Transformative lightsheet microscopy techniques for subcellular imaging in physiologically relevant 3D environments

University Of New Mexico, Albuquerque NM

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Abstract

Abstract Optical imaging enables fast and minimally invasive observation of biological processes within living cells and organisms. However, current state-of-the-art imaging instruments have limitations in acquisition speed, spatial resolution and light-penetration depth that restrict the types of biological questions that can be addressed. This is particularly problematic for biological samples that span several orders of magnitude in spatiotemporal scale. For example, cell-cell interactions within the tumor microenvironment and their response to treatment can occur over seconds to days and be heterogeneous throughout an entire tissue volume. Coupling these physiological outcomes to the underlying molecular mechanisms (and potential therapeutic targets) requires a transformation in not only the technologies we use, but also the combination of methods to cross the spatiotemporal scales from cells to tissues. Recent developments in emerging techniques like cleared-tissue-imaging coupled with lightsheet microscopy (LSM) has enabled researchers to probe deeper into the tissue without needing to section them. Illumination with lightsheet offers a much faster and less phototoxic alternative in comparison to point scanning microscopes. However, all LSM (including Lattice lightsheet) struggled with a number of fundamental limitations: (a) the maximum number of possible labels that can be imaged, (b) the size of the samples that they can handle, and, (c) poor spatial and temporal resolution. In order to fill these gaps my research program will engineer new optics that will not only improve the spatiotemporal resolution of the current state-of-the-art but also enable researchers to probe multiple simultaneous cellular phenotypes within the 3D architecture of the tissue microenvironment. By employing multiple scanning lightsheets we will develop a large volume hyperspectral LSM that will be able to unmix (segmentation and classification) 12+ fluorophores and image at 300 nm XYZ resolution to quantify the complex spatiotemporal interactions between various cell-types in tissue microenvironment. Additionally, we will develop a next generation LSM that will provide users a seamless transition from an organ/organism level imaging to 300 nm XYZ resolution. It will be proficient in identifying events-of-interest at lower resolution in large organs and intelligently adapt to high-resolution imaging, thus reducing imaging-time and generated-data burden. We will also design a new sample scanning strategy that will minimize light loss within the tissues. In order to prevent out-of-focus blur while imaging inside the tissue we will implement an autofocus routine that will enable users to carry out prolonged and unsupervised imaging of large specimens. Finally, we will develop a lattice lightsheet fluorescence microscope that will be able to perform fast, high-resolution multicolor imaging of live cells and spheroids. A configurable emission path will augment LSM with adaptive optics to counter sample induced aberrations. This will allow us to dynamically observe and quantify morphological phenotypes characteristic for highly metastatic cancer cells, which will be staged in organoids. I believe these have the potential to determine statistically significant patterns within the intact tissue that are bound to uncover novel biological questions.