@article {49173, title = {Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms.}, journal = {Science (New York, N.Y.)}, volume = {360}, year = {2018}, month = {2018 Apr 20}, abstract = {

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.

}, issn = {1095-9203}, doi = {10.1126/science.aaq1392}, author = {Liu, Tsung-Li and Upadhyayula, Srigokul and Milkie, Daniel E and Singh, Ved and Wang, Kai and Swinburne, Ian A and Mosaliganti, Kishore R and Collins, Zach M and Hiscock, Tom W and Shea, Jamien and Kohrman, Abraham Q and Medwig, Taylor N and Dambournet, Daphne and Forster, Ryan and Cunniff, Brian and Ruan, Yuan and Yashiro, Hanako and Scholpp, Steffen and Meyerowitz, Elliot M and Hockemeyer, Dirk and Drubin, David G and Martin, Benjamin L and Matus, David Q and Koyama, Minoru and Megason, Sean G and Kirchhausen, Tom and Betzig, Eric} } @article {48417, title = {Near-infrared fluorescent protein iRFP713 as a reporter protein for optogenetic vectors, a transgenic Cre-reporter rat, and other neuronal studies.}, journal = {Journal of Neuroscience Methods}, volume = {284}, year = {2017}, month = {2017 Apr 02}, pages = {1-14}, abstract = {

BACKGROUND: The use of genetically-encoded fluorescent reporters is essential for the identification and observation of cells that express transgenic modulatory proteins. Near-infrared (NIR) fluorescent proteins have superior light penetration through biological tissue, but are not yet widely adopted.

NEW METHOD: Using the near-infrared fluorescent protein, iRFP713, improves the imaging resolution in thick tissue sections or the intact brain due to the reduced light-scattering at the longer, NIR wavelengths used to image the protein. Additionally, iRFP713 can be used to identify transgenic cells without photobleaching other fluorescent reporters or affecting opsin function. We have generated a set of adeno-associated vectors in which iRFP713 has been fused to optogenetic channels, and can be expressed constitutively or Cre-dependently.

RESULTS: iRFP713 is detectable when expressed in neurons both in vitro and in vivo without exogenously supplied chromophore biliverdin. Neuronally-expressed iRFP713 has similar properties to GFP-like fluorescent proteins, including the ability to be translationally fused to channelrhodopsin or halorhodopsin, however, it shows superior photostability compared to EYFP. Furthermore, electrophysiological recordings from iRFP713-labeled cells compared to cells labeled with mCherry suggest that iRFP713 cells are healthier and therefore more stable and reliable in an ex vivo preparation. Lastly, we have generated a transgenic rat that expresses iRFP713 in a Cre-dependent manner.

CONCLUSIONS: Overall, we have demonstrated that iRFP713 can be used as a reporter in neurons without the use of exogenous biliverdin, with minimal impact on viability and function thereby making it feasible to extend the capabilities for imaging genetically-tagged neurons in slices and in vivo.

}, issn = {1872-678X}, doi = {10.1016/j.jneumeth.2017.03.020}, author = {Richie, Christopher T and Whitaker, Leslie R and Whitaker, Keith W and Necarsulmer, Julie and Baldwin, Heather A and Zhang, Yajun and Fortuno, Lowella and Hinkle, Josh J and Koivula, Pyry and Henderson, Mark J and Sun, Wenzhi and Wang, Kai and Smith, Jeffrey C and Pickel, Jim and Ji, Na and Hope, Bruce T and Harvey, Brandon K} } @article {47548, title = {in vivo brain imaging with adaptive optical microscope.}, journal = {Conference on Lasers and Electro-Optics (CLEO): Applications and Technology}, year = {2016}, month = {2016 Jun }, pages = {AM40.1}, abstract = {

The diffraction limited resolution of two photon and confocal microscope can be recovered using adaptive optics to explore the detailed neuronal network in the brains of zebrafish and mouse in vivo.

}, doi = {10.1364/CLEO_AT.2016.AM4O.1}, author = {Wang, Kai and Sun, Wenzhi and Ji, Na and Betzig, Eric} } @article {46849, title = {Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue.}, journal = {Nature Communications}, volume = {6}, year = {2015}, month = {2015-Jun-15}, pages = {7276}, abstract = {

Adaptive optics by direct imaging of the wavefront distortions of a laser-induced guide star has long been used in astronomy, and more recently in microscopy to compensate for aberrations in transparent specimens. Here we extend this approach to tissues that strongly scatter visible light by exploiting the reduced scattering of near-infrared guide stars. The method enables in vivo two-photon morphological and functional imaging down to 700 μm inside the mouse brain.

}, issn = {2041-1723}, doi = {10.1038/ncomms8276}, author = {Wang, Kai and Sun, Wenzhi and Richie, Christopher T and Harvey, Brandon K and Betzig, Eric and Ji, Na} } @article {45617, title = {Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.}, journal = {Science}, volume = {346}, year = {2014}, month = {2014 Oct 24}, pages = {1257998}, abstract = {

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

}, issn = {1095-9203}, doi = {10.1126/science.1257998}, author = {Chen, Bi-Chang and Legant, Wesley R and Wang, Kai and Shao, Lin and Milkie, Daniel E and Davidson, Michael W and Janetopoulos, Chris and Wu, Xufeng S and Hammer, John A and Liu, Zhe and English, Brian P and Mimori-Kiyosue, Yuko and Romero, Daniel P and Ritter, Alex T and Lippincott-Schwartz, Jennifer and Fritz-Laylin, Lillian and Mullins, R Dyche and Mitchell, Diana M and Bembenek, Joshua N and Reymann, Anne-Cecile and B{\"o}hme, Ralph and Grill, Stephan W and Wang, Jennifer T and Seydoux, Geraldine and Tulu, U Serdar and Kiehart, Daniel P and Betzig, Eric} } @article {6938, title = {Rapid adaptive optical recovery of optimal resolution over large volumes.}, journal = {Nature Methods}, volume = {11}, year = {2014}, month = {2014 Apr}, pages = {625-8}, abstract = {

Using a descanned, laser-induced guide star and direct wavefront sensing, we demonstrate adaptive correction of complex optical aberrations at high numerical aperture (NA) and a 14-ms update rate. This correction permits us to compensate for the rapid spatial variation in aberration often encountered in biological specimens and to recover diffraction-limited imaging over large volumes (\>240 mm per side). We applied this to image fine neuronal processes and subcellular dynamics within the zebrafish brain.

}, doi = {10.1038/nmeth.2925}, author = {Wang, Kai and Milkie, Daniel E and Saxena, Ankur and Engerer, Peter and Misgeld, Thomas and Bronner, Marianne E. and Mumm, Jeff and Betzig, Eric} }