• What is RPOC?

RPOC stands for 'Real-time precision opto-control'. It is an optical technology enabling precise spatial and temporal control of chemical processes within biological samples while avoiding unintended impact on other locations. 

The prototype of RPOC was first published in 2022 (Nat. Commun. 2022, 13, 4343). This original prototype utilized stimulated Raman scattering modality for the selection of chemical targets. It facilitated opto-control by utilizing femtosecond lasers at 405 nm or 520 nm wavelengths. Subsequently, in 2023, a continuous-wave (CW) version of RPOC was developed, significantly reducing the overall system cost (Adv. Sci. 2024, 2307342). Later in 2023, a software-enabled iteration of RPOC was introduced, enhancing the flexibility of target selection and optical control capabilities (ACS Photonics, 2025, 12, 3421–3434). A femtosecond laser was also used as the action laser for precise target perturbation based on multiphoton processes (Small Science, 2025, 2500166). Femtosecond laser RPOC enabled single organelle microsurgery, perturbing molecular targets using multiphoton absorption and low-density plasma generation. 

• Terminology

Action laser: The lasers that are employed to manage and regulate chemical activities within the sample precisely.

Excitation laser: The lasers that excite chemically selective optical signals from the sample.

APX: active pixels. The specific pixels on which the action lasers are activated.

Comparator circuit: The circuit that compares the intensities of optical signals with user-defined criteria, such as an intensity threshold.

• Key components of RPOC

RPOC has three key components

1. Chemical detection

Various optical modalities can be used for chemical detection for RPOC. For example: single-photon fluorescence, two-photon fluorescence, stimulated Raman scattering, transient absorption, and harmonic generation. 

2. Opto-control

Current opto-control methods include:

CW-laser in the visible range: inducing reactive oxygen species (405 nm); regulating photoswitchable inhibitors (blue and green lasers); Photobleaching using different laser wavelengths; Adaptive FRAP and FLIP; 

Femtosecond lasers: Low-density plasma generation; multiphoton absorption

Infrared lasers: organelle heating.

3. Readout

Short-term: Fluorescence signal changes, protein dynamics, intracellular organelle dynamics, cell morphology, ROS generation, mitochondria membrane potential change, calcium signaling, etc. 

Long-term: Cell migration, cell viability, cell division, and microenvironment-related changes such as hypoxia or nutrition deprivation. 

• Application examples of RPOC

1. Activate photoswitchable or photochromic compounds associated with specific organelles or at any desired subcellular location. References: (Nat. Commun. 2022, 13, 4343) (Adv. Sci. 2024, 2307342)

2. Induce ROS using blue light at selected organelles or subcellular compartments. (Adv. Sci. 2024, 2307342)

3. Allow inhibition of microtubule polymerization at desired locations. (Adv. Sci. 2024, 2307342)

4. Evaluate the influence of ROS on different organelles and perform long-term monitoring of cell responses. (ACS Photonics 2025, 12, 3421–3434). 

5. Control cell division by precisely perturbing centrosomes. (ACS Photonics 2025, 12, 3421–3434) 

6. Evaluation of phototoxicity, the synergy effect of photons of fluorescent dyes. (VIEW 2024, 20240013)

7. Single organelle microsurgery and perturbation of cellular compartments with femtosecond lasers with different average and peak power (Small Sci. 2025, 2500166)

8. Real-time feedback for cell sorting based on Raman signals. (Sci. Adv. 2024, 10, eadp2438)