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Liquid crystalline polymers are attractive materials for untethered miniature soft robots. When they contain azo dyes, they acquire light-responsive actuation properties. However, the manipulation of such photoresponsive polymers at the micrometer scale remains largely unexplored. Here, uni- and bidirectional rotation and speed control of polymerized azo-containing chiral liquid crystalline photonic microparticles powered by light is reported. The rotation of these polymer particles is first studied in an optical trap experimentally and theoretically. The micro-sized polymer particles respond to the handedness of a circularly polarized trapping laser due to their chirality and exhibit uni- and bidirectional rotation depending on their alignment within the optical tweezers. The attained optical torque causes the particles to spin with a rotation rate of several hertz. The angular speed can be controlled by small structural changes, induced by ultraviolet (UV) light absorption. After switching off the UV illumination, the particle recovers its rotation speed. The results provide evidence of uni- and bidirectional motion and speed control in light-responsive polymer particles and offer a new way to devise light-controlled rotary microengines at the micrometer scale.

Liquid crystalline polymers are attractive materials for untethered miniature soft robots. When they contain azo dyes, they acquire light-responsive actuation properties. However, the manipulation of such photoresponsive polymers at the micrometer scale remains largely unexplored. Here, uni- and bidirectional rotation and speed control of polymerized azo-containing chiral liquid crystalline photonic microparticles powered by light is reported. The rotation of these polymer particles is first studied in an optical trap experimentally and theoretically. The micro-sized polymer particles respond to the handedness of a circularly polarized trapping laser due to their chirality and exhibit uni- and bidirectional rotation depending on their alignment within the optical tweezers. The attained optical torque causes the particles to spin with a rotation rate of several hertz. The angular speed can be controlled by small structural changes, induced by ultraviolet (UV) light absorption. After switching off the UV illumination, the particle recovers its rotation speed. The results provide evidence of uni- and bidirectional motion and speed control in light-responsive polymer particles and offer a new way to devise light-controlled rotary microengines at the micrometer scale. © 2023 Wiley-VCH GmbH.

Interacting with an evanescent wave, an achiral particle can experience a lateral optical force (LOF) in a direction which is perpendicular to that of the illuminating photon momentum while the system apparently has the left-right symmetry. Here, using the Cartesian multipole expansion theory, a general relationship between LOF and spin and momentum is established. Most importantly, it is demonstrated that an anomalous LOF exerted on an isotropic achiral particle of arbitrary size and composition is proportional to the optical transverse spin in the incident evanescent wave that carries pure transverse spin associated with spin-momentum locking. Manifesting itself as a mechanical consequence of the Berry phase effect, this anomalous LOF suggests a direct detectability of the transverse spin of light, as well as mimicking the transverse shift of light in the spin Hall effect of photons.

Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects, ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in the life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nano-particle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration.

Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects, ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in the life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nano-particle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration.

Soft matter exhibits a multitude of intrinsic physico-chemical attributes. Their mechanical properties are crucial characteristics to define their performance. In this context, the rigidity of these systems under exerted load forces is covered by the field of biomechanics. Moreover, cellular transduction processes which are involved in health and disease conditions are significantly affected by exogenous biomechanical actions. In this framework, atomic force microscopy (AFM) and optical tweezers (OT) can play an important role to determine the biomechanical parameters of the investigated systems at the single-molecule level. This review aims to fully comprehend the interplay between mechanical forces and soft matter systems. In particular, we outline the capabilities of AFM and OT compared to other classical bulk techniques to determine nanomechanical parameters such as Young’s modulus. We also provide some recent examples of nanomechanical measurements performed using AFM and OT in hydrogels, biopolymers and cellular systems, among others. We expect the present manuscript will aid potential readers and stakeholders to fully understand the potential applications of AFM and OT to soft matter systems.

We demonstrate an optofluidic device which utilizes the optical scattering and gradient forces for particle trapping in microchannels featuring 300 nm thick membranes. On-chip waveguides are used to direct light into microfluidic trapping channels. Radiation pressure is used to push particles into a protrusion cavity, isolating the particles from liquid flow. Two different designs are presented: the first exclusively uses the optical scattering force for particle manipulation, and the second uses both scattering and gradient forces. Trapping performance is modeled for both cases. The first design, referred to as the orthogonal force design, is shown to have a 80% capture efficiency under typical operating conditions. The second design, referred to as the gradient force design, is shown to have 98% efficiency under the same conditions.

Tuberculosis (TB) is an airborne disease that kills 1.5 million people and affects ~ 9 million worldwide. Its etiological agent, Mycobacterium tuberculosis (Mtb), persists in a third of the general population. There is also an emergent problem of drug and multidrug resistance in Mtb that creates the need for novel drugs. In the treatment against TB, one target of antibiotics is the prokaryotic RNA polymerase (RNAP), an essential multitask enzyme that catalyzes RNA synthesis during transcription. Rifampicin, the first-line antibiotic anti-TB, binds and inhibits the MtbRNAP.Consequently, combatting TB requires a better understanding of the primary mechanism of transcription by MtbRNAP. In fact, during Mtb transcription, unique and essential factors might affect the MtbRNAP dynamics and regulate its activity. That regulation plays a significant role in the adaptation and pathogenesis of the parasite. Therefore, understanding how these factors and new antibiotics modulate together the MtbRNAP activity will contribute valid information to address TB. To this end, we use a single-molecule approach based on high-resolution optical tweezers to study transcription elongation by MtbRNAP in the context of its DNA elements, transcriptional factors, and antibiotics.

