Abstract B012: Developing applications for a new, fully automated, low-coherence, label free, time lapse holotomographic analysis platform-TomoCube HT-X1: Preparations for AI based analysis
Authors: Luther Ed A. , Filipczak Nina , Ataide Janaina Artem , Torchilin Vladimir P. 
Published: February 2024

Abstract
Our groups are involved in the development of multifunctional nanoparticles and combinations primarily for applications in cancer research is one aspect, we are developing multi-functional for better targeting of the cancer cells. An important part of our efforts is to visualize the effects of the compounds either by visualization, or by quantitative measurements such as the generated by flow cytometry and by laser scanning cytometry. Label free holographic imaging is a new technology that employs laser interference to calculate the refractive index of the pixels. Deconvolution routines create 3-dimensional holograms, and we have developed techniques for 4-dimensional imaging. In Holotomographic imaging there is either a rotating mirror (Nanolive), or a multi-mirror display module (Tomocube) We currently have a Program of Excellence Agreement with Tomocube and are serving as beta testers for their new HT-X1 system. This new instrument abandons laser illumination in favor of low coherence LED illumination. It is a large instrument designed for completely automated operation including autofocus and detector settings, has an automated stage, and is coupled to a high-performance environmental control system. The HT-X1 will have AI capabilities. Some applications. such as accurate cell segmentation and lipid enumeration have already been developed, but have not been released yet. We were told that if we develop our own truth sets, they will be able to process them for us. To illustrate the imaging capabilities, we will use an experiment based on repurposing of an anti-microbial rifamycin as a breast cancer therapeutic. Breast cancer cells were seeded into a 24 well microtiter plate and allowed to attach. Row A was untreated, Row B was treated with free rifamycin, and Row C was treated with liposomal rifamycin. Two-fold dilutions of the drugs were applied starting with column 2. Plates were incubated overnight and imaged on the HT-X1. This experiment showed that at the higher concentration levels, the free rifamycin precipitated leaving rod shaped crystals which presumably reduce its efficacy. The effects of the liposomal rifamycin on the cell morphology is striking. A complete set of the images will be included in the presentation. One of our areas of research is the brain cancer glioblastoma. In previous work using label free tomographic imaging and videography, we identified what we believe to be proto-stem cells, and we made the observation that they are closely associated with nucleoli. In follow-up experiments, we are using a combination of fluorescent dyes, Hoechst 33342 for DNA, Mito tracker green and mitosox red to observe the expression patterns of ROS appearing first in the nucleoli, then spreading to the nucleus, and eventually to the entire cell and the microenvironment. We know that nucleoli are generated from ribosomal proteins at the start of each cell cycle round and disassembled before exiting the G2 phase in a tightly orchestrated sequence. Our hope is that we will be able to further elucidate this using AI.
Citation Format: Ed A. Luther, Nina Filipczak, Janaina Artem Ataide, Vladimir P. Torchilin. Developing applications for a new, fully automated, low-coherence, label free, time lapse holotomographic analysis platform-TomoCube HT-X1: Preparations for AI based analysis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B012.