Cellular DNA is regularly subject to torsional stress during genomicprocesses, such as transcription and replication, resulting in arange of supercoiled DNA structures. For this reason, methods toprepare and study supercoiled DNA at the single-molecule levelare widely used, including magnetic, angular-optical, micropipette,and magneto-optical tweezers. However, it is currently challeng-ing to combine DNA supercoiling control with spatial manipulationand fluorescence microscopy. This limits the ability to studycomplex and dynamic interactions of supercoiled DNA. Here wepresent a single-molecule assay that can rapidly and controllablygenerate negatively supercoiled DNA using a standard dual-trapoptical tweezers instrument. This method, termed Optical DNASupercoiling (ODS), uniquely combines the ability to study super-coiled DNA using force spectroscopy, fluorescence imaging of thewhole DNA, and rapid buffer exchange. The technique can be usedto generate a wide range of supercoiled states, with between<5and 70% lower helical twist than nonsupercoiled DNA. Highlight-ing the versatility of ODS, we reveal previously unobserved effectsof ionic strength and sequence on the structural state of under-wound DNA. Next, we demonstrate that ODS can be used to di-rectly visualize and quantify protein dynamics on supercoiledDNA. We show that the diffusion of the mitochondrial transcrip-tion factor TFAM can be significantly hindered by local regions ofunderwound DNA. This finding suggests a mechanism by whichsupercoiling could regulate mitochondrial transcription in vivo.Taken together, we propose that ODS represents a powerfulmethod to study both the biophysical properties and biologicalinteractions of negatively supercoiled DNA.