Cells are known to constantly interact with their local extracellular matrix (ECM) and respond to a variety of biochemical and mechanical cues received from the ECM. Nonetheless, comprehensive understanding of cell-ECM interactions has been elusive. Many studies rely on analysis of cell behavior on 2D substrates, which do not reflect a natural cell environment. Further, lack of dynamic control over local stiffness anisotropies and fiber alignment hinders progress in studies in naturally derived fibrous 3D cultures. Here, we present a cell-safe method of patterned photocrosslinking, which can aid in studying biological hypotheses related to mechanotransduction in 3D hydrogels. As previously described by our group, ruthenium-catalyzed photocrosslinking (RCP) of selected ECM regions promotes localized increase in stiffness mediated by focused blue laser light in a confocal microscope. In this study, we further demonstrate that RCP can induce localized strain stiffening and fiber alignment outside of the selected crosslinked region and induce stiffness anisotropy biased towards the direction of fiber alignment. MDA-MB-231 cells are shown to respond to RCP-induced changes in local ECM architecture and display directional bias towards the direction of fiber alignment, as compared to control cells. Further, the effect of patterned crosslinking on a stiffness landscape is measured using multi-axes optical tweezers active microrheology (AMR) with backscattered laser beam illumination. AMR validates RCP as a suitable tool for creating distinct stiffness anisotropies which promote directed migration of cells, further underscoring the usefulness of RCP in cell-ECM studies. STATEMENT OF SIGNIFICANCE: Studies on cell-ECM interactions in 3D cultures have often been hindered by the lack of available tools to dynamically alter local ECM stiffness and fiber alignment. Here, we present a non-invasive, cell-safe and easily applicable method of patterned photocrosslinking, which can aid

Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins. Upon binding to a specific sequence within centromeric regions, the DNA-binding protein CENP-B compacts centromeres by forming pronounced DNA loops between the repeats, which favor inter-chromosomal centromere compaction and clustering. This DNA-loop-mediated organization of centromeric chromatin participates in maintaining centromere position and integrity upon microtubule pulling during mitosis. Our findings emphasize the importance of DNA topology in centromeric regulation and stability., Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public., BN/Cees Dekker Lab

Manipulating particles with optical tweezers in combination with the ability to measure force and torque provides a powerful way to investigate objects on the microscale. In this thesis we present an optical-tweezers-based generally applicable holographic measurement method that overcomes many limitations and issues of existing techniques. Specifically, in the presence of several trapping spots it enables one to simultaneously measure all components of the optical force and the optical (spin and orbital) torque applied at an individual trapping site. Our approach uses the scattered light in the farfield to deduce the force and torque from the change of linear and angular momentum of light induced by the trapped particle. The holographic measurement relies on a detailed analysis of a single interference pattern formed in the back focal plane of a high-NA condenser lens to recover amplitude and phase of the far-field by means of an iterative algorithm, using the known positions of the optical traps as constraints., Franziska Maria Strasser, Kumulative Dissertation aus drei Artikeln, Dissertation University Innsbruck 2022

Artificially engineered structures, namely metamaterials and their two-dimensional (2D) counterpart –metasurfaces, have been proven as promising platforms to realize unusual light-matter interactions. Such structures have enabled the efficient manipulation of electromagnetic waves in unprecedented ways that cannot be obtained using conventional materials and thus have triggered exciting applications such as hyperlensing, canalization of light, negative refraction, hyperbolic dispersion, cloaking, or the enhancement of the spontaneous emission rate of dipole emitters, amongst many others. Moreover, the discovery of graphene and other 2D materials, and their electrical tunability, have enabled the use of surface plasmon polaritons at terahertz and infrared frequencies in numerous nanophotonic applications.In this thesis, I propose novel nano-optical plasmonic tweezers based on hyperbolic and nonreciprocalmetasurfaces to efficiently trap and manipulate nanoparticles in the near field with superior performance compared to the state of the art. To this purpose, I develop a rigorous theoretical framework able to compute optical forces on dipolar Rayleigh nanoparticles located near the metasurfaces. The theoretical model is based on Lorentz force within the dipole approximation combined with the scattered dyadic Green’s function of the system. Analytical expressions show that the force strength is directly proportional to the fourth power of wavenumber of the supported surface plasmons. This tells that the strength can be dramatically enhanced by the proper choice of metasurfaces that support ultra-confined surface plasmons with larger wavenumber. One potential candidate to achieve such response is the use of hyperbolic metasurface that supports surface plasmons with wavenumbers up to ~200 times larger than the ones supported in free space. My theoretical and numerical results using full wave simulations show that the use of hyperbolic metasurfaces enables unusual enhancem

Optical tweezers have been used for a number of applications since their invention by Arthur Ashkin in 1986, and are particularly useful for biological and biophysical studies due to their exceptionally high spatial and force-based resolution. The same intense laser focus that allows light to be used as a tool for micro-nanoscale manipulation also has the potential to damage the objects being studied, and the extremely high force resolution is coupled with the limitation of very low forces. There is potential to overcome these drawbacks of optical manipulation through making use of another laser based technique: two-photon absorption polymerisation (TPAP). This thesis has brought these together to demonstrate the uses of optical nanomachines as helpful tools for optical tweezer studies. The project was highly interdisciplinary, concerning the intersection of optical trapping, 3D micromachine design and development, and DNA stretching. The thesis was based around the strategy of first developing microrobots and demonstrating their manipulation using optical tweezers, then adjusting the design for specific applications. Microlevers were developed for lever-assisted DNA stretching and amplification of optical forces. The influence of design features and TPAP parameters on microlever functionality was investigated; particularly the influence of overlapping area and presence of supports, and the effects of differently shaped "trapping handles". These features were important as lever functionality was tested in solutions of different ionic strength, and stable trapping of the levers was required for force amplification. DNA stretching was chosen as a target application for distanced-application of optical forces due to its status as a well-known and characterised example of single-molecule studies with optical tweezers. Amplification of optical forces was also seen as an application that could demonstrate the utility of optical micromachines, and microlevers with a 2:1 le

The work is aimed at the development of the experimental device to study of the motion of different colloidal objects in the combined force field composed of the optical-trapping force originating in the light-intensity gradient and the thermophoretic force caused by the temperature gradient in aqueous solvents. Polystyrene microscopic beads of submicrometer diameters are used as a simple model of the optical tweezers calibration. The methods of generating temperature gradient are under development. A dual optical trap is used as a tool for the investigation of the thermophoretic force acting on the observed particles. Also, the generated optical trap's force constant is calculated by various methods, including Fast Fourier Transform, and the influence of differences in the sample environment and light intensity is compared. Results of these experiments should contribute to the theory of the optical trapping effect, so far not understood in detail, and should open a way for developing novel methods of the investigation of thermophoretic phenomena in a water environment.

