Laboratory for the Physics of Life Thomas Gregor, Professor of Physics, Princeton University

My laboratory is interested in problems at the interface of physics and biology. The main thrust of our research is the design of new experimental approaches and performing high-precision physics-style measurements in living animals, such that our data allows for direct validation of mathematical models. This program is aimed at the generation of theories describing biological phenomena which are derived from general principles in the physics tradition. Currently, our primary interests lie in the self-organization and collective behaviors of eukaryotic cell aggregates, from microbes to embryonic tissue, and in how the control of gene expression in early fly embryos leads to the formation of an animal body plan. We are making experimental and theoretical progress on the biological questions in each of these areas but the long-term goal is to find theories inspired by experimental data that go beyond the specifics of the biological systems hoping to find in the living world new physics, which has been hidden and cannot quickly be revealed in the inanimate world. The approach outlined here extends the traditional physics approach, which my colleagues used to study the stars or inert matter on Earth, to the complex processes underlying the living world.

The laboratory is currently composed of two research facilities working on similar topics and questions. One at Princeton University, since 2009, and since recently (2018) a second laboratory at the Institut Pasteur in Paris (France). The laboratory in Princeton works with Drosophila melanogaster models for which we have developed many highly quantitative imaging technologies to perform precision measurements in living embryos. At the unit at Institut Pasteur, we implement derivatives of these technologies in mammalian model systems such as mouse embryonic stem cells and stem cell-derived embryoid cell aggregates (i.e. gastruloids). Once these perform at similar levels of precision to what we achieve with the fly system, we will assess physical concepts such as developmental precision and reproducibility, system size scaling, or self-organization phenomena across both model systems. 

Activities at Princeton will continue with the Drosophila model along the following directions: studying the flow of information from molecules to macroscopic patterns, where cells determine their identity by interpreting this information from a genetic network of interconnected transcription factors. With the gene regulatory sequence, the enhancer, still at the core of our investigation, we identified three central questions that we will continue to address during the next cycle:

1) How do individual enhancers read information from a genetic network of interconnected transcription factors?

2) How is this information transferred from the activated enhancer to its target promoter, often over large genomic distances?

3) How is information eventually transformed into a transcriptional output at the level of the promoter?

To answer these questions, several technologies are being developed, namely live single-molecule imaging and optogenetic perturbation of single cells and individual gene loci in living embryos.

We are developing a novel microscopy modality, a single-objective light-sheet microscope with a high numerical aperture. It is an entirely novel design for a light-sheet microscope that combines the convenience of conventional sample mounting with sensitive subcellular and super-resolution imaging of cells and tissues. This single objective light-sheet fluorescence microscope started acquiring from biological samples only a few months ago and already several researchers across campus claimed interest in obtaining their own version, and there are plans to build a clone of the microscope for the imaging facility.

In an OPM, a single primary objective is used to both create the excitation light sheet and capture emitted light from the sample. Excitation light enters the objective sideways, resulting in an oblique light sheet on the sample, with an angle between 30°–45°. Emitted light from the tilted plane is collected by the same objective and optically refocused to a secondary objective, without introducing any relevant aberrations. It is subsequently re-imaged by a tilted tertiary objective onto a camera. The beauty of this approach lies in having a single high NA objective close to the sample, allowing for traditional sample mounting geometry (microscope slides, glass-bottom dish), and leaving accessible space around the sample for other perturbations and manipulations, such as microfluidic devices or optogenetic light stimulation.

As a proof of concept, we acquired images of Drosophila embryos, mESCs, and mouse gastruloids in their optimal growth conditions, showing that we achieve diffraction-limited resolution that, e.g., allows us to discriminate sister chromatids, follow their dynamics over time, and measure how they are transcriptionally, spatially, and temporally correlated. Moreover, we achieve a combination of high resolution, high contrast, and high speed that enables us to identify and track single mRNA molecules as they are released at the transcription site. We are currently developing analysis tools that will allow us to extract quantitative data from these images, e.g., to measure the diffusion and reach of individual mRNA molecules.

David B. Brückner, Hongtao Chen, Lev Barinov, Benjamin Zoller, Thomas Gregor. Science 380, 1357-1362 (2023).

Mircea R. Davidescu, Pavel Romanczuk, Thomas Gregor, Iain D. Cousin. Proc Natl Acad Sci (USA) 120 (11) e2206163120 (2023).