Multiplex Magnify-enabled Nanoscale Imaging and Analysis of Microparticles, Pathogens and Infected Tissues.

 Magnify, a pioneering expansion microscopy method, employs a universal labeling strategy  to retain a wide array of biomolecules within a mechanically robust hydrogel. Distinguishing  itself from most expansion methods, Magnify combines gelling solution penetration with  molecular anchoring in a single step. This method enables the expansion of cells and tissues up  to 11-fold, achieving an effective resolution of approximately 25 nm using a diffraction-limited  objective lens on a confocal microscope, and around 15 nm when paired with super-resolution optical fluctuation imaging. The primary focus of my PhD research has been to broaden the  application scope of Magnify: 1) developing an optimized Magnify protocol specifically for  pathogens and infected tissues; 2) establishing a multiplexed imaging pipeline for different  biomolecules in infected cell cultures and cancer tissues; 3) prototyping sampling and image  analysis workflows for infected tissues and liquid biopsies. 

I developed microMagnify, a nanoscale multiplexed imaging method for pathogens and  infected tissues based on Magnify. This method found a combination of heat denaturation and  enzyme cocktails essential for robust cell wall digestion and expansion of microbial cells and  infected tissues without distortion. This approach enabled up to an 8-fold expansion in a diverse  array of pathogen-containing specimens, including bacterial and fungal biofilms, infected culture  cells, infected mouse lung/tongue tissue, and human corneal samples fixed in formalin and  embedded in paraffin, infected by various pathogens.

 I established a high-plex nanoscale fluorescence imaging pipeline with effective preservation of proteins, DNA, and RNA. We demonstrated its versatility through applications such as cyclic  immunostaining in infected cell cultures (10-plex) using overlapping spectrum unmixing, and  cyclic fluorescence in situ hybridization for cancer genomic loci amplification (9-plex) and RNA  expression (9-plex) in formalin-fixed paraffin-embedded (FFPE) cancer tissues. The resultant  high-content data can be interactively explored and manipulated using virtual reality tools,  enhancing immersive and collaborative research experiences for scientists globally. 

I also prototyped the utility of Magnify in detecting and quantifying microbial cells and  microparticles in their natural environment. In situ polymerized hydrogel captured microbes  within biofilms on glaucoma devices and cancer-secreted extracellular vesicles for simultaneous  immunostaining and fluorescence in situ hybridization. Additionally, an image analysis pipeline was developed to deep mine high-resolution fluorescence microscopy images for particle  quantification, co-localization, and classification 

 My work has significantly diversified the utility of Magnify, making it compatible with microparticles, pathogen and infected tissues. The demonstration of multiplex imaging and its downstream image analysis underscores its potential to investigate these biological specimens with a higher resolution and depth