NIR in, far-red out: developing a two-photon fluorescent probe for tracking nitric oxide in deep tissue

Area of Science:

• Chemical Biology
• Biomedical Imaging
• Fluorescent Probes

Background:

• Nitric oxide (NO) is a crucial signaling molecule in physiological and pathological processes.
• Existing fluorescent probes for NO imaging face challenges in deep-tissue applications due to short excitation/emission wavelengths, leading to background interference and low collection efficiency.
• In situ tracking of NO in biological events remains difficult.

Purpose of the Study:

• To develop a novel far-red emissive two-photon (TP) excitable probe for sensitive and specific detection of nitric oxide (NO).
• To enable deep-tissue imaging of NO in vitro and in vivo.
• To explore the potential of Nile Red derivatives for creating advanced far-red emissive TP probes.

Main Methods:

• Development of the NRNO probe utilizing Nile Red as the TP fluorophore, emitting at 650 nm.
• Evaluation of the probe's response to NO, including speed and limit of detection (LOD).
• Testing the probe's performance in detecting both exogenous and endogenous NO in living cells and deep tissues, including visualization of NO generation during LPS-mediated inflammation.

Main Results:

• The NRNO probe exhibits a fast (within 180 s) and specific fluorescence response to NO with a low LOD of 46 nM.
• The probe demonstrates sensitive detection of NO in living cells and improved penetration depth for deep-tissue imaging due to its far-red emission.
• NRNO successfully visualized NO generation in a lipopolysaccharide (LPS)-mediated inflammation model for the first time.

Conclusions:

• The NRNO probe is a practical and effective tool for the sensitive detection and imaging of nitric oxide in biological systems, including deep tissues.
• The probe's properties facilitate the study of NO-related biological events, such as inflammation.
• This work highlights the potential of Nile Red derivatives for developing novel far-red emissive two-photon probes for advanced bioimaging applications.