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.