Single molecule biophysics experiments for the study of DNA-protein interactions usually require production of a homogeneous population of long DNA molecules with controlled sequence content and/or internal tertiary structures. Traditionally, Lambda phage DNA has been used for this purpose, but it is difficult to customize. In this article, we provide a detailed and simple protocol for cloning large (~ 25 kbp) plasmids with bespoke sequence content, which can be used to generate custom DNA constructs for a range of single-molecule experiments. In particular, we focus on a procedure for making long single-stranded DNA (ssDNA) molecules, ssDNA-dsDNA hybrids and long DNA constructs with flaps, which are especially relevant for studying the activity of DNA helicases and translocases. Additionally, we describe how the modification of the free ends of such substrates can facilitate their binding to functionalized surfaces allowing immobilization and imaging using dual optical tweezers and confocal microscopy. Finally, we provide examples of how these DNA constructs have been applied to study the activity of human DNA helicase B (HELB). The techniques described herein are simple, versatile, adaptable, and accessible to any laboratory with access to standard molecular biology methods.

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was-60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results. (c) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Diseases caused by pathogenic agents such as bacteria and viruses result in devastating costs on personal and societal levels. However, it is not just the emergence of new diseases that is problematic. Antibiotic resistance among bacteria makes uncomplicated infections difficult and lethal. Resilient disease-causing spores spread in hospitals, the food industry, and water supplies requiring effective detection and disinfection methods. Further, we face complex neurological diseases where no effective treatment or diagnostic methods exist. Thus, we must increase our fundamental understanding of these diseases to develop effective diagnostic, detection, disinfection, and treatment methods. Classically, the methods used for detecting and studying the underlying mechanics of pathogenic agents work on a large scale, measuring the average macroscopic behavior and properties of these pathogens. However, just as with humans, the average behavior is not always representative of individual behavior. Therefore, it is also essential to investigate the characteristics of these pathogens on a single cell or particle level. This thesis develops and applies optical techniques to characterize pathogenic biomaterial on a single cell or particle level. At the heart of all these studies is our Optical Tweezers (OT) instrument. OT are a tool that allows us to reach into the microscopic world and interact with it. Finally, by combining OT with other experimental techniques, we can chemically characterize biomaterials and develop assays that mimic different biological settings. Using these tools, we investigate bacterial adhesion, disinfection, and detection of pathogenic spores and proteins. Hopefully, the insights of these studies can lessen the burden on society caused by diseases by helping others develop effective treatment, diagnostic, detection, and disinfection methods in the future.

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was-60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results. (c) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Nucleosome DNA unwrapping and its disassembly into hexasomes and tetrasomes is necessary for genomic access and plays an important role in transcription regulation. Previous single-molecule mechanical nucleosome unwrapping revealed a low- and a high-force transitions, and force-FRET pulling experiments showed that DNA unwrapping is asymmetric, occurring always first from one side before the other. However, the assignment of DNA segments involved in these transitions remains controversial. Here, using high-resolution optical tweezers with simultaneous single-molecule FRET detection, we show that the low-force transition corresponds to the undoing of the outer wrap of one side of the nucleosome (∼27 bp), a process that can occur either cooperatively or noncooperatively, whereas the high-force transition corresponds to the simultaneous unwrapping of ∼76 bp from both sides. This process may give rise stochastically to the disassembly of nucleosomes into hexasomes and tetrasomes whose unwrapping/rewrapping trajectories we establish. In contrast, nucleosome rewrapping does not exhibit asymmetry. To rationalize all previous nucleosome unwrapping experiments, it is necessary to invoke that mechanical unwrapping involves two nucleosome reorientations: one that contributes to the change in extension at the low-force transition and another that coincides but does not contribute to the high-force transition.

Understanding complex interactions between a cell and its extracellular matrix (ECM) lies at the core of mechanobiology. For instance, stiffness of the ECM was previously shown to be highly heterogenous and deregulated in wound healing or during cancer progression. However, tools available for measuring and dynamically altering peri-cellular stiffness have been lacking and bulk measurements do not probe stiffness sensed by the cells. Thus, we have developed an optical tweezers active microrheology (AMR) system capable of multi-axes stiffness measurements in the peri-cellular region. In this thesis, multi-axes AMR system was used to investigate highly heterogenous and anisotropic stiffness landscapes established by dermal fibroblasts, human breast cancer and fibrosarcoma cells. Peri-cellular stiffness and anisotropy are shown to vary between the tested cell lines and with different treatments modifying cell behavior. Further, stiffness landscape and cell response to ECM vary with hydrogel source, concentration and fiber architecture of the local ECM. These studies underscore the need for peri-cellular and not bulk stiffness measurements in studies on cellular mechanotransduction. Additionally, the method of patterned crosslinking is used to alter ECM topography by inducing fiber alignment and peri-cellular stiffness landscape. Following localized crosslinking, human breast cancer cells undergo contact guidance and durotaxis, as indicated by cell migration in a direction of fiber alignment and along an off-axis stiffness gradient, respectively. While these phenomena are widely known and studied on 2D substrates or in 3D synthetic hydrogels, patterned crosslinking allows for better understanding of processes governing directed migration of individual cells embedded inside naturally-derived fibrous hydrogels. Collectively, this thesis work investigates how cells respond to and regulate their local ECM stiffness based on a variety of different factors. Local ECM stiffnes

