Real-time precision opto-control of chemical processes in live cells

Precise control of molecular activities and chemical reactions within living cells is a significant challenge in biological research. Existing technologies lack the ability to simultaneously identify and control specific molecular targets with high spatial accuracy. This limitation hinders a deeper understanding of spatial target distributions, off-target effects, organelle-specific enzyme activities, and individual organelle functions. Conventional chemical treatments lack spatial selectivity, while optical tweezers and trapping methods can only manipulate a limited number of pre-identified targets. Laser ablation and optogenetics techniques, while offering some control, suffer from limitations in subcellular precision and real-time adaptability to dynamic cellular environments. This research addresses this need by developing a real-time system for precise, selective opto-control of targeted molecules within living cells.

The paper reviews existing technologies for controlling molecular activities in live cells, highlighting their limitations. Conventional chemical methods lack spatial precision, causing off-target effects. Optical tweezers and trapping are limited in the number of targets they can manipulate. Laser ablation and current optogenetic methods require pre-imaging and lack subcellular precision, making them unsuitable for dynamic systems. The absence of a technology capable of simultaneous real-time detection and control of selected molecular targets with high chemical selectivity and spatial accuracy motivated the development of RPOC.

The researchers developed a real-time precision opto-control (RPOC) technology. During laser scanning, a chemical-specific optical signal (e.g., fluorescence, Raman) from target molecules triggers an acousto-optic modulator (AOM). This, in turn, couples a separate control laser beam to interact with the same pixel, achieving precise control. Digital comparator circuits process the optical signal, activating the AOM only when the signal meets preset conditions. The system uses a dual-output femtosecond laser for both signal excitation and opto-control, with the ability to use various optical signals such as fluorescence and stimulated Raman scattering (SRS). The response time of the system is <50 ns using fluorescence signals. Active pixels (APXs), the locations where the control laser is activated, are tracked to visualize the opto-control locations. The selection of APXs is controlled by parameters such as thresholds and optical signal intensity. The incorporation of a second comparator box allows for digital logic operations (AND, OR, NAND, NOR) to further refine APX selection based on multiple signal channels. The system’s capabilities are demonstrated using a photochromic molecule (CMTE) and a synthesized photoswitchable microtubule polymerization inhibitor (PST-1). CMTE’s state is precisely controlled in different cellular regions, while PST-1 allows site-specific control of microtubule polymerization and lipid droplet trafficking.

The RPOC platform demonstrates real-time, precise control of molecular activities and chemical reactions in live cells. Using a photochromic molecule (CMTE), the researchers achieved precise control of its isomerization states in different cellular regions. The system’s ability to select active pixels (APXs) based on various thresholds and signal intensities was shown. Digital logic functions were implemented, enabling the selection of APXs based on combinations of signals from multiple detectors. The researchers synthesized a photoswitchable microtubule polymerization inhibitor (PST-1) and used RPOC to achieve site-specific inhibition of microtubule polymerization. This resulted in altered lipid droplet trafficking, showing the impact on cellular dynamics. The response time of the system is fast (<50 ns for fluorescence signals), and the spatial precision is submicron. Quantitative analysis of CMTE conversion showed a quadratic dependence of integrated SRS intensity changes on the number of APXs, and a near-linear dependence of mean SRS intensity changes on the number of APXs. The results demonstrate the capability of RPOC to selectively control chemical changes in space and potentially quantify reaction rates. Control of CMTE within specific organelles, such as the endoplasmic reticulum (ER), was achieved using the AND logic function from two detectors. The study also showed that RPOC could be used to control the dynamics of microtubules and lipid droplets in live cells.

The RPOC technology significantly advances the field of biophotonics by providing a method for precise, real-time control of molecular activities and chemical processes in live cells. The ability to selectively target and control molecules with submicron precision and fast response times opens new possibilities for studying cellular processes. The use of digital logic functions expands the versatility of RPOC, enabling complex control strategies and enhancing the chemical selectivity of the system. The successful application of RPOC to control both a photochromic molecule and a photoswitchable inhibitor demonstrates its broad applicability to diverse biological systems and research questions. This technology offers significant advancements over existing methods for controlling cellular processes, paving the way for new discoveries in cell biology and related fields.

The development of real-time precision opto-control (RPOC) provides a powerful new tool for manipulating cellular processes. RPOC offers submicron spatial accuracy, fast response times, and high chemical specificity, enabling precise control of molecular activities and chemical reactions. Future directions include optimizing the control laser beam for improved precision, developing a more cost-effective platform based on continuous-wave lasers, and integrating the technology with commercial microscopes. Expansion of the RPOC capabilities with programmable acousto-optic tunable filters will further enhance its versatility and applications.

While RPOC demonstrates significant advancements, certain limitations exist. The current system uses a relatively expensive femtosecond laser. The oversampling condition used for RPOC can lead to some non-specific effects at the edges of the APXs. Further optimization of the control laser beam and the use of a more cost-effective laser source are areas for future improvement. The applicability of RPOC may be limited to molecules or processes that can be modulated by light. The complexity of the system might be a barrier for widespread adoption.