Our research interests lie at the intersection of biophysics and instrument engineering. We are creating new tools to decipher how biomachines function using advanced-microscopy techniques to capture the behaviors of individual molecules at work inside living cells. Using these capabilities, we are exploring new strategies for novel diagnostic devices that can rapidly assess the identity of cell types, efficacy of drugs, and cellular phenotypes associated with a variety of maladies.
Localization microscopy is used to recover information from the image shape that is formed on the detector. Most commonly, this is used to extract a precise x-y position that is obscured by the finite numerical aperture of the imaging system. The potential goes far beyond! For example, it can also be used to extract information like z position, emitter color, orientation, and more.
"Multicolor localization microscopy and point-spread-function engineering by deep learning," Hershko & Weiss, et al. Optics Express, (2019). (doi.org/10.1364/OE.27.006158);
One key application of localization microscopy is for single-molecule tracking. Here we show the movements of a single protein traversing a micro-scale signaling organelle known as the primary cilium.
"The nuclear basket regulates distribution and mobility of nuclear pore complexes in budding yeast," Zsok et al. BiorXiv, (2023).
"Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol," Weiss & Milenkovic et al. PNAS, (2019). (doi.org/10.1073/pnas.1816747116);
Yet another application of localization microscopy is for super-resolution microscopy. Here, many different molecules are recorded asynchronously by exploiting the photophysical tendency of molecules to convert between fluorescent and non-fluorescent states.
The tradeoff between field-of-view and magnification makes it difficult to image large populations of cells. This poses an especially critical challenge when cells are undergoing dynamic changes that prevent the collection of sufficient statistics. We use flow-based imaging methods to solve that problem and have demonstrated 3D localization microscopy in 1000 cells each minute.
"Three-dimensional localization microscopy in live flowing cells," Weiss et al., Nature Nanotechnology (2020). (doi.org/10.1038/s41565-020-0662-0);
"High-throughput Imaging of CRISPR- and Recombinant Adeno-associated Virus–induced DNA Damage Response in Human Hematopoietic Stem and Progenitor Cells ," Allen & Weiss et al. The CRISPR journal (2022). (doi.org/10.1089/crispr.2021.0128);
Disposables have become an entrenched mainstay in the laboratory and form a sizeable percentage of global plastic waste. By design, many pieces of equipment are single-use, and recycling is made difficult by biocontamination. Our objective is to create reusable devices by incorporating reset switches, thus creating a more sustainable design.
Optical microscopy is a powerful tool for diagnostics and thus single-molecule imaging would seemingly be well positioned for ultrasensitive diagnostics. The longstanding challenge is that single-molecule data is often very heterogeneous and thus insufficiently robust as a critical detection metric. Our work combines high-throughput modalities (microfluidics) with ultrasensitive instruments (single-molecule microscopy) to bridge this gap.
We are using these approaches for biosensing and to characterize the next generation of gene therapies.
Analysis code from our team