only can be reduced to tens to hundreds of micrometers, thus limiting the spatial resolution in detection. On the other hand, molecular imaging methods can noninvasively monitor the level of biomarkers in the animal body by using appropriate imaging probes. [4] The spatial resolution of molecular imaging varies with the imaging modality, for example, sub-micrometer resolution can be easily reached by using a fluorescence microscope. But unlike needle-type microelectrode whose sensor tip is fully controlled by the physical handle, the molecular imaging probes are usually passively delivered in the animal body after administration, [5] even after modification with targeting groups. [6] So far, an in vivo detection technique that not only allows the active control of the detecting probes but also enables noninvasive detection at sub-micrometer resolution has been rarely reported.To address this dilemma, we propose a technique that combines the design concepts of needle-type microelectrode and fluorescence imaging method. Using needle-type microelectrode as the reference (Figure 1a1), we first substitute a fluorescent nanoprobe for the sensor tip to reduce its size to the nanoscale. Then, the physical handle is replaced by a near-infrared (NIR) laser beam with specially modulated optical field for the optical trapping and manipulation of the nanoprobe (Figure 1a2). [7] To make these two parts compatible, the nanoprobe will be carefully designed and synthesized so that it will be suitable for optical manipulation and also excitable by the NIR laser beam. [8] When the nanoprobe is applied with the laser beam, it will be trapped at the beam focus and meanwhile excited to generate fluorescence signal to report the level of biomarkers. In addition, multipoint detection can be realized by scanning the laser beam to shift a nanoprobe to multiple positions or trap multiple nanoprobes simultaneously. In this way, a new detection technique is established, which is named as "optically controlled virtual microsensor" (OCViM). In OCViM, the noninvasive detection is realized by employing the laser beam as the virtual handle, the high spatial resolution is obtained by using a nanoscale probe as the sensor tip, and the active control of the sensor tip is achieved by the action of optical force.According to this design, we prepared OCViMs for the detection of various biomarkers, including O 2 , pH, and reactive oxygen species (ROS). Their quantitative, multipoint and high-resolution Current technologies for the real-time analysis of biomarkers in vivo, such as needle-type microelectrodes and molecular imaging methods based on exogenous contrast agents, are still facing great challenges in either invasive detection or lack of active control of the imaging probes. In this study, by combining the design concepts of needle-type microelectrodes and the fluorescence imaging method, a new technique is developed for detecting biomarkers in vivo, named as "optically controlled virtual microsensor" (OCViM). OCViM is established by the organi...