How biological fibrous materials respond to forces and changes at the cellular scale is important for understanding what cells `feel' from their extracellular environment. The local mechanical property within a fibrin fiber network is dependent on the local structure of the network. Elastic force transmission through hydrogel depends on the arrangement of the fiber. A novel method for visualizing force transmission through this network is measuring the pixel fluctuation of individual fibers in the presence of an actively applied oscillation using embedded microbeads and optical tweezers. Optical tweezers can also be used for measuring local stiffness through active microrheology. After micropatterned crosslinking, fibrin hydrogels were shown to produce local stiffening directly and indirectly through strain hardening. This stiffening resulted in stiffness anisotropy and fiber alignment which MDA-231 cells were shown to respond to. Finally, a model of elastically connected nodes recapitulates some of the experimental observations such as fiber force transmission, strain hardening, and stiffness anisotropy when strained. Manipulating, measuring, and modelling of local stiffness changes in response to applied forces offers further insight into how cells sense their surroundings.

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ("reverse topology"), but they can also be directed toward the cytosol ("normal topology"). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.

Cells sense and respond to mechanical stimuli for activation, proliferation, migration, and differentiation. The associated mechanosensing and biomechanical properties of cells and tissues are significantly implicated in the context of cancer, fibrosis, dementia, and cardiovascular diseases. To gain more mechanobiology insights, dynamic force spectroscopies (DFSs), particularly optical tweezers (OT), have been further advanced to enable in situ force measurement and subcellular manipulation from the outer cell membrane to the organelles inside of a cell. In this review, we first explain the classic OT-DFS rationales and discuss their applications to protein biophysics, extracellular biomechanics, and receptor-mediated cell mechanosensing. As a non-invasive technique, optical tweezers’s unique advantages in probing cytoplasmic protein behaviors and manipulating organelles inside living cells have been increasingly explored in recent years. Hereby, we then introduce and highlight the emerging OT rationales for intracellular force measurement including refractive index matching, active–passive calibration, and change of light momentum. These new approaches enable intracellular OT-DFS and mechanical measurements with respect to intracellular motor stepping, cytosolic micro-rheology, and biomechanics of irregularly shaped nuclei and vesicles. Last but not least, we foresee future OT upgrades with respect to overcoming phototoxicity and system drifting for longer duration live-cell measurements; multimodal integration with advanced imaging and nanotechnology to obtain higher spatiotemporal resolution; and developing simultaneous, automated, and artificial intelligence–inspired multi-trap systems to achieve high throughput. These further developments will grant unprecedented accessibility of OT-DFS and force measurement nanotools to a wider biomedical research community, ultimately opening the floodgates for exciting live-cell mechanobiology and novel therapeutic discoveri

Spatial manipulation of a precise number of viruses for host cell infection is essential for the extensive studies of virus pathogenesis and evolution. Albeit optical tweezers have been advanced to the atomic level via optical cooling, it is still challenging to efficiently trap and manipulate arbitrary number of viruses in an aqueous environment, being restricted by insufficient strength of optical forces and a lack of multifunctional spatial manipulation techniques. Here, by employing the virus hopping and flexibility of moving the laser position, multifunctional virus manipulation with a large trapping area is demonstrated, enabling single or massive (a large quantity of) virus transporting, positioning, patterning, sorting, and concentrating. The enhanced optical forces are produced by the confinement of light in engineered arrays of nanocavities by fine tuning of the interference resonances, and this approach allows trapping and moving viruses down to 40 nm in size. The work paves the way to efficient and precise manipulation of either single or massive groups of viruses, opening a wide range of novel opportunities for virus pathogenesis and inhibitor development at the single-virus level.

The chaperone heat shock protein 90 (Hsp90) is well known to undergo important conformational changes, which depend on nucleotide and substrate interactions. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally yet is central to its function. Here, using optical tweezers, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded, and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable clients and cooperates with Hsp70., BN/Sander Tans Lab

Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins. Upon binding to a specific sequence within centromeric regions, the DNA-binding protein CENP-B compacts centromeres by forming pronounced DNA loops between the repeats, which favor inter-chromosomal centromere compaction and clustering. This DNA-loop-mediated organization of centromeric chromatin participates in maintaining centromere position and integrity upon microtubule pulling during mitosis. Our findings emphasize the importance of DNA topology in centromeric regulation and stability.

