Introduction to RPOC

• 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 history of RPOC

The prototype of RPOC was invented in the Zhang lab at Purdue University in 2021 and was first published in 2022 (Nat. Commun. 2022, 13, 4343). This original prototype utilized a femtosecond laser, enabling the acquisition of chemical contrasts through stimulated Raman scattering. Additionally, 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 manipulation capabilities (https://www.biorxiv.org/content/10.1101/2024.02.09.579709v1). 

• Terminology

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

Excitation laser: The lasers that are employed to 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 is utilized to compare 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

Plenty of optical modalities can be used for chemical detections for RPOC. For example: single-photon fluorescence, two-photon fluorescence, stimulated Raman scattering, transient absorption, and harmonic generation. Among these modalities, single-photon fluorescence is the most powerful one. 

2. Optical manipulation

Current manipulation methods include:

405 nm CW-laser, inducing reactive oxygen species (ROS); 405 and 532 nm laser, regulating photoswitchable inhibitors; Photobleaching using different laser wavelengths; Adaptive FRAP and FLIP; Low-density plasma generation by femtosecond lasers; Infrared lasers for organelle heating.

For cell manipulation, it allows for:

Selective perturbing cell functions at selected organelles using ROS, local heating, or low-density plasma; Inhibit polymerization of microtubule or actin; Study protein dynamics by adaptive FRAP and FLIP; Controlling cell division; Controlling cell migration; Selective killing specific cell types in a population

3. Readout

Short-term: Fluorescence signal changes, protein dynamics, intracellular organelle dynamics, cell morphology, ROS generation

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

• Applications of RPOC

RPOC can turn on photoswitchable compounds associated with specific organelles or at any desired subcellular locations. It allows inhibition of microtubule polymerization at desired locations. References: (Nat. Commun. 2022, 13, 4343) (Adv. Sci. 2024, 2307342)

RPOC can induce ROS using blue light at selected organelles or subcellular compartments. (Adv. Sci. 2024, 2307342)

RPOC can evaluate the influence of ROS on different organelles and perform long-term monitoring of cell responses. (https://www.biorxiv.org/content/10.1101/2024.02.09.579709v1). 

RPOC can achieve adaptive photobleaching and analysis of protein dynamics. It also enables manually selected and signal-guided automated target selection. The optical treatment and imaging are performed simultaneously. (https://www.biorxiv.org/content/10.1101/2024.02.09.579709v1). 

RPOC can control cell division by precisely perturbing centrosomes. (https://www.biorxiv.org/content/10.1101/2024.02.09.579709v1). 

RPOC gives real-time feedback for cell sorting based on Raman signals. (https://doi.org/10.1101/2023.10.16.562526)