Combination Of Fluorescence Imaging Research Articles

A versatile bioluminescent probe with tunable color.

Bioluminescence is a powerful method for imaging in vivo, but applications at the microscale are far from routine. This is due, in part, to a lack of versatile tools for visualizing dynamic events. To address this void, we developed a new platform-Bioluminescence Resonance Energy mAKe over with a Fluorescence-Activating absorption-Shifting Tag (BREAKFAST). BREAKFAST features a bright luciferase combined with a chemogenetic tag (pFAST) for rapid color switching. In the presence of luciferin and a discrete fluorogenic ligand, signal is observed via resonance energy transfer. We evaluated spectral outputs with various fluorogens and established the utility of BREAKFAST for combined fluorescence and bioluminescence imaging. Dynamic, four-color visualization was achieved with sequential ligand addition and spectral phasor analysis. We further showed selective signal quenching with a dark fluorogen. Collectively, this work establishes a new method for bioluminescence imaging at the cellular scale and sets the stage for continued probe development.

The Usefulness of Fluorescence Imaging in Laparoscopic Liver Resection

AbstractIndocyanine green (ICG) has been used extensively in medical applications after being approved by the Food and Drug Administration (FDA) in 1954. In the beginning, this substance was originally employed for evaluating cardiac output and quantifying liver function. The widespread acceptance of the technique was delayed until the early 2000s, when advancements in digital imaging resolution provided more satisfactory images compared with those of film-based photography. ICG-based fluorescence imaging has since been used in various aspects of liver surgery. In 2008, Aoki et al first reported an intraoperative technique for subsegmental and segmental fluorescent staining during open hepatectomy, achieving a success rate of 93% in identifying the anatomical plane. At about the same time, laparoscopic liver resections (LLRs) were increasingly used for liver malignancies but were still in a developmental phase with yet to be standardized surgical protocols. Combined ICG fluorescence imaging in LLR has since been developed to improve the quality and precision of anatomical liver resections. This article is a case presentation and review of the clinical applications of ICG fluorescence imaging in liver surgery.

Distribution of xylan linked glucuronic acid labelled by molecularly imprinted polymers on pulp fiber surface

Efficiently utilization of plant resources is heavily restricted by the resistance of lignocellulose in plant cells, which is related to the interlinkages of lignocellulose components. Hemicellulose in plant cell wall is bound to cellulose by hydrogen bond and linked with lignin in lignin-carbohydrate complex (LCC). In the xylan chain of hemicellulose, glucuronic acid (GA) is a typical side-group, which provides clues for us to label and locate hemicellulose. The way to label GA on the surface of pulp fibers obtained from pulping process is benefit to explore the deconstruction of lignocellulose. Herein, a new visualization method, fluorescence modified molecularly imprinted polymers (MIP) were applied to recognize and locate GA on the pulp fiber surface. The method combining fluorescence imaging and integrated 3D fiber structure verified the feasibility of the MIP for specific GA recognition. The results showed that xylan (represented by GA) was closely attached to lignin, distributed along the inner wall of pulp fiber cells, and gradually taken off from the inside edge of fiber cells with the deconstruction of lignocellulose. This research provided a basis to develop visualization bioimaging technology to identify biomass components.

Development of optical probes with excellent intracellular retention

Small-molecule based activatable fluorescence probes for detecting specific enzyme activity with high sensitivity can visualize the expression site of marker genes and cancers where the enzyme is highly expressed. However, the enzyme-catalyzed fluorescent hydrolysis product easily leaks out and diffuses from the reaction site, making it difficult to perform long-term tracking and immunohistochemical analysis which needs washing/fixation procedure. Our group have focused on quinone methide chemistry and developed series of activatable fluorescence probes with excellent intracellular retention that are converted to quinone-methide or aza-quinone-methide intermediates upon reaction with enzymes, which are then react with intracellular nucleophiles such as proteins and glutathione to be retained in cells and to exhibit significant increase in fluorescence. Based on this molecular design, we have developed fluorescence probes targeting β-galactosidase and γ-glutamyltranspeptidase with different colors. We also developed photo-functional probes such as activatable photosensitizers and caged fluorophores. These probes can visualize or kill target enzyme-expressing cells with high selectivity by suppressing the leakage of hydrolysis products from target cells, and fluorescence imaging in combination with immunostaining was possible due to the high tolerance of the obtained fluorescence signal even after washing and fixation.

