Crosstalk‐Free Real‐Time Precision Opto‐Control of Biochemical Processes Through Intra‐Pixel Optical Decoupling

The ability to regulate biochemical processes in live cells and whole organisms is critical when addressing fundamental biological questions. However, conventional chemical treatment methods overlook the spatial heterogeneity of these processes, offering only the ability to perturb a sample globally. Optical approaches, in contrast, can regulate biochemical activities with submicron spatial precision. Real‐time precision opto‐control (RPOC) is a recently developed technique that integrates laser scanning, chemical imaging, real‐time decision making, and spatially precise optical regulation. RPOC uses chemically specific optical signals to trigger an action laser for selective regulation of dynamic chemical species. In RPOC, optical readout and treatment occur simultaneously, enabling continuous monitoring of chemical changes during perturbation. However, activation of the action laser can enhance fluorescent signals in the readout channel, creating a form of crosstalk that obscures the readout of chemical changes during optical treatment. To overcome this limitation, we introduce intra‐pixel optical decoupling, a method that separates optical control and readout within each image pixel. This strategy preserves the simultaneous treatment‐and‐readout capabilities of RPOC while eliminating action‐laser‐induced crosstalk. As a result, chemical changes at the active pixels or surrounding areas can be accurately quantified during treatment, improving RPOC's ability to probe local chemical changes over time.