New Technologies for Light and Electron Microscopy

Low light level 3D deconvolution: We continue collaborations with Sedat (UCSF). New studies raise awareness that extreme photosensitivity plagues in vivo fluorescence imaging – e.g. cell cycle progression is severely perturbed at photon doses below 1% of typical. While SIM hardware developments at UCSF and JFRC now allow dynamic processes to be captured with unprecedented resolution, the utility of such breakthrough performance is severely compromised by phototoxicity. To surmount this, we developed a powerful deconvolution method (ER-DECON) that provides high quality reconstructions at previously unworkably low light levels, promising leaps in our understanding of dynamic biological systems.

Electron counting detector for cryoEM: For large viruses it is now possible to obtain 3-3.5Å resolution by single particle cryoEM, sufficient to trace the polypeptide within experimental maps. The big challenge is obtaining comparable resolutions with smaller, lower symmetry samples. Funded initially by HHMI and an NSF grant (Agard), we have been pushing the development (collaborating with LBNL and Gatan) of a direct detection camera, that most importantly, is optimized for single electron counting; allowing individual electron events to be detected and centroided to sub-pixel accuracy. By counting primary electrons rather than integrating the charge, as in other direct detection and CCD cameras, the readout noise and, more importantly, noise arising from the statistical deposition of energy by the primary electron (Landau noise) can be essentially eliminated and the DQE dramatically improved. Because readout rates are high and there is no penalty for multiple readouts, stacks of dose-fractionated frames can be recorded throughout a single “exposure”. Recently Grigorieff showed that the electron beam causes particle images to move during the exposure, significantly blurring the recorded image. Together with Yifan Cheng (UCSF), we developed an image alignment algorithm allowing frames to be re-registered to sub-pixel accuracy, resulting in dramatic image improvement; effectively turning every image into that rare “perfect” image. This allowed us to determine the structure of the 700KDa proteasome core to an unprecedented 3.3Å resolution, heralding a new phase in cryoEM. 

Relevant Publications

"Influence of electron dose rate on electron counting images recorded with the K2 camera," Li X, Zheng SQ, Egami K, Agard DA, Cheng Y., J Struct Biol. 2013 Aug 20 (html, pdf)

"Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM," Li, Xueming; Mooney, Paul; Zheng, Shawn; Booth, Christopher R.; Braunfeld, Michael B.; Gubbens, Sander; Agard, David A.; Cheng, Yifan, Nature Methods (2013), 10(6), 584-590. (html, pdf)

"Fast multicolor 3D imaging using aberration-corrected multifocus microscopy," Sara Abrahamsson, Jiji Chen, Bassam Hajj, Sjoerd Stallinga, Alexander Y Katsov, Jan Wisniewski, Gaku Mizuguchi, Pierre Soule, Florian Mueller, Claire Dugast Darzacq, Xavier Darzacq, Carl Wu , Cornelia I Bargmann , David A Agard , Maxime Dahan, & Mats G L Gustafsson, Nature Methods, 2013, 10(1), 60-3 (pdf)

"A Method for Integrative Structure Determination of Protein-Protein Complexes," Schneidman-Duhovny Dina; Rossi Andrea; Avila-Sakar Agustin; Kim Seung Joong; Velazquez-Muriel Javier; Strop Pavel; Liang Hong; Krukenberg Kristin A; Liao Maofu; Kim Ho Min; Sobhanifar Solmaz; Dötsch Volker; Rajpal Arvind; Pons Jaume; Agard David A; Cheng Yifan; and Sali Andrej, Bioinformatics (Oxford, England) (2012) (html, pdf).

"Effect of depth dependent spherical aberrations in 3D structured illumination microscopy." Arigovindan M, Sedat JW, Agard DA. Opt Express. (2012), 20(6), 6527-41. (pdf)

Interferometer-based structured-illumination microscopy utilizing complementary phase relationship through constructive and destructive image detection by two cameras, Shao L; Winoto L; Agard D A; Gustafsson M G L; Sedat J W, Journal of microscopy (2012), 246(3), 229-36.

