Image simulation for biological microscopy: microlith

Image simulation remains under-exploited for the most widely used biological phase microscopy methods, because of difficulties in simulating partially coherent illumination. We describe an open-source toolbox, microlith (https://code.google.com/p/microlith), which accurately predicts three-dimensional images of a thin specimen observed with any partially coherent imaging system, including coherently illuminated and incoherent, self-luminous specimens. Its accuracy is demonstrated by comparing simulated and experimental bright-field and dark-field images of well-characterized amplitude and phase targets, respectively. The comparison provides new insights about the sensitivity of the dark-field microscope to mass distributions in isolated or periodic specimens at the length-scale of 10nm. Based on predictions using microlith, we propose a novel approach for detecting nanoscale structural changes in a beating axoneme using a dark-field microscope.Comment: current: 17 pages, 8 figures, expanded to include biological simulations; previous version: 7 pages, 2 figures; related website: https://code.google.com/p/microlit

Equivalent of the point spread function for partially coherent imaging

Angularly diverse or partially coherent illumination is widely used for optical, x-ray, and electron microscopy. A long-standing challenge in developing new partially coherent approaches is that the nonlinear image formation model does not allow physical intuition into how the imaging and illumination pupils impact contrast and resolution. We report a phase-space model, the phase-space imaging kernel, for partially coherent systems that describes image formation in terms of a convolution and is analogous to the point spread function model for coherent imaging. We simulate phase-space imaging kernels for brightfield and differential interference contrast (DIC) microscopes to explain a seemingly paradoxical experimental result that the DIC image of a point depends on the coherence of the illumination. We discuss interpretation of the spatial and spatial-frequency marginals of the kernel. We expect this intuitive model and simulations to facilitate design of novel computational schemes for phase imaging and optical lithography

Polarized light imaging of birefringence and diattenuation at highresolution and high sensitivity

Polarized light microscopy provides unique opportunities for analyzing the molecular order in man-made and natural materials, including biological structures inside living cells, tissues, and whole organisms. 20 years ago, the LC-PolScope was introduced as a modern version of the traditional polarizing microscope enhanced by liquid crystal devices for the control of polarization, and by electronic imaging and digital image processing for fast and comprehensive image acquisition and analysis. The LC- PolScope is commonly used for birefringence imaging, analyzing the spatial and temporal variations of the differential phase delay in ordered and transparent materials. Here we describe an alternative use of the LC-PolScope for imaging the polarization dependent transmittance of dichroic materials. We explain the minor changes needed to convert the instrument between the two imaging modes, discuss the relationship between the quantities measured with either instrument, and touch on the physical connection between refractive index, birefringence, transmittance, diattenuation, and dichroism.Comment: 21 pages, 5 figures, accepted for publication in Journal of Optic

Polarized light microscopy in reproductive and developmental biology

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of John Wiley & Sons for personal use, not for redistribution. The definitive version was published in Molecular Reproduction and Development (2013), doi:10.1002/mrd.22221.The polarized light microscope reveals orientational order in native molecular structures inside living cells, tissues, and whole organisms. Therefore, it is a powerful tool to monitor and analyze the early developmental stages of organisms that lend themselves to microscopic observations. In this article we briefly discuss the components specific to a traditional polarizing microscope and some historically important observations on chromosome packing in sperm head, first zygote division of the sea urchin, and differentiation initiated by the first uneven cell division in the sand dollar. We then introduce the LC-PolScope and describe its use for measuring birefringence and polarized fluorescence in living cells and tissues. Applications range from the enucleation of mouse oocytes to analyzing the polarized fluorescence of the water strider acrosome. We end by reporting first results on the birefringence of the developing chick brain, which we analyzed between developmental stages of days 12 through 20.This work was supported by funds from the National Institute of General Medical Sciences (grant 1R01GM100160-01A1 awarded to TT) and the National Institute of Biomedical Imaging and Bioengineering (grant EB002045 awarded to RO)

Single-fluorophore orientation determination with multiview polarized illumination : modeling and microscope design

Author Posting. © Optical Society of America, 2017. This article is posted here by permission of Optical Society of America for personal use, not for redistribution. The definitive version was published in Optics Express 25 (2017): 31309-31325, doi:10.1364/OE.25.031309.We investigate the use of polarized illumination in multiview microscopes for determining the orientation of single-molecule fluorescence transition dipoles. First, we relate the orientation of single dipoles to measurable intensities in multiview microscopes and develop an information-theoretic metric—the solid-angle uncertainty—to compare the ability of multiview microscopes to estimate the orientation of single dipoles. Next, we compare a broad class of microscopes using this metric—single- and dual-view microscopes with varying illumination polarization, illumination numerical aperture (NA), detection NA, obliquity, asymmetry, and exposure. We find that multi-view microscopes can measure all dipole orientations, while the orientations measurable with single-view microscopes is halved because of symmetries in the detection process. We also find that choosing a small illumination NA and a large detection NA are good design choices, that multiview microscopes can benefit from oblique illumination and detection, and that asymmetric NA microscopes can benefit from exposure asymmetry.National Institute of Health (NIH) (R01GM114274, R01EB017293)

pH dependent isotropic to nematic phase transitions in graphene oxide dispersions reveal droplet liquid crystalline phases

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Royal Society of Chemistry for personal use, not for redistribution. The definitive version was published in Chemical Communications 50 (2014): 6668-6671, doi:10.1039/C4CC00970C.Size fractionation, amplified by the surface charge density of graphene oxide (GO) sheets, broadens the pH dependent isotropic (I) to nematic (N) phase transition in aqueous dispersions of graphene oxide (GO). In this biphasic region, a highly organized droplet nematic phase of uniform size (20 ± 2.8 μm diameter) with an isotropic interior is observed.Supports from the Australian Research Council (LP110100612 to MM), National Institute of Biomedical Imaging and Bioengineering (R01EB002045 to RO) and HFSP fellowship (to SM) are acknowledged.2015-05-0

Septin assemblies form by diffusion-driven annealing on membranes

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of National Academy of Sciences for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences of the United States of America 111 (2014): 2146-2151, doi:10.1073/pnas.1314138111.Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knit together into higher-order assemblies. Using fluorescence correlation spectroscopy (FCS), we determined that cytosolic septins are in small complexes suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence (TIRF) microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends and do not exchange subunits in the middle of filaments. This work shows for the first time that annealing is an intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.This project was supported with funding from by NSF (MCB-507511, ASG) and NIH (GM100160, TT and ASG), and Colwin, Lemann and Spiegel summer fellowships and The Nikon Award for summer investigation at MBL in Woods Hole, MA (ASG) and instrument support from Micro Video Instruments (MVI).2014-07-2

Dissection of Molecular Assembly Dynamics by Tracking Orientation and Position of Single Molecules in Live Cells

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells

The effect of activity and novelty on response frequency in a two-choice learning situation.

Originally published in Optics Express on 23 March 2015 (oe-23-6-7734