Abstract
Fluorescence imaging, due to its high sensitivity and specificity, has been widely used for molecular detection, visualization of bioactivity and disease diagnosis. However, traditional fluorescence imaging is limited by shallow penetration depth, low resolution, and susceptibility to photobleaching. As a result, many novel imaging technologies have been developed, requiring more advanced probes to meet the demands of further biological studies. For example, photoacoustic (PA) imaging is an emerging technique that enables deep tissue penetration. By combining PA and fluorescence imaging, it is possible to bridge the gap between optical sensitivity and ultrasound depth. Additionally, super-resolution imaging can overcome the diffraction limit of light (approximately 200 nm for visible light), allowing for the visualization of structures at the nanometer scale. This technology has been widely applied in the imaging of subcellular structures and other nanoscale biological processes.Chapter 1 provides an overview of PA imaging and super-resolution imaging. In the section on PA imaging, the classification of fluorescence PA probes and existing PA or fluorescence molecular platforms will be introduced. In the section on super-resolution imaging, the different types of super-resolution technologies will be classified, and advanced fluorescent probes for various types of super-resolution imaging will be highlighted.
In Chapter 2, we described the successful development of CySN, a ratiometric near-infrared fluorescent (NIRF) and photoacoustic (PA) dual-modality platform derived from a conventional NIRF dye, cyanine. The CySN platform exhibited remarkable wavelength-shifting properties, including a large fluorescence shift (68 nm) and a substantial PA shift (145 nm) after the decaging reaction. These significant changes resulted in an exceptionally high ratiometric NIRF change of 603-fold and a ratiometric PA change of 261-fold. Utilizing the CySN platform, dual-channel NIRF/PA probes were successfully developed for detecting both small-molecule biomarkers (H₂O₂) and enzyme biomarkers (esterase). These probes demonstrated the ability to detect their respective targets via dual-channel NIRF/PA detection with high sensitivity and selectivity in vitro. Notably, the probes showed the capability to accurately diagnose tumors by detecting tumor markers (H₂O₂ and esterase), revealing a 3.6- to 7-fold ratiometric PA enhancement over normal tissue. Therefore, the CySN platform holds significant potential to further advance the development of dual-channel NIRF/PA probes for biomolecule detection in disease diagnosis.
In chapter 3, we developed a series of high-specificity endoplasmic reticulum (ER) probes SRB-Cn by systematically selecting aliphatic chains based with the aggregation-disaggregation turn-on mechanism. Among these probes, SRB-C8 exhibited excellent fluorogenic, high specificity and pH-resistance when binding to ER. SRB-C8 enabled long-term visualizing the dynamic change of ER-nanostructures and observing the change in ER morphology induced by ferroptosis throughout stimulated emission depletion (STED) imaging. SRB-C8 was suitable for single-molecule localization (SMLM) imaging in different cell lines as well. Therefore, SRB-C8 is a promising tool not only for super resolution imaging, but also for ER-related disease monitoring.
In Chapter 4, we further expanded our self-assembling fluorogenic strategy based on sulforhodamine B (SRB) to create a modular platform, designing both self-labeling (SRB-Halo) and bioorthogonal (SRB-Tz) probes. With SRB-Halo, we successfully visualized the nanostructure of the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM) using STED imaging, an achievement not feasible with traditional techniques. Using SRB-Tz, we conducted single-particle tracking (SPT) studies of lysosomes under various conditions by STED. This platform integrates synthetic simplicity, pH resistance, and photostability, making it suitable for multiple types of super-resolution imaging. We envision that our strategy will inspire the development of a robust foundation for next-generation imaging probes.
Chapter 5 summarizes the key highlights of our work. It outlines the limitations of current PA and super-resolution probes and discusses future research directions in PA and super-resolution imaging.
| Date of Award | 24 Dec 2025 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Hongyan SUN (Supervisor) |