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was-60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results. (c) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was-60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results. (c) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Nanomaterials are an essential part of modern life due to their extensive use in industrial and commercial products and future personalized medicine. In recent years, new nanoparticles (NPs) have been introduced in bioimaging, diagnostics, drug delivery, and therapy. Along with the production growth of NPs, concerns about NPs safety for human health have been raised. As blood is an inevitable target for NP-based pharmaceutics at systemic NP-based drug administration, the investigation of the poorly understood effects of NPs on blood properties is of high demand. The present thesis focuses on the assessment of commercial and novel synthesized NPs towards the haemorheologica l properties of the main blood cellular component — red blood cells (RBCs). In the research for this thesis, RBC morphology, deformability and mutual interactions were examined at non-haemolytic concentrations of NPs. A revolutionary optical tweezers technique revealed subtle effects of indirect toxicity towards mutual RBCs interactions and deformability. Further, additional conventional optical microscopy analysis revealed the influence of NPs on RBC interactions on a multicellular level, while scanning electron microscopy (SEM) enabled high-resolution monitoring of RBCs surfaces and morphological alterations triggered by NPs. Another aim of the study addressed the unclear mechanism behind RBC interactions. Experimental evidence of the definitive role of the size and proportion of macromolecules in RBC interactions was provided. The mixture of natural polymer dextran mimicking plasma protein composition induced an RBC interaction mode similar to one observed in blood plasma. Thus, the new hybrid model combining “cross-bridges” and “depletion” effects was proposed. The reported findings contribute to the fundamental understanding of RBC interactions and can help to facilitate the design and clinical validation of polymer-based plasma expanders and novel NPs for safe and beneficial use., Tiivistelmä Nanomateriaalit ovat olennainen osa nykyaikaista elämäntapaa, sillä niitä käytetään laajasti teollisissa ja kaupallisissa tuotteissa sekä tulevaisuuden yksilöllisessä lääketieteessä. Viime vuosina uusia nanopartikkeleita (NP) on otettu käyttöön biolääketieteellisessä kuvantamisessa ja diagnostiikassa sekä lääkkeiden antamisessa ja terapiassa. NP:iden tuotannon kasvun myötä on noussut esiin huoli niiden turvallisuudesta ihmisten terveydelle. Koska veri on väistämätön kohde NP-pohjaisille lääkkeille systeemisessä NP-pohjaisessa lääkkeen annossa, on nanopartikkelien vähän tutkittujen, veren ominaisuuksiin kohdistuvien vaikutusten selvittämiselle suuri tarve. Kyseinen opinnäytetyö keskittyy kaupallisten ja uusien syntetisoitujen nanaopartikkelien arviointiin verisolujen pääkomponentin eli punasolujen (RBC) hemorheologisten ominaisuuksien osalta. Tämän työn tutkimuksessa tarkasteltiin punasolujen morfologiaa, muodonmuutoksia ja keskinäisiä vuorovaikutuksia ei-hemolyyttisissä NP-pitoisuuksissa. Vallankumouksellinen optinen pinsettitekniikka paljasti epäsuoran myrkyllisyyden hienovaraiset vaikutukset punasolujen keskinäiseen vuorovaikutukseen ja muodonmuutokseen. Lisäksi tavanomainen optinen mikroskopia-analyysi paljasti NP:iden ja RBC:n vuorovaikutukset monisoluisella tasolla, kun taas pyyhkäisyelektronimikroskopia (SEM) mahdollisti punasolujen pintojen ja NP:iden laukaisemien morfologisten muutosten korkearesoluutioisen seurannan. Tutkimuksen toisena tavoitteena oli selvittää punasolujen vuorovaikutuksen taustalla olevaa epäselvää mekanismia. Työssä esitettiin kokeellisia todisteita makromolekyylien koon ja osuuden lopullisesta roolista punasolujen vuorovaikutuksessa. Plasmaproteiinikoostumusta jäljittelevän luonnollisen polymeerin dekstraanin seos indusoi punasolujen vuorovaikutusmuodon, joka on samanlainen kuin veriplasmassa havaittu. Siten työssä ehdotettiin uutta "ristisiltoja" ja "tyhjennystä” yhdistävää hybridimallia. Raportoidut löydökset auttavat ymmä

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was-60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results. (c) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Diseases caused by pathogenic agents such as bacteria and viruses result in devastating costs on personal and societal levels. However, it is not just the emergence of new diseases that is problematic. Antibiotic resistance among bacteria makes uncomplicated infections difficult and lethal. Resilient disease-causing spores spread in hospitals, the food industry, and water supplies requiring effective detection and disinfection methods. Further, we face complex neurological diseases where no effective treatment or diagnostic methods exist. Thus, we must increase our fundamental understanding of these diseases to develop effective diagnostic, detection, disinfection, and treatment methods. Classically, the methods used for detecting and studying the underlying mechanics of pathogenic agents work on a large scale, measuring the average macroscopic behavior and properties of these pathogens. However, just as with humans, the average behavior is not always representative of individual behavior. Therefore, it is also essential to investigate the characteristics of these pathogens on a single cell or particle level. This thesis develops and applies optical techniques to characterize pathogenic biomaterial on a single cell or particle level. At the heart of all these studies is our Optical Tweezers (OT) instrument. OT are a tool that allows us to reach into the microscopic world and interact with it. Finally, by combining OT with other experimental techniques, we can chemically characterize biomaterials and develop assays that mimic different biological settings. Using these tools, we investigate bacterial adhesion, disinfection, and detection of pathogenic spores and proteins. Hopefully, the insights of these studies can lessen the burden on society caused by diseases by helping others develop effective treatment, diagnostic, detection, and disinfection methods in the future.

Diseases caused by pathogenic agents such as bacteria and viruses result in devastating costs on personal and societal levels. However, it is not just the emergence of new diseases that is problematic. Antibiotic resistance among bacteria makes uncomplicated infections difficult and lethal. Resilient disease-causing spores spread in hospitals, the food industry, and water supplies requiring effective detection and disinfection methods. Further, we face complex neurological diseases where no effective treatment or diagnostic methods exist. Thus, we must increase our fundamental understanding of these diseases to develop effective diagnostic, detection, disinfection, and treatment methods. Classically, the methods used for detecting and studying the underlying mechanics of pathogenic agents work on a large scale, measuring the average macroscopic behavior and properties of these pathogens. However, just as with humans, the average behavior is not always representative of individual behavior. Therefore, it is also essential to investigate the characteristics of these pathogens on a single cell or particle level. This thesis develops and applies optical techniques to characterize pathogenic biomaterial on a single cell or particle level. At the heart of all these studies is our Optical Tweezers (OT) instrument. OT are a tool that allows us to reach into the microscopic world and interact with it. Finally, by combining OT with other experimental techniques, we can chemically characterize biomaterials and develop assays that mimic different biological settings. Using these tools, we investigate bacterial adhesion, disinfection, and detection of pathogenic spores and proteins. Hopefully, the insights of these studies can lessen the burden on society caused by diseases by helping others develop effective treatment, diagnostic, detection, and disinfection methods in the future.