Dual imaging technique for a real-time inspection system of foreign object detection in fresh-cut vegetables

Fresh-cut vegetables are a food product susceptible to contamination by foreign materials (FMs). To detect a range of potential FMs in fresh-cut vegetables, a dual imaging technique (fluorescence and color imaging) with a simple and effective image processing algorithm in a user-friendly software interface was developed for a real-time inspection system. The inspection system consisted of feeding and sensing units, including two cameras positioned in parallel, illuminations (white LED and UV light), and a conveyor unit. A camera equipped with a long-pass filter was used to collect fluorescence images. Another camera collected color images of fresh-cut vegetables and FMs. The feeding unit fed FMs mixed with fresh-cut vegetables onto a conveyor belt. Two cameras synchronized programmatically in the software interface simultaneously collected fluorescence and color image samples based on the region of interest as they moved through the conveyor belt. Using simple image processing algorithms, FMs could be detected and depicted in two different image windows. The results demonstrated that the dual imaging technique can effectively detect potential FMs in two types of fresh-cut vegetables (cabbage and green onion), as indicated by the combined fluorescence and color imaging accuracy. The test results showed that the real-time inspection system could detect FMs measuring 0.5 mm in fresh-cut vegetables. The results showed that the combined detection accuracy of FMs in the cabbage (95.77%) sample was superior to that of green onion samples (87.89%). Therefore, the inspection system was more effective at detecting FMs in cabbage samples than in green onion samples.

Multimodal Imaging-Guided Strategy for Developing 177Lu-Labeled Metal-Organic Framework Nanomedicine with Potential in Cancer Therapy.

Nano-metal-organic frameworks (nano-MOFs) labeled with radionuclides have shown great potential in the anticancer field. In this work, we proposed to combine fluorescence imaging (FI) with nuclear imaging to systematically evaluate the tumor inhibition of new nanomedicines from living cancer cells to the whole body, guiding the design and application of a high-performance anticancer radiopharmaceutical to glioma. An Fe-based nano-MOF vector, MIL-101(Fe)/PEG-FA, was decorated with fluorescent sulfo-cyanine7 (Cy7) to investigate the binding affinity of the targeting nanocarriers toward glioma cells in vitro, as well as possible administration modes for in vivo cancer therapy. Then, lutetium-177 (177Lu)-labeled MIL-101(Fe)/PEG-FA was prepared for high-sensitive imaging and targeted radiotherapy of glioma in vivo. It has been demonstrated that the obtained 177Lu-labeled MIL-101(Fe)/PEG-FA can work as a complementary probe to rectify the cancer binding affinity of the prepared nanocarrier given by fluorescence imaging, providing more precise biodistribution information. Besides, 177Lu-labeled MIL-101(Fe)/PEG-FA has excellent antitumor effect, leading to cell proliferation inhibition, upregulation of intracellular reactive oxygen species, tumor growth suppression, and immune response-related protein and cytokine upregulation. This work reveals that optical imaging and nuclear imaging can work complementarily as multimodal imaging in the design and evaluation of anticancer nanomedicine, offering a MIL-101(Fe)/PEG-FA-based pharmaceutical with potential in tumor endoradiotherapy.

Design of Water-Soluble Rotaxane-Capped Superparamagnetic, Ultrasmall Fe3O4 Nanoparticles for Targeted NIR Fluorescence Imaging in Combination with Magnetic Resonance Imaging.

Integrating an NIR fluorescent probe with a magnetic resonance imaging (MRI) agent to harvest complementary imaging information is challenging. Here, we have designed water-soluble, biocompatible, noncytotoxic, bright-NIR-emitting, sugar-functionalized, mechanically interlocked molecules (MIMs)-capped superparamagnetic ultrasmall Fe3O4 NPs for targeted multimodal imaging. Dual-functional stoppers containing an unsymmetrical NIR squaraine dye interlocked within a macrocycle to construct multifunctional MIMs are developed with enhanced NIR fluorescence efficiency and durability. One of the stoppers of the axle is composed of a lipophilic cationic TPP+ functionality to target mitochondria, and the other stopper comprises a dopamine-containing catechol group to anchor at the surface of the synthesized Fe3O4 NPs. Fe3O4 NPs surface-coated with targeted NIR rotaxanes help to deliver ultrasmall magnetic NPs specifically inside the mitochondria. Two carbohydrate moieties are conjugated with the macrocycle of the rotaxane via click chemistry to improve the water solubility of MitoSQRot-(Carb-OH)2-DOPA-Fe3O4 NPs. Water-soluble, rotaxane-capped Fe3O4 NPs are used for live-cell mitochondria-targeted NIR fluorescence confocal imaging, 3D and multicolor imaging in combination with T2-weighted MRI on a 9.4 T MR scanner with a high relaxation rate (r2) of 180.7 mM-1 s-1. Biocompatible, noncytotoxic, ultrabright NIR rotaxane-capped superparamagnetic ultrasmall monodisperse Fe3O4 NPs could be a promising agent for targeted multimodal imaging applications.

