@article {47418, title = {Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging.}, journal = {Biomedical Optics Express}, volume = {7}, year = {2016}, month = {2016 Feb 16}, pages = {855-69}, abstract = {

Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a {\textquotedblleft}precise color{\textquotedblright} MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is ≈90\% and we have obtained efficiencies of \> 80\% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living\ C. elegans.

Additional authors include:

Xin Jin, Joan Pulupa, Christine Cho, Mustafa Mir, Mohamed El Beheiry, Xavier Darzacq, Marcelo Nollmann, Maxime Dahan, Carl Wu, Timoth{\'e}e Lionnet, J. Alexander Liddle, and Cornelia I. Bargmann

}, doi = {10.1364/BOE.7.000855}, author = {Abrahamsson, Sara and Ilic, Rob and Wisniewski, Jan and Mehl, Brian and Yu, Liya and Chen, Lei and Davanco, Marcelo and Oujedi, Laura and Fiche, Jean-Bernard and Hajj, Bassam} } @article {46925, title = {Nuclear accessibility of β-actin mRNA is measured by 3D single-molecule real-time tracking.}, journal = {Journal of Cell Biology}, volume = {209}, year = {2015}, month = {2015 May 25}, pages = {609-19}, abstract = {

Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that β-actin mRNAs freely access the entire nucleus and fewer than 60\% of mRNAs are more than 0.5 {\textmu}m away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization.

}, issn = {1540-8140}, doi = {10.1083/jcb.201411032}, author = {Smith, Carlas S and Preibisch, Stephan and Joseph, Aviva and Abrahamsson, Sara and Rieger, Bernd and Myers, Eugene and Singer, Robert H and Grunwald, David} } @article {5522, title = {Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.}, journal = {Nature Methods}, volume = {10}, year = {2013}, month = {2013}, pages = {60-3}, abstract = {

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.

}, issn = {1548-7105}, doi = {10.1038/nmeth.2277}, author = {Abrahamsson, Sara and Chen, Jiji and Hajj, Bassam and Stallinga, Sjoerd and Katsov, Alexander Y and Wisniewski, Jan and Mizuguchi, Gaku and Soule, Pierre and Mueller, Florian and Darzacq, Claire Dugast and Darzacq, Xavier and Wu, Carl and Bargmann, Cornelia I and Agard, David A and Dahan, Maxime and Gustafsson, Mats G L} }