Proper rheological properties of red blood cells (RBC) including flexibility and aggregability are essential for healthy blood microcirculation. Excessive RBC aggregation has been observed to be associated with many pathological conditions and is crucial in acute circulatory problems. Low-level laser radiation (LLLR) has been found to have positive effects on the rheology of human blood, however, the detailed mechanisms of blood photobiomodulation remains unclear. In this study, utilizing the single-cell technique optical tweezers (OT) and traditional light microscopy, the influence of photobiomodulation of human RBC was examined under different conditions of laser irradiation. The results revealed that high radiant exposure (over 170.5 J/cm² radiant fluence) caused enhanced RBC aggregation and cell shape transformation while the aggregation force between single RBC remained unchanged. LLLR with radiant fluence below 9.5 J/cm² by 450 nm wavelength improved the RBC deformability, weakened the strength of cell-cell interaction in the RBC disaggregation process, and showed rejuvenating effects on RBC suspended in a harsh cell environment.

This thesis reports on studies of the fundamental interaction dynamics of human red blood cells (RBC) by optical tweezers (OT) and the application of the OT-based RBC-investigation protocol in facilitating blood photobiomodulation research at a single-cell level. The motivation for the present study arises from the scientific and clinical significance of examining RBC interaction mechanisms as a model for studying general cell interaction in cytological science. Exploring the phenomenon and mechanism of the photobiomodulation of laser radiation on the rheological properties of RBC has a great potential in the field of laser blood therapy. Interaction dynamics and the role of intercellular interaction time and mutual contact in RBC aggregation and OT-induced disaggregation in autologous plasma are presented as new evidence, clarifying the RBC interaction mechanism. The rheological alterations of RBC induced by laser radiation with various irradiation conditions were thoroughly explored at a single-cell level for the first time to provide a better understanding of the underlying mechanism of photobiomodulation on blood. This study demonstrated the beneficial effects of low-level laser irradiation by a 450-nm wavelength with a radiant exposure below 9.5 J/cm2 of improving RBC deformability and preserving cell shape in a harsh environment. Additionally, irradiated RBC aggregates were easily destroyed by the external influence (i.e., optical force), which could be the flow shearing force in a blood vessel and geometrical resistance by vasculature for in vivo conditions. The current work is important for optimizing the technique of the OT-based RBC evaluation system. It will contribute to the development of effective approaches for improving blood cell viability and blood microcirculation based on the reported photobiomodulation effects., Tiivistelmä Tässä opinnäytetyössä raportoidaan ihmisen punasolujen (RBC) perustavanlaatuisen vuorovaikutusdynamiikan tutkimuksista optisilla pinseteillä (OT) ja OT-pohjaisen RBC-tutkimusprotokollan soveltamisesta veren fotobiomodulaatiotutkimuksen helpottamiseksi yksisolutasolla. Motivaatio nykyiselle tutkimukselle syntyy punasolujen vuorovaikutusmekanismien tutkimisen tieteellisestä ja kliinisestä merkityksestä mallina yleisen soluvuorovaikutuksen tutkimiseen sytologiassa. Kun lasersäteilyn fotobiomodulaation ilmiötä ja mekanismia tutkitaan punasolujen reologisissa ominaisuuksissa, siinä on paljon potentiaalia laserveriterapian alalla. Interaktiodynamiikka ja solunvälisen interaktioajan ja keskinäisen kontaktin rooli punasolujen aggregaatiossa ja OT:n aiheuttamassa hajoamisessa autologisessa plasmassa esitetään uutena todisteena punasolujen yhteisvaikutusmekanismia selventävänä näyttönä. Punasoluissa tapahtuneita, lasersäteilyn erilaisissa valaistusolosuhteissa aiheuttamia reologisia muutoksia tarkasteltiin nyt ensimmäistä kertaa perusteellisesti yksittäisten solujen tasolla, minkä seurauksena saatiin aiempaa parempi ymmärrys veren fotobiomodulaation taustalla olevasta mekanismista. Tutkimus havainnollisti matalaenergisen, teholtaan alle 9,5 J/cm2:n säteilyaltistuksen ja aallonpituudeltaan 450 nm:n lasersäteilyn hyödylliset vaikutukset punasolujen muodonmuutoksen parantamisessa ja solujen kyvyssä säilyttää muotonsa ankarissakin olosuhteissa. Lisäksi säteilytetyt RBC-aggregaatit tuhoutuivat helposti ulkoisen vaikutuksen vaikutuksesta (optinen voima), joka voi olla virtauksen leikkausvoima verisuonessa ja geometrinen vastus verisuonissa in vivo -olosuhteissa. Tutkimuksen saavutukset ovat merkittäviä optimoitaessa optiseen pinsettiin perustuvan punasolujen arviointijärjestelmän tekniikoita. Raportoitujen fotobiomodulaatio vaikutusten kautta työ edistää verisolujen elinkelpoisuuden ja veren mikrovirtauksen parantamiseen tähtäävien tehokkaiden lähestymistapojen kehittäm