Multifunctional Nanotheranostics for Dual-Modal Imaging-Guided Precision Therapy of Nasopharyngeal Carcinoma.

Currently, the low survival rate and poor prognosis of patients with nasopharyngeal carcinoma are ascribed to the lack of early and accurate diagnosis and resistance to radiotherapy. In parallel, the integration of imaging-guided diagnosis and precise treatment has gained much attention in the field of theranostic nanotechnology. However, constructing dual-modal imaging-guided nanotheranostics with desired imaging performance as well as great biocompatibility remains challenging. Therefore, we developed a simple but multifunctional nanotheranostic GdCPP for the early and accurate diagnosis and efficient treatment of nasopharyngeal carcinoma (NPC), which combined fluorescence imaging and magnetic resonance imaging (MRI) onto a single nanoplatform for imaging-guided subsequent photodynamic therapy (PDT). GdCPP had an appropriate particle size (81.93 ± 0.69 nm) and was highly stable, resulting in sufficient tumor accumulation, which along with massive reactive oxygen species (ROS) generation upon irradiation further significantly killed tumor cells. Moreover, GdCPP owned much stronger r1 relaxivity (9.396 mM-1 s-1) compared to clinically used Gd-DTPA (5.034 mM-1 s-1) and exhibited better T1WI MRI performance. Under dual-modal imaging-guided PDT, GdCPP achieved efficient therapeutic outcomes without causing any noticeable tissue damage. The results of in vitro and in vivo studies indicated that GdCPP may be a suitable candidate for dual-modal imaging-guided precision tumor therapy.

Force generation in human blood platelets by filamentous actomyosin structures

Blood platelets are central elements of the blood clotting response after wounding. Upon vessel damage, they bind to the surrounding matrix and contract the forming thrombus, thus helping to restore normal blood circulation. The hemostatic function of platelets is directly connected to their mechanics and cytoskeletal organization. The reorganization of the platelet cytoskeleton during spreading occurs within minutes and leads to the formation of contractile actomyosin bundles, but it is not known if there is a direct correlation between the emerging actin structures and the force field that is exerted to the environment. In this study, we combine fluorescence imaging of the actin structures with simultaneous traction force measurements in a time-resolved manner. In addition, we image the final states with superresolution microscopy. We find that both the force fields and the cell shapes have clear geometrical patterns defined by stress fibers. Force generation is localized in a few hotspots, which appear early during spreading, and, in the mature state, anchor stress fibers in focal adhesions. Moreover, we show that, for a gel stiffness in the physiological range, force generation is a very robust mechanism and we observe no systematic dependence on the amount of added thrombin in solution or fibrinogen coverage on the substrate, suggesting that force generation after platelet activation is a threshold phenomenon that ensures reliable thrombus contraction in diverse environments.

Multiclass classifier based on deep learning for detection of citrus disease using fluorescence imaging spectroscopy

In this work, we have combined fluorescence imaging spectroscopy (FIS) and supervised learning methods to identify and discriminate between citrus canker, Huanglongbing, and other leaf symptoms. Our goal is to differentiate these diseases and nutrient conditions without prior eye assessment of symptoms. Five supervised learning methods were evaluated. Our results show that by combining FIS with a convolutional neural network (AlexNet), it is possible to identify the disease of a sample with up to 95% accuracy. An enormous gain of time and a substantial cost reduction were achieved by this approach compared to polymerase chain reaction-based methods.

Tet-Regulated Expression and Optical Clearing for In Vivo Visualization of Genetically Encoded Chimeric dCas9/Fluorescent Protein Probes.

