Optical microrobots are activated by a laser in a liquid medium using optical tweezers. To create visual control loops for robotic automation, this work describes a deep learning-based method for orientation estimation of optical microrobots, focusing on detecting 3-D rotational movements and localizing microrobots and trapping points (TPs). We integrated and fine-tuned You Only Look Once (YOLOv7) and Deep Simple Online Real-time Tracking (DeepSORT) algorithms, improving microrobot and TP detection accuracy by $\sim 3$% and $\sim 11$%, respectively, at the 0.95 Intersection over Union (IoU) threshold in our test set. Additionally, it increased mean average precision (mAP) by 3% at the 0.5:0.95 IoU threshold during training. Our results showed a 99% success rate in trapping events with no false-positive detection. We introduced a model that employs EfficientNet as a feature extractor combined with custom convolutional neural networks (CNNs) and feature fusion layers. To demonstrate its generalization ability, we evaluated the model on an independent in-house dataset comprising 4,757 image frames, where microrobots executed simultaneous rotations across all three axes. Our method provided mean rotation angle errors of $1.871^\circ$, $2.308^\circ$, and $2.808^\circ$ for X (yaw), Y (roll), and Z (pitch) axes, respectively. Compared to pre-trained models, our model provided the lowest error in the Y and Z axes while offering competitive results for X-axis. Finally, we demonstrated the explainability and transparency of the model’s decision-making process. Our work contributes to the field of microrobotics by providing an efficient 3-axis orientation estimation pipeline, with a clear focus on automation.

Molecular motors are machines essential for life since they convert chemical energy into mechanical work. However, the precise mechanism by which nucleotide binding, catalysis, or release of products is coupled to the work performed by the molecular motor is still not entirely clear. This is due, in part, to a lack of understanding of the role of force in the mechanical–structural processes involved in enzyme catalysis. From a mechanical perspective, one promising hypothesis is the Haldane–Pauling hypothesis which considers the idea that part of the enzymatic catalysis is strain-induced. It suggests that enzymes cannot be efficient catalysts if they are fully complementary to the substrates. Instead, they must exert strain on the substrate upon binding, using enzyme-substrate energy interaction (binding energy) to accelerate the reaction rate. A novel idea suggests that during catalysis, significant strain energy is built up, which is then released by a local unfolding/refolding event known as ‘cracking’. Recent evidence has also shown that in catalytic reactions involving conformational changes, part of the heat released results in a center-of-mass acceleration of the enzyme, raising the possibility that the heat released by the reaction itself could affect the enzyme’s integrity. Thus, it has been suggested that this released heat could promote or be linked to the cracking seen in proteins such as adenylate kinase (AK). We propose that the energy released as a consequence of ligand binding/catalysis is associated with the local unfolding/refolding events (cracking), and that this energy is capable of driving the mechanical work.

Biomolecular interactions are at the base of all physical processes within living organisms; the study of these interactions has led to the development of a plethora of different methods. Among these, single-molecule (in singulo) experiments have become relevant in recent years because these studies can give insight into mechanisms and interactions that are hidden for ensemble-based (in multiplo) methods. The focus of this review is on optical tweezer (OT) experiments, which can be used to apply and measure mechanical forces in molecular systems. OTs are based on optical trapping, where a laser is used to exert a force on a dielectric bead; and optically trap the bead at a controllable position in all three dimensions. Different experimental approaches have been developed to study protein–protein interactions using OTs, such as: (1) refolding and unfolding in trans interaction where one protein is tethered between the beads and the other protein is in the solution; (2) constant force in cis interaction where each protein is bound to a bead, and the tension is suddenly increased. The interaction may break after some time, giving information about the lifetime of the binding at that tension. And (3) force ramp in cis interaction where each protein is attached to a bead and a ramp force is applied until the interaction breaks. With these experiments, parameters such as kinetic constants (koff, kon), affinity values (KD), energy to the transition state ΔG≠, distance to the transition state Δx≠ can be obtained. These parameters characterize the energy landscape of the interaction. Some parameters such as distance to the transition state can only be obtained from force spectroscopy experiments such as those described here.

The advent of single-molecule force spectroscopy represents the introduction of forces, torques, and displacements as controlled variables in biochemistry. These methods afford the direct manipulation of individual molecules to interrogate the forces that hold together their structure, the forces and torques that these molecules generate in the course of their biochemical reactions, and the use of force, torque, and displacement as tools to investigate the mechanisms of these reactions. Because of their microscopic nature, the signals detected in these experiments are often dominated by fluctuations, which, in turn, play an important role in the mechanisms that underlie the operation of the molecular machines of the cell. Their direct observation and quantification in single-molecule experiments provide a unique window to investigate those mechanisms, as well as a convenient way to investigate fundamental new fluctuation theorems of statistical mechanics that bridge the equilibrium and non-equilibrium realms of this discipline. In this review we have concentrated on the developments that occurred in our laboratory on the characterization of biopolymers and of molecular machines of the central dogma. Accordingly, some important areas like the study of cytoskeletal motors have not been included. While we adopt at times an anecdotal perspective with the hope of conveying the personal circumstances in which these developments took place, we have made every effort, nonetheless, to include the most important developments that were taking place at the same time in other laboratories.

Optical tweezers have been used as versatile tools for non-contact manipulation of micrometer-sized entities. This paper proposes a hybrid micro/nanoscale manufacturing system using optical tweezers and chemical linkages for fabricating 2D and 3D micro/nanostructures. A holographic multiple trap optical tweezers system is first used to trap particles in a desired pattern. The particles are then connected to form rigid units using suitable chemistry. Connection schemes based on gold seeding, complementary-DNA linkage and streptavidin-biotin chemistry are presented and possible applications of this technique are explored. This method combines the advantages of top-down and bottom-up approaches and is compatible with organic and inorganic materials.

A general method is presented for determining and correcting nonlinear position detector responses in single particle tracking as used in three-dimensional scanning probe microscopy based on optical tweezers. The method uses locally calculated mean square displacements of a Brownian particle to detect spatial changes in the sensitivity of the detector. The method is applied to an optical tweezers setup, where the position fluctuations of a microsphere within the optical trap are measured by an interferometric detection scheme with nanometer precision and microsecond temporal resolution. Detector sensitivity profiles were measured at arbitrary positions in solution with a resolution of approximately 6 nm and 20 nm in the lateral and axial directions, respectively. Local detector sensitivities are used to reconstruct the real positions of the particle from the measured position signals.

Optical tweezers, or the single-beam optical gradient force trap, is becoming a major tool in biology for noninvasive micromanipulation on an optical microscope. The principles and practical aspects that influence construction are presented in an introductory primer. Quantitative theories are also reviewed but have yet to supplant user calibration. Various biological applications are summarized, including recent quantitative force and displacement measurements. Finally, tantalizing developments for new, nonimaging microscopy techniques based on optical tweezers are included.