Understanding complex interactions between a cell and its extracellular matrix (ECM) lies at the core of mechanobiology. For instance, stiffness of the ECM was previously shown to be highly heterogenous and deregulated in wound healing or during cancer progression. However, tools available for measuring and dynamically altering peri-cellular stiffness have been lacking and bulk measurements do not probe stiffness sensed by the cells. Thus, we have developed an optical tweezers active microrheology (AMR) system capable of multi-axes stiffness measurements in the peri-cellular region. In this thesis, multi-axes AMR system was used to investigate highly heterogenous and anisotropic stiffness landscapes established by dermal fibroblasts, human breast cancer and fibrosarcoma cells. Peri-cellular stiffness and anisotropy are shown to vary between the tested cell lines and with different treatments modifying cell behavior. Further, stiffness landscape and cell response to ECM vary with hydrogel source, concentration and fiber architecture of the local ECM. These studies underscore the need for peri-cellular and not bulk stiffness measurements in studies on cellular mechanotransduction. Additionally, the method of patterned crosslinking is used to alter ECM topography by inducing fiber alignment and peri-cellular stiffness landscape. Following localized crosslinking, human breast cancer cells undergo contact guidance and durotaxis, as indicated by cell migration in a direction of fiber alignment and along an off-axis stiffness gradient, respectively. While these phenomena are widely known and studied on 2D substrates or in 3D synthetic hydrogels, patterned crosslinking allows for better understanding of processes governing directed migration of individual cells embedded inside naturally-derived fibrous hydrogels. Collectively, this thesis work investigates how cells respond to and regulate their local ECM stiffness based on a variety of different factors. Local ECM stiffnes

How biological fibrous materials respond to forces and changes at the cellular scale is important for understanding what cells `feel' from their extracellular environment. The local mechanical property within a fibrin fiber network is dependent on the local structure of the network. Elastic force transmission through hydrogel depends on the arrangement of the fiber. A novel method for visualizing force transmission through this network is measuring the pixel fluctuation of individual fibers in the presence of an actively applied oscillation using embedded microbeads and optical tweezers. Optical tweezers can also be used for measuring local stiffness through active microrheology. After micropatterned crosslinking, fibrin hydrogels were shown to produce local stiffening directly and indirectly through strain hardening. This stiffening resulted in stiffness anisotropy and fiber alignment which MDA-231 cells were shown to respond to. Finally, a model of elastically connected nodes recapitulates some of the experimental observations such as fiber force transmission, strain hardening, and stiffness anisotropy when strained. Manipulating, measuring, and modelling of local stiffness changes in response to applied forces offers further insight into how cells sense their surroundings.

Cells sense and respond to mechanical stimuli for activation, proliferation, migration, and differentiation. The associated mechanosensing and biomechanical properties of cells and tissues are significantly implicated in the context of cancer, fibrosis, dementia, and cardiovascular diseases. To gain more mechanobiology insights, dynamic force spectroscopies (DFSs), particularly optical tweezers (OT), have been further advanced to enable in situ force measurement and subcellular manipulation from the outer cell membrane to the organelles inside of a cell. In this review, we first explain the classic OT-DFS rationales and discuss their applications to protein biophysics, extracellular biomechanics, and receptor-mediated cell mechanosensing. As a non-invasive technique, optical tweezers’s unique advantages in probing cytoplasmic protein behaviors and manipulating organelles inside living cells have been increasingly explored in recent years. Hereby, we then introduce and highlight the emerging OT rationales for intracellular force measurement including refractive index matching, active–passive calibration, and change of light momentum. These new approaches enable intracellular OT-DFS and mechanical measurements with respect to intracellular motor stepping, cytosolic micro-rheology, and biomechanics of irregularly shaped nuclei and vesicles. Last but not least, we foresee future OT upgrades with respect to overcoming phototoxicity and system drifting for longer duration live-cell measurements; multimodal integration with advanced imaging and nanotechnology to obtain higher spatiotemporal resolution; and developing simultaneous, automated, and artificial intelligence–inspired multi-trap systems to achieve high throughput. These further developments will grant unprecedented accessibility of OT-DFS and force measurement nanotools to a wider biomedical research community, ultimately opening the floodgates for exciting live-cell mechanobiology and novel therapeutic discoveri

Spatial manipulation of a precise number of viruses for host cell infection is essential for the extensive studies of virus pathogenesis and evolution. Albeit optical tweezers have been advanced to the atomic level via optical cooling, it is still challenging to efficiently trap and manipulate arbitrary number of viruses in an aqueous environment, being restricted by insufficient strength of optical forces and a lack of multifunctional spatial manipulation techniques. Here, by employing the virus hopping and flexibility of moving the laser position, multifunctional virus manipulation with a large trapping area is demonstrated, enabling single or massive (a large quantity of) virus transporting, positioning, patterning, sorting, and concentrating. The enhanced optical forces are produced by the confinement of light in engineered arrays of nanocavities by fine tuning of the interference resonances, and this approach allows trapping and moving viruses down to 40 nm in size. The work paves the way to efficient and precise manipulation of either single or massive groups of viruses, opening a wide range of novel opportunities for virus pathogenesis and inhibitor development at the single-virus level.

Diseases caused by pathogenic agents such as bacteria and viruses result in devastating costs on personal and societal levels. However, it is not just the emergence of new diseases that is problematic. Antibiotic resistance among bacteria makes uncomplicated infections difficult and lethal. Resilient disease-causing spores spread in hospitals, the food industry, and water supplies requiring effective detection and disinfection methods. Further, we face complex neurological diseases where no effective treatment or diagnostic methods exist. Thus, we must increase our fundamental understanding of these diseases to develop effective diagnostic, detection, disinfection, and treatment methods. Classically, the methods used for detecting and studying the underlying mechanics of pathogenic agents work on a large scale, measuring the average macroscopic behavior and properties of these pathogens. However, just as with humans, the average behavior is not always representative of individual behavior. Therefore, it is also essential to investigate the characteristics of these pathogens on a single cell or particle level. This thesis develops and applies optical techniques to characterize pathogenic biomaterial on a single cell or particle level. At the heart of all these studies is our Optical Tweezers (OT) instrument. OT are a tool that allows us to reach into the microscopic world and interact with it. Finally, by combining OT with other experimental techniques, we can chemically characterize biomaterials and develop assays that mimic different biological settings. Using these tools, we investigate bacterial adhesion, disinfection, and detection of pathogenic spores and proteins. Hopefully, the insights of these studies can lessen the burden on society caused by diseases by helping others develop effective treatment, diagnostic, detection, and disinfection methods in the future.