The catalytically inactive mutant of Cas9 (dCas9) endonuclease has multiple biomedical applications, with the most useful being the activation/repression of transcription. dCas9 family members are also emerging as potential experimental tools for gene mapping at the level of individual live cells and intact tissue. We performed initial testing on a set of tools for Cas9-mediated visualization of nuclear compartments. We investigated doxycycline (Dox)-inducible (Tet-On) intracellular distribution of constructs encoding dCas9 orthologs from St. thermophilus (St) and N. meningitides (Nm) fused with EGFP and mCherry fluorescent proteins (FP) in human A549 cells. We also studied time-dependent expression of these chimeric fluorescent constructs (dCas9-FP) after Tet-On induction in live cells and compared it with the time course of dCas9-FP expression in experimental dCas9-FP-expressing tumor xenografts using a combination of fluorescence imaging and in vivo contrast-assisted magnetic resonance imaging for assessing the extent of tumor perfusion. In vivo Dox-induction of mCherry-chimera expression occurred in tumor xenografts as early as 24 h post-induction and was visualized by using optical clearing (OC) of the skin. OC via topical application of gadobutrol enabled high-contrast imaging of FP expression in tumor xenografts due to a 1.1-1.2-fold increase in FI in both the red and green channels.

Fluorescence guidance improves the accuracy of radiological imaging-guided surgical navigation.

Imaging-based navigation technologies require static referencing between the target anatomy and the optical sensors. Imaging-based navigation is therefore well suited to operations involving bony anatomy; however, these technologies have not translated to soft-tissue surgery. We sought to determine if fluorescence imaging complement conventional, radiological imaging-based navigation to guide the dissection of soft-tissue phantom tumors. Using a human tissue-simulating model, we created tumor phantoms with physiologically accurate optical density and contrast concentrations. Phantoms were dissected using all possible combinations of computed tomography (CT), magnetic resonance, and fluorescence imaging; controls were included. The data were margin accuracy, margin status, tumor spatial alignment, and dissection duration. Margin accuracy was higher for combined navigation modalities compared to individual navigation modalities, and accuracy was highest with combined CT and fluorescence navigation (p = 0.045). Margin status improved with combined CT and fluorescence imaging. At present, imaging-based navigation has limited application in guiding soft-tissue tumor operations due to its inability to compensate for positional changes during surgery. This study indicates that fluorescence guidance enhances the accuracy of imaging-based navigation and may be best viewed as a synergistic technology, rather than a competing one.

LSSmScarlet2 and LSSmScarlet3, Chemically Stable Genetically Encoded Red Fluorescent Proteins with a Large Stokes' Shift.

Red fluorescent proteins with a large Stokes’ shift (LSSRFPs) are genetically encoded and efficiently excited by 488 nm light, allowing simultaneous dual-color one- and two-photon fluorescence imaging and fluorescence correlation spectroscopy in combination with green fluorescent proteins FPs. Recently, based on the conventional bright mScarlet RFP, we developed the LSSRFP LSSmScarlet. LSSmScarlet is characterized by two pKa values at pH values of 1.9 and 5.8. In this study, we developed improved versions of LSSmScarlet, named LSSmScarlet2 and LSSmScarlet3, which are characterized by a Stokes’ shift of 128 nm and extreme pH stability with a single pKa value of 2.2. LSSmScarlet2 and LSSmScarlet3 had 1.8-fold faster and 3-fold slower maturation than LSSmScarlet, respectively. In addition, both LSSRFPs were 1.5- to 1.6-fold more photostable and more chemically resistant to denaturation by guanidinium chloride and guanidinium thiocyanate. We also compared the susceptibility of the LSSmScarlet2, LSSmScarlet3, and other LSSRFPs to the reagents used for whole-mount imaging, expansion microscopy, and immunostaining techniques. Due to higher pH stability and faster maturation, the LSSmScarlet3-LAMP3 fusion was 2.2-fold brighter than LSSmScarlet-LAMP3 in lysosomes of mammalian cells. The LSSmScarlet3-hLAMP2A fusion was similar in brightness to LSSmScarlet-hLAMP2A in lysosomes. We successfully applied the monomeric LSSmScarlet2 and LSSmScarlet3 proteins for confocal imaging of structural proteins in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet2 protein at a resolution of 1.41 Å. Site-directed mutagenesis of the LSSmScarlet2 protein demonstrated the key role of the T74 residue in improving the pH and chemical stability of the LSSmScarlet2 protein.

A Small-Molecule Based Organic Nanoparticle for Photothermal Therapy and Near-Infrared-IIb Imaging.

