Zohreh Azizi PhD Proposal Review

Name:
Zohreh Azizi

Title:
Exploring SIM for monomer orientation

Date:
8/22/2024

Time:
1:00:00 PM

Location: https://northeastern.zoom.us/j/7318775019
Meeting ID: 731 877 5019

Committee Members:
1. Prof. Charles DiMarzio (Advisor)
2. Prof. Carey Rappaport
3. Dr. Sangyeon (Fred) Cho

Abstract:
Collagen fibrils, the most abundant protein polymers in animals, protect cells from mechanical forces such as stress, tension, compression, and shear. Each collagen molecule, approximately 300 nm long and 1.5 nm in diameter, consists of three polypeptide chains forming a supercoiled triple helix. These fibrils self-assemble, with diameters ranging from 20 nm to several hundred nanometers. Large collagen fibrils are visible with scanning electron microscopy (SEM) and optical microscopy. Electron microscopy damages samples during preparation, limiting observations to static, non-living conditions. The natural self-assembly behavior, spatial arrangement, and dimensions of collagen fibrils play a vital role in shaping the structure, strength, and function of tissues. These factors are essential in determining how tissues are organized, how they withstand physical forces, and how effectively they perform their biological functions.

Detecting the orientation and location of collagen monomers is essential for understanding their role in collagen spontaneous formation and their interactions with fibril surfaces. To study collagen monomers in a dynamic, living state, high-resolution optical microscopy is preferred, as it allows for detailed imaging beyond the diffraction limit of light.We introduce a new technique using structured illumination to determine the spatial separation of punctuate objects to super–resolution limits that is amenable to both scattering and fluorescent objects. We call the technique Structured–Illumination Point–Separation (SIPS) Microscopy. We apply it to determine the orientation of a collagen monomer by imaging two fluorescent tags at different locations on the monomer. Experimentally, we show that our approach effectively resolves the orientation of collagen monomers with a resolution surpassing the diffraction limit.

In this illumination technique we are employing time-multiplexed binary patterns with a DMD based SIM while the camera shutter is open, mitigating undesired diffractions from the DMD. Three different phases of sinusoidal patterns generated by the DMD 3000 DLP series are projected onto the fluorescent sample in a 4f system. After data acquisition of the effect of the structured patterns on sample in three different phases images multiplied by a phase factor (1, 𝑒−𝑖2𝜋/3,𝑒−𝑖4𝜋/3 ) and then combined. By implementing the Radon transform of Fourier transform of phase of complex image, and evaluate the Radon transform in center of x’=0 , direction of pairs obtain. we enhance image reconstruction and analysis, utilizing their characteristics for detailed imaging and data extraction in Structured–Illumination Point–Separation. With this strategy a non-destructive and effective technique that allows researchers to measure orientation of collagen monomer in a way that preserves the sample and offers high-resolution imaging, which is crucial for studying the structure and properties of collagen.