Diseases caused by pathogenic agents such as bacteria and viruses result in devastating costs on personal and societal levels. However, it is not just the emergence of new diseases that is problematic. Antibiotic resistance among bacteria makes uncomplicated infections difficult and lethal. Resilient disease-causing spores spread in hospitals, the food industry, and water supplies requiring effective detection and disinfection methods. Further, we face complex neurological diseases where no effective treatment or diagnostic methods exist. Thus, we must increase our fundamental understanding of these diseases to develop effective diagnostic, detection, disinfection, and treatment methods. Classically, the methods used for detecting and studying the underlying mechanics of pathogenic agents work on a large scale, measuring the average macroscopic behavior and properties of these pathogens. However, just as with humans, the average behavior is not always representative of individual behavior. Therefore, it is also essential to investigate the characteristics of these pathogens on a single cell or particle level. This thesis develops and applies optical techniques to characterize pathogenic biomaterial on a single cell or particle level. At the heart of all these studies is our Optical Tweezers (OT) instrument. OT are a tool that allows us to reach into the microscopic world and interact with it. Finally, by combining OT with other experimental techniques, we can chemically characterize biomaterials and develop assays that mimic different biological settings. Using these tools, we investigate bacterial adhesion, disinfection, and detection of pathogenic spores and proteins. Hopefully, the insights of these studies can lessen the burden on society caused by diseases by helping others develop effective treatment, diagnostic, detection, and disinfection methods in the future.

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was ∼60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results.

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was ∼60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results.

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was ∼60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results.

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was ∼60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results.

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was ∼60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results.

Ultrasensitive, versatile sensors for molecular biomarkers are a critical component of disease diagnostics and personalized medicine as the COVID-19 pandemic has revealed in dramatic fashion. Integrated electrical nanopore sensors can fill this need via label-free, direct detection of individual biomolecules, but a fully functional device for clinical sample analysis has yet to be developed. Here, we report amplification-free detection of SARS-CoV-2 RNAs with single molecule sensitivity from clinical nasopharyngeal swab samples on an electro-optofluidic chip. The device relies on optically assisted delivery of target carrying microbeads to the nanopore for single RNA detection after release. A sensing rate enhancement of over 2,000x with favorable scaling towards lower concentrations is demonstrated. The combination of target specificity, chip-scale integration and rapid detection ensures the practicality of this approach for COVID-19 diagnosis over the entire clinically relevant concentration range from 104-109 copies/mL.

We propose an experiment to demonstrate spontaneous ordering and symmetry breaking of kinesin-driven microtubules confined to an optical trap. Calculations involving the feasibility of such an experiment are first performed which analyze the power needed to confine microtubules and address heating concerns. We then present the results of first-principles simulations of active microtubules confined in such a trap and analyze the types of motion observed by the microtubules as well as the velocity of the surrounding fluid, both near the trap and in the far-field. We find three distinct phases characterized by breaking of distinct symmetries and also analyze the power spectrum of the angular momenta of polymers to further quantify the differences between these phases. Under the correct conditions, microtubules were found to spontaneously align with one another and circle the trap in one direction.

Vital biological processes, such as trafficking, sensing, and motility, are facilitated by cellular lipid membranes, which interact mechanically with surrounding fluids. Such lipid membranes are only a few nanometers thick and composed of a liquid crystalline structure known as the lipid bilayer. Here, we introduce an active, noncontact, two-point microrheology technique combining multiple optical tweezers probes with planar freestanding lipid bilayers accessible on both sides. We use the method to quantify both fluid slip close to the bilayer surface and transmission of fluid flow across the structure, and we use numerical simulations to determine the monolayer viscosity and the intermonolayer friction. We find that these physical properties are highly dependent on the molecular structure of the lipids in the bilayer. We compare ordered-phase with liquid disordered-phase lipid bilayers, and we find the ordered-phase bilayers to be 10 to 100 times more viscous but with 100 times less intermonolayer friction. When a local shear is applied by the optical tweezers, the ultralow intermonolayer friction results in full slip of the two leaflets relative to each other and as a consequence, no shear transmission across the membrane. Our study sheds light on the physical principles governing the transfer of shear forces by and through lipid membranes, which underpin cell behavior and homeostasis., BN/Marie-Eve Aubin-Tam Lab, Fluid Mechanics

The aim of this thesis was to explore the utility of different biophysical techniques, particularly optical tweezers (OT), in the investigation of the mechanical properties and interactions of biological samples. Specifically, MCF7 cells and their extracted nuclei were investigated mechanically, while the adhesion property of selected bacteria to the milk fat globule was also used as an exemplar. Biological cells have the ability to actively respond to external mechanical forces exerted by the microenvironment. The cellular response can be viscous, elastic, or viscoelastic in nature depending on the nature of the applied forces and the mechanical stresses applied. Changes in the mechanical properties of cells and nuclei have emerged as a prominent hallmark of many human diseases, particularly in neurodegenerative and metastatic diseases. In this thesis, to understand the application of these techniques to biological systems better, bulk rheology and microrheology studies were first performed on a model viscoelatic fluid (PEO). Particularly, the passive and active microrheology of this model viscoelastic material was characterized using optical tweezers and video particle tracking to develop the prerequisite experimental and analytical methods. Using the experimental knowledge gained from applying optical tweezers to standard materials, a mechanistic approach was developed in order to better understand how the mechanical properties of MCF7 cells change when the amount of heterochromatin protein (HP1a) present inside the nuclei was reduced. (HP1a) is an architectural protein that establishes and maintains heterochromatin, ensuring genome fidelity and nuclear integrity. While the mechanical effects of changes in the relative amount of euchromatin and heterochromatin brought about by inhibiting chromatin modifying enzymes have been studied previously, here we measure how the material properties of the cells are modified following the knockdown HP1a. Indentation experime