Near-infrared window IIb (NIR-IIb, 1500-1700 nm) fluorescence imaging demonstrates attractive properties including low scattering, low absorption, and deep tissue penetration, and photothermal therapy (PTT) is also a promising modality for cancer treatment. However, until now, there is no report on theranostic systems based on small organic molecules combining fluorescence imaging in the NIR-IIb and PTT, highlighting the challenge and strong need for development of such agents. Herein, we report a novel small molecule NIR-IIb dye IT-TQF with a D-A-D structure, which exhibited high fluorescence intensity in the NIR-IIb window. To further translate IT-TQF into an effective theranostic agent, IT-TQF was encapsulated into DSPE-PEG2000 to construct IT-TQF NPs. The physical and photochemical properties of the nanoprobe were investigated in vitro, and the in vivo NIR-IIb imaging and PTT performance were evaluated in normal, subcutaneous, orthotopic, and metastatic tumor mice models. IT-TQF NP-based NIR-IIb imaging demonstrated high spatial resolution and high tissue penetration depth, and small normal blood vessels (55.3 μm) were successfully imaged in the NIR-IIb window. Subcutaneous, orthotopic, and metastatic tumors were all clearly delineated. A high tumor signal-to-background ratio (SBR) of 9.42 was achieved for orthotopic osteosarcoma models, and the erosions of bone tissue caused by tumor cells were precisely visualized. Moreover, NIR-II image-guided surgery was successfully performed to completely remove the orthotopic tumor. Importantly, IT-TQF NPs displayed high PTT efficacy (photothermal conversion efficiency: 47%) for effective treatment of tumor mice. In conclusion, IT-TQF NPs are a novel and promising phototheranostic agent in the NIR-IIb window, and the nanoprobe has high potential for a broad range of biomedical applications.

Evaluating spatial and network properties of NMDA-dependent neuronal connectivity in mixed cortical cultures

A technique combining fluorescence imaging with Ca2+ indicators and single-cell laser scanning photostimulation of caged glutamate (LSPS) allowed identification of functional connections between individual neurons in mixed cultures of rat neocortical cells as well as observation of synchronous spontaneous activity among neurons. LSPS performed on large numbers of neurons yielded maps of functional connections between neurons and allowed calculation of neuronal network parameters. LSPS also provided an indirect measure of excitability of neurons targeted for photostimulation. By repeating LSPS sessions with the same neurons, stability of connections and change in the number and strength of connections were also determined. Experiments were conducted in the presence of bicuculline to study in detail the properties of excitatory neurotransmission. The AMPA receptor inhibitor, 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), abolished synchronous neuronal activity but had no effect on connections mapped by LSPS. In contrast, the NMDA receptor inhibitor, 2-Amino-5-phosphono-pentanoic acid (APV), dramatically decreased the number of functional connections between neurons while also affecting synchronous spontaneous activity. Functional connections were also decreased by increasing extracellular Mg2+ concentration. These data demonstrated that LSPS mapping interrogates NMDA receptor-dependent connectivity between neurons in the network. In addition, a GluN2A-specific inhibitor, NVP-AAM077, decreased the number and strength of connections between neurons as well as neuron excitability. Conversely, the GluN2A-specific positive modulator, GNE-0723, increased these same properties. These data showed that LSPS can be used to directly study perturbations in the properties of NMDA receptor-dependent connectivity in neuronal networks. This approach should be applicable in a wide variety of in vitro and in vivo experimental preparations.

Saturated-excitation image scanning microscopy.

Image scanning microscopy (ISM) overcomes the trade-off between spatial resolution and signal volume in confocal microscopy by rearranging the signal distribution on a two-dimensional detector array to achieve a spatial resolution close to the theoretical limit achievable by infinitesimal pinhole detection without sacrificing the detected signal intensity. In this paper, we improved the spatial resolution of ISM in three dimensions by exploiting saturated excitation (SAX) of fluorescence. We theoretically investigated the imaging properties of ISM, when the fluorescence signals are nonlinearly induced by SAX, and show combined SAX-ISM fluorescence imaging to demonstrate the improvement of the spatial resolution in three dimensions. In addition, we confirmed that the SNR of SAX-ISM imaging of fluorescent beads and biological samples, which is one of the challenges in conventional SAX microscopy, was improved.

