Single-Molecule and Single-Cell Optical 'Omics
Recent advances in optical imaging have been so dramatic that light microscopy can now be used to directly quantify protein or nucleic acid numbers, modifications and interactions. This has given rise to the new fields of ‘optical proteomics’ and ‘optical genomics’.
We are developing quantitative approaches to single-molecule microscopy that will help us to understand the organizational principles of bacteria by mapping out their internal structure and quantifying the intracellular abundance of proteins and nucleic acids. This information can provide insight into stochastic varation among populations of bacteria at the single-cell level.
Molecular Counting with Localization Microscopy: A Bayesian estimate based on fluorophore statistics,
D. Nino, N. Rafiei, Y. Wang, A. Zilman and J. N. Milstein, Biophys. J. 112, 1777-1785 (2017).
Quantitative localization microscopy reveals a novel organization of a high-copy number plasmid,
Y. Wang, P. Penkul and J. N. Milstein, Biophys. J. 111, 467-479 (2016).
Uncovering the Biophysical Mechanisms Behind Gene Silencing
Bacteria can rapidly adapt to a changing environment by acquiring genes from viruses or other bacteria. Expressing these genes, however, may entail a fitness cost putting the bacteria at a competitive disadvantage or, in the worst case, lead to cell death. The incorporation and eventual expression of foreign DNA is, therefore, carefully controlled. Newly acquired, foreign DNA must, at least initially, be recognized as such and silenced.
The protein H-NS, found in common bacteria like E. coli and Salmonella, and Lsr2, found in the pharmaceutical factories Streptomyces, are examples of silencing proteins that target foreign DNA. These proteins are known to somehow interfere with genetic transcription. Bulk biochemical and genomic approaches to understanding gene silencing by H-NS and Lsr2 have been employed for decades. Only recently, with the advent of single-molecule measurements, have researchers begun to gain a fundamental understanding of the biophysical mechanisms through which H-NS and Lsr2, and associated co-regulatory proteins, function to regulate gene expression.Xenogeneic silencing and its impact on bacterial genomes,
K. Singh, J. N. Milstein and W. W. Navarre, Annu. Rev. Microbiol. 70, 199-213 (2016).
A biomechanical mechanism for initiating DNA packaging,
H. Wang, S. Yehoshua, S. S. Ali, W. W. Navarre, and J. N. Milstein, Nucl. Acids Res. 42, 11921 (2014).
Optical Engineering and Force Spectroscopy
We are developing a next generation of axial optical tweezers that can manipulate small segments of DNA with an extremely high degree of spatial and temporal precision. These tools will enable us to explore novel regulatory mechanisms such as sequence dependent effects on protein-DNA interactions, which are inaccessible via current technology.
Our focus is on using light-shaping techniques, resulting in what are often called holographic optical tweezers, to correct for aberrations that would otherwise interfere with single-molecule measurements and to engineer the focal spot for novel applications of optical trapping.Accounting for polarization in the calibration of a donut beam axial optical tweezers,
R. Pollari and J. N. Milstein PLoS ONE 13(2): e0193402 (2018).
Improved axial trapping with holographic optical tweezers,
R. Pollari and J. N. Milstein, Opt. Express 23, 28857-28867 (2015).
Axial optical traps: A new direction for optical tweezers,
S. Yehoshua, R. Pollari and J. N. Milstein, Biophys. J. 108, 2759-2766 (2015).
With the advent of single-molecule localization microscopy (SMLM), images of cellular structure and organization can be acquired with visible light at a spatial resolution well surpassing the diffraction limit. SMLM is increasingly being employed in cells to image and quantitatively analyze various protein complexes forming tens to hundreds of nm assemblies, from membrane receptors to nucleosome bundles.
We are developing clustering algorithms for detecting and analyzing protein aggregates in SMLM datasets and have already applied the technique to study transcription factories within cell nuclei. The software package is currently made available on our lab website and, while it only works on 2-dimensional datasets, we will soon be releasing a more extensive version that directly clusters 3D SMLM data.Fast Optimized Cluster Algorithm for Localizations (FOCAL): A spatial cluster analysis optimized for super-resolved microscopy,
A. Mazouchi and J. N. Milstein, Bioinformatics 32(5), 747–754 (2016).