RPOC stands for Real-Time Precision Opto-Control. It enables unprecedented flexibility, spatial precision, and chemical selectivity for regulating chemical processes within live biological samples.
RPOC can be integrated into any custom laser-scanning microscopes or stage-scanning microscope platforms.
How does RPOC work?
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The imaging laser excites chemical signals
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Chemical signals then activate action lasers.
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Action lasers modulate and control the chemical processes.
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The readout lasers record the resulting changes.
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All of these steps are performed simultaneously in real time within the same pixel.
Terminology
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Action laser: The lasers that are employed to precisely manage and regulate chemical activities within the sample.
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Excitation laser: The lasers that are employed to excite chemically selective optical signals from the sample.
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APX: active pixels. The specific pixels on which the action lasers are activated.
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Comparator circuit: The circuit that is utilized to compare intensities of optical signals with user-defined criteria, such as an intensity threshold.
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S-RPOC: software-assisted RPOC, provides the maximum flexibility and throughout
Key components of RPOC
1. Chemical detection (imaging)
RPOC is based on chemical imaging. Various optical modalities can be used for chemical detection in RPOC. Examples include:
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Single-photon fluorescence (most widely used)
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Two-photon fluorescence
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Stimulated Raman scattering
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Transient absorption
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Harmonic generation
2. Opto-control
Optical means to provide precision control:
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CW-laser: inducing reactive oxygen species (ROS); regulating photoswitchable inhibitors; photobleaching; adaptive FRAP and FLIP; small molecular uncaging
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Femtosecond lasers: ROS generation; low-density plasma generation; multiphoton absorption; small molecular uncaging; localized wounding
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Near or mid-infrared lasers: localized heating
3. Readout (imaging)
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Short-term: Fluorescence signal changes, protein dynamics, intracellular organelle dynamics, cell morphology, ROS generation, etc.
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Long-term: Cell migration, cell viability, cell division, and microenvironment-related changes such as hypoxia or nutrition deprivation.
Hardware
Key hardware to convert laser-scanning microscope to RPOC system including:
Comparator circuits
Enables real-time target tracking and decision-making, which is essential for precise control of highly dynamic chemical targets.
Acousto-optic modulators
Enables rapid activation of action lasers in response to detected chemical signals. First-order diffraction is used to ensure complete laser cutoff at TTL = 0.
Software
Lab-VIEW based RPOC software
For Lab-VIEW users:
Standard Lab-VIEW interface. Up to 8 input signal channels. RPOC functions integrated with laser-scanning and image-acquisition capabilities. Automated RPOC ROI selection, flexible action laser selection, and dosage monitoring functions available.
Requires LabVIEW installation (version 2023 or newer).
Fully compatible with comparator circuit integration.
Python based RPOC software
For Python users and developers:
The software provides core laser-scanning and image-acquisition capabilities, along with advanced RPOC features for ROI selection and action-laser control. It also enables intra-pixel optical decoupling to suppress or exclude signal enhancement from the action laser.
Fully compatible with comparator circuit integration.
References:
Clark, et al. Nat Commun, 2022, 13, 4343
Dong, et al. Adv Sci, 2024, 2307342
Ma, et al. Small Sci, 2025, 2500166
Dong, et al. ACS Photonics, 12, 7, 3421-3434