Combined TPEF and SHG Imaging for the Microstructural Characterization of Different Wood Species Used in Artworks

The morphological and chemical conformation of wood microstructures is characteristic of individual species and strongly influences the macromechanical properties of the material, as well as its sensitivity to deterioration factors. Noninvasive techniques enabling the visualization of wood microstructures, while simultaneously providing compositional information, can significantly facilitate the analysis of wooden artworks for conservation purposes. In this paper, we present the application of combined two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) imaging as a versatile diagnostic tool for the microcharacterization of three hardwood species never analyzed by this method. Multimodal mapping of the molecular constituents based on the detected nonlinear signals provides useful information for studying the biological and biochemical deterioration of wood, opening a new field of application for a well-established and widely used imaging technology.

Cryptochrome proteins regulate the circadian intracellular behavior and localization of PER2 in mouse suprachiasmatic nucleus neurons

The ∼20,000 cells of the suprachiasmatic nucleus (SCN), the master circadian clock of the mammalian brain, coordinate subordinate cellular clocks across the organism, driving adaptive daily rhythms of physiology and behavior. The canonical model for SCN timekeeping pivots around transcriptional/translational feedback loops (TTFL) whereby PERIOD (PER) and CRYPTOCHROME (CRY) clock proteins associate and translocate to the nucleus to inhibit their own expression. The fundamental individual and interactive behaviors of PER and CRY in the SCN cellular environment and the mechanisms that regulate them are poorly understood. We therefore used confocal imaging to explore the behavior of endogenous PER2 in the SCN of PER2::Venus reporter mice, transduced with viral vectors expressing various forms of CRY1 and CRY2. In contrast to nuclear localization in wild-type SCN, in the absence of CRY proteins, PER2 was predominantly cytoplasmic and more mobile, as measured by fluorescence recovery after photobleaching. Virally expressed CRY1 or CRY2 relocalized PER2 to the nucleus, initiated SCN circadian rhythms, and determined their period. We used translational switching to control CRY1 cellular abundance and found that low levels of CRY1 resulted in minimal relocalization of PER2, but yet, remarkably, were sufficient to initiate and maintain circadian rhythmicity. Importantly, the C-terminal tail was necessary for CRY1 to localize PER2 to the nucleus and to initiate SCN rhythms. In CRY1-null SCN, CRY1Δtail opposed PER2 nuclear localization and correspondingly shortened SCN period. Through manipulation of CRY proteins, we have obtained insights into the spatiotemporal behaviors of PER and CRY sitting at the heart of the TTFL molecular mechanism.

Bi-modal near-infrared fluorescence and ultrasound imaging via a transparent ultrasound transducer for sentinel lymph node localization.

Sentinel lymph node biopsy with an indocyanine green-based near-infrared fluorescence imaging system avoids the shortcomings of using a radioisotope or a combination of a blue dye and a radioactive tracer. To improve surgical precision, recent research has provided a depth profile of the sentinel lymph node by fusing fluorescence and ultrasound imaging. Here, we present a combined near-infrared fluorescence and ultrasound imaging system based on a transparent ultrasound transducer. The transparent ultrasound transducer enables seamless coaxial alignment of the fluorescence and ultrasound beam paths, allowing bi-modal observation of a single region of interest. Further, we demonstrate that the sentinel lymph node of mice injected with indocyanine green can be successfully localized and dissected based on information from the bi-modal imaging system.

Generating Negatively Supercoiled DNA Using Dual-Trap Optical Tweezers.

Many genomic processes lead to the formation of underwound (negatively supercoiled) or overwound (positively supercoiled) DNA. These DNA topological changes regulate the interactions of DNA-binding proteins, including transcription factors, architectural proteins and topoisomerases. In order to advance our understanding of the structure and interactions of supercoiled DNA, we recently developed a single-molecule approach called Optical DNA Supercoiling (ODS). This method enables rapid generation of negatively supercoiled DNA (with between <5% and 70% lower helical twist than nonsupercoiled DNA) using a standard dual-trap optical tweezers instrument. ODS is advantageous as it allows for combined force spectroscopy, fluorescence imaging, and spatial control of the supercoiled substrate, which is difficult to achieve with most other approaches. Here, we describe how to generate negatively supercoiled DNA using dual-trap optical tweezers. To this end, we provide detailed instructions on the design and preparation of suitable DNA substrates, as well as a step-by-step guide for how to control and calibrate the supercoiling density produced.