"A distributed multi-GPU system for high speed electron microscopic tomographic reconstruction," Zheng, Shawn Q.; Branlund, Eric; Kesthelyi, Bettina; Braunfeld, Michael B.; Cheng, Yifan; Sedat, John W.; Agard, David A., Ultramicroscopy (2011), 111(8), 1137-1143. (pdf)

Zheng, Shawn Q.; Sedat, J. W.; Agard, D. A.. "Automated data collection for electron microscopic tomography." Methods in Enzymology (2010), 481(Cryo-EM, Part A), 283-315. (pdf)

Carlton PM, Boulanger J, Kervrann C, Sibarita JB, Salamero J, Gordon-Messer S, Bressan D, Haber JE, Haase S, Shao L, Winoto L, Matsuda A, Kner P, Uzawa S, Gustafsson M, Kam Z, Agard DA, Sedat JW., "Inaugural Article: From the Cover: Fast live simultaneous multiwavelength four-dimensional optical microscopy," Proc Natl Acad Sci U S A. (2010), 107(37), 16016-22. (html, pdf, commentary)

Kner, P; Winoto, L; Agard, DA.; Sedat, JW.; Conchello, J-A; Cogswell, CJ.; Wilson, T; Brown, TG., "Closed loop adaptive optics for microscopy without a wavefront sensor," Proceedings of SPIE (2010), 7570, 757006-757006-9. (pdf)

Arigovindan M, Shaevitz J, McGowan J, Sedat JW, Agard DA., "A Parallel Product-Convolution approach for representing the depth varying Point Spread Functions in 3D widefield microscopy based on principal component analysis," Opt Express., 2010 Mar 29, 18(7), 6461-76. (pdf)

Kner P, Sedat JW, Agard DA, Kam Z., "High-resolution wide-field microscopy with adaptive optics for spherical aberration correction and motionless focusing," J Microsc., 2010, 237(2), 136-47. (html, pdf)

Xu F, Xu W, Jones M, Keszthelyi B, Sedat J, Agard D, Mueller K, "On the efficiency of iterative ordered subset reconstruction algorithms for acceleration on GPUs," Comp Met Prog Biomed, 2009. (html, pdf)

Suloway C, Shi J, Cheng A, Pulokas J, Carragher B, Potter CS, Zheng SQ, Agard DA, Jensen GJ, "Fully automated, sequential tilt-series acquisition with Leginon," J Struct Biol. (2009) Jul, 167(1):11-18. (html, pdf)

Shao L, Isaac B, Uzawa S, Agard DA, Sedat JW, Gustafsson MG., "I5S: wide-field light microscopy with 100-nm-scale resolution in three dimensions," Biophys J. (2008), 94(12), 4971-83 (html, pdf)

Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, Kner P, Burke B, Cardoso MC, Agard DA, Gustafsson MG, Leonhardt H, Sedat JW., "Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy." Science (2008), 320(5881), 1332-6. (html, pdf)

E.F. Hom, F. Marchis, T.K. Lee, S. Haase, D.A. Agard, and J.W. Sedat, "AIDA: an adaptive image deconvolution algorithm with application to multi-frame and three-dimensional data," J Opt Soc Am A Opt Image Sci Vis. 2007 Jun;24(6):1580-600 (pdf).

Zheng SQ, Kollman JM, Braunfeld MB, Sedat JW, Agard DA, "Automated acquisition of electron microscopic random conical tilt sets," J Struct Biol. 2007 Jan;157(1):148-55.161.(html or pdf).

Zheng SQ, Keszthelyi B, Branlund E, Lyle JM, Braunfeld MB, Sedat JW, and Agard DA,"UCSF tomography: An integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction," J Struct Biol. 2006 Jun 23 (html or pdf).

W. C. Moss, S. Haase, J. M. Lyle, D. A. Agard & J. W. Sedat, "A novel 3D wavelet-based filter for visualizing features in noisy biological data," J. Microscopy, (2005), 219, 43-49. (pdf).

Koenig P, Braunfeld M, Agard DA., "Use of surface affinity enrichment and cryo-embedding to prepare in vitro reconstituted mitotic chromosomes for EM tomography." Ultramicroscopy. 2005 Jul;103(4):261-74. (html or pdf).

Hanser BM, Gustafsson MG, Agard DA, Sedat JW, "Phase-retrieved pupil functions in wide-field fluorescence microscopy." J Microsc. 2004 Oct;216(Pt 1):32-48 (html or pdf).

Zheng, Q.S., Braunfeld, M.B., Sedat, J.W, and Agard, DA. "An improved strategy for automated electron microscopic tomography," J. Struct. Biol., (2004) 147(2):91-101 (html or pdf).

Hanser BM, Gustafsson MG, Agard DA, Sedat JW. (2003). Phase retrieval for high-numerical-aperture optical systems. Opt Lett. 2003 May 15;28(10):801-3 (pdf).

Kam Z., Hanser, B., Gustafsson, M.G., Agard, D.A., Sedat, J.W. (2001) Computational adaptive optics for live three-dimensional biological imaging. Proc Natl Acad Sci U S A. 98(7):3790-5 (pdf).

Gustafsson, M.G., Agard, D.A., Sedat, J.W. (1999) I5M: 3D widefield light microscopy with better than 100 nm axial resolution. J Microsc. 195 ( Pt 1):10-6 (pdf).

Han K F; Sedat J W; Agard D A Practical image restoration of thick biological specimens using multiple focus levels in transmission electron microscopy. Journal of Structural Biology (1997), 120(3), 237-44. (pdf).

Kam, Z., Margolis, H.J., Agard, D.A., and Sedat, J.W. (1996). Three-dimensional microscopy in thick biological samples: a fresh approach for adjusting focus and correcting spherical aberration. Bioimaging 5: 40-49. (pdf).

Gustafsson, Mats G. L.; Sedat, John W.; Agard, David A.. Method and apparatus for three-dimensional microscopy with enhanced depthresolution. PCT Int. Appl. (1996), No pp. given.

Han, K.F., Gubbens, A.J., Sedat, J.W., and Agard, D.A. (1996). Optimal strategies for imaging thick biological specimens: exit wavefront reconstruction and energy-filtered imaging. J. Microscopy 183 (2): 124-132.(pdf).

Fung, J.C., De Ruijter, W.J., Chen, H., Abbey, C.K., Sedat, J.W., and Agard, D.A. (1996). Toward fully automated high-resolution electron tomography. J. Struct. Biol. 116(1): 181-9 (pdf).

Chen, H., Hughes, D.D., Chan, T.-A., Sedat, J.W. and Agard, D.A. (1996). IVE (image visualization environment): a software platform for all three-dimensional microscopy applications. J. Struct. Biol. 116: 56-60 (pdf).

Scalettar, B.A., Swedlow, J.R., Sedat, J.W., and Agard, D.A. (1996). Dispersion, aberration and deconvolution in multi-wavelength fluorescence images. J. Microscopy 182 (1): 55-60.(pdf).

Gustafsson, M.G.L., Agard, D.A., and Sedat, J.W. (1995). Seven-fold improvement of axial resolution in 3-D widefield microscopy using two objective lenses. proc. SPIE 2412: 145-156.

Han, K.F., Sedat, J.W., and Agard, D.A. (1995). Mechanism of image formation for thick biological specimens: exit wavefront reconstruction and electron energy-loss spectroscopic imaging. J. Microscopy 178 (2): 107-119. (pdf).

Han, K. F.; Sedat, J. W.; Agard, D. A.. Image restoration for thick biological specimens using a through focus series as applied to electron tomography. Electron Microscopy 1994, Proceedings of the International Congress on Electron Microscopy, 13th, Paris, July 17-22, 1994 (1994), 1 503-504.

Braunfeld, M.B., Koster, A.J., Sedat, J.W., and Agