Posts Tagged ‘microscopy’

Precise and scalable self-organization in mammalian pseudo-embryos

Mélody Merle, Leah Friedman, Corinne Chureau, Armin Shoushtarizadeh & Thomas Gregor.
Nature Structural & Molecular Biology, doi: 10.1038/s41594-024-01251-4 (2024)

A single-objective light-sheet microscope

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.

Stochastic motion and transcriptional dynamics of pairs of distal DNA loci on a compacted chromosome

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

Growth produces coordination trade-offs in Trichoplax adhaerens, an animal lacking a central nervous system

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


Structured foraging of soil predators unveils functional responses to bacterial defenses

Fernando W. Rossine, Gabriel Vercelli, Corina Tarnita, and Thomas Gregor. Proceedings of the National Academy of Science (USA) 119(52): e2210995119 (2022).


Transcriptional coupling of distant regulatory genes in living embryos

Michal Levo, João Raimundo, Xin Yang Bing, Zachary Sisco, Philippe J. Batut, Sergey Ryabichko, Thomas Gregor & Michael S. Levine (2022). Nature 605, 754–760 (2022).

Optogenetic control of the Bicoid morphogen reveals fast and slow modes of gap gene regulation

Anand P. Singh, Ping Wu, Sergey Ryabichko, Joao Raimundo, Michael Swan, Eric Wieschaus,
Thomas Gregor, and Jared E. Toettcher (2022). Cell Reports 38, 110543.

The Impact of Space and Time on the Functional Output of the Genome

Marcello Nollmann, Isma Bennabi, Markus Götz, and Thomas Gregor (2021). Cold Spring Harb Perspect Biol.: a040378. doi: 10.1101/cshperspect.a040378.


Using RNA Tags for Multicolor Live Imaging of Chromatin Loci and Transcription in Drosophila Embryos

Chen H and Gregor T. In: Heinlein M. (eds) RNA Tagging. Methods in Molecular Biology, vol 2166. Humana, New York, NY (2020). (more…)

Eco-evolutionary significance of “loners”

Fernando W. Rossine, Ricardo Martinez-Garcia, Allyson E. Sgro, Thomas Gregor, Corina E. Tarnita. Eco-evolutionary significance of “loners”. PLoS Biol 18(3): e3000642 (2020). (more…)

Diverse spatial expression patterns emerge from unified kinetics of transcriptional bursting

Benjamin Zoller, Shawn C. Little, and Thomas Gregor. Cell 175(3), 835–847 (2018). (more…)

Dynamic interplay between enhancer-promoter topology and gene activity

Hongtao Chen, Michal Levo, Lev Barinov, Miki Fujioka, James B. Jaynes and Thomas Gregor. Nature Genetics 50, 1296–1303 (2018). (more…)

Single mRNA Molecule Detection in Drosophila

Shawn C Little and Thomas Gregor. In: Gaspar I. (eds) RNA Detection. Methods in Molecular Biology, vol 1649. Humana Press, New York, NY (2018) (more…)

Live Imaging of mRNA Synthesis in Drosophila

Hernan G Garcia and Thomas Gregor. In: Gaspar I. (eds) RNA Detection. Methods in Molecular Biology, vol 1649. Humana Press, New York, NY (2018) (more…)

Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo

Jacques Bothma, Hernan Garcia, Samuel Ng, Michael W. Perry, Thomas Gregor, and Michael S. Levine. eLife 2015; 4:e07956 (2015). (more…)

Dynamic Regulation of Eve Stripe 2 Expression Reveals Transcriptional Bursts in Living Drosophila Embryos

Jacques P. Bothma, Hernan G. Garcia, Emilia Esposito, Gavin Schlissel, Thomas Gregor, Michael S. Levine. PNAS 111 (29): 10598–10603 (2014).

Quantitative imaging of transcription in living Drosophila embryos links polymerase activity to patterning

Hernan G. Garcia, Mikhail Tikhonov, Albert Lin and Thomas Gregor. Current Biology 23, 2140–2145 (2013).

Quantifying the Bicoid morphogen gradient in living fly embryos

Alexander H. Morrison, Martin Scheeler Julien O. Dubuis and Thomas GregorCold Spring Harb Protoc. 2012(4): 398-406 (2012).

Researchers Develop Improved Method to Visualize Biologic Molecules

PLoS Press Release for our first paper on mRNA quantification in whole embryos.

“How are biologic molecules arranged inside the embryo so that embryonic development occurs reliably every time? Princeton researchers, led by Thomas Gregor, an assistant professor of physics and the Lewis-Sigler Institute for Integrative Genomics, and Shawn Little, a postdoctoral fellow in the laboratory of Professor Eric Wieschaus in the Department of Molecular Biology, have developed a new method to better understand how an embryo’s basic molecular makeup helps ensure that the embryo’s development occurs reliably every time. The results of this research into the fruit fly Drosophila introduce a method for making precise measurements of biologic units (so-called mRNA molecules) that play a key role in development. The findings are published in the March 1st issue of  in the online, open access journal PLoS Biology.”