly amplified when cells move in groups. From colony formation in bacteria to wound healing and embryonic development in multicellular organisms, groups of living cells must often move collectively. Although considerable study has probed the biophysical mechanisms of how eukaryotic cells generate forces during migration, little such study has been devoted to bacteria, in particular with regard to the question of how bacteria generate and coordinate forces during collective motion. This question is addressed here using traction force microscopy. We study two distinct motility mechanisms of Myxococcus xanthus, namely, twitching and gliding. For twitching, powered by type-IV pilus retraction, we find that individual cells exert local traction in small hotspots with forces on the order of 50 pN. Twitching bacterial groups also produce traction hotspots, but with forces around 100 pN that fluctuate rapidly on timescales of <1.5 min. Gliding, the second motility mechanism, is driven by lateral transport of substrate adhesions. When cells are isolated, gliding produces low average traction on the order of 1 Pa. However, traction is amplified approximately fivefold in groups. Advancing protrusions of gliding cells push, on average, in the direction of motion. Together, these results show that the forces generated during twitching and gliding have complementary characters, and both forces have higher values when cells are in groups.

Single microtubules and small networks become significantly stiffer on short time-scales upon mechanical stimulation

The transfer of mechanical signals through cells is a complex phenomenon. To uncover a new mechanotransduction pathway, we study the frequency-dependent transport of mechanical stimuli by single microtubules and small networks in a bottom-up approach using optically trapped beads as anchor points. We interconnected microtubules to linear and triangular geometries to perform micro-rheology by defined oscillations of the beads relative to each other. We found a substantial stiffening of single filaments above a characteristic transition frequency of 1–30 Hz depending on the filament’s molecular composition. Below this frequency, filament elasticity only depends on its contour and persistence length. Interestingly, this elastic behavior is transferable to small networks, where we found the surprising effect that linear two filament connections act as transistor-like, angle dependent momentum filters, whereas triangular networks act as stabilizing elements. These observations implicate that cells can tune mechanical signals by temporal and spatial filtering stronger and more flexibly than expected.

Introduction to Optical Tweezers

Thirty years after their invention by Arthur Ashkin and colleagues at Bell Labs in 1986 [1], optical tweezers (or traps) have become a versatile tool to address numerous biological problems. Put simply, an optical trap is a highly focused laser beam that is capable of holding and applying forces to micron-sized dielectric objects. However, their development over the last few decades has converted these tools from boutique instruments into highly versatile instruments of molecular biophysics. This introductory chapter intends to give a brief overview of the field, highlight some important scientific achievements, and demonstrate why optical traps have become a powerful tool in the biological sciences. We introduce a typical optical setup, describe the basic theoretical concepts of how trapping forces arise, and present the quantitative position and force measurement techniques that are most widely used today.

Bakterien gefangen im Licht

Bacterial locomotion is often related to pathogenicity and the spread of diseases. Bacteria are able to move under various environmental conditions, and their complex motions are often too fast and minute to be quantitatively imaged with conventional microscopy techniques. The study of their locomotion principles requires new, advanced photonic measurement techniques, capable of extracting forces and energetics exerted by the cell on time scales ranging from milliseconds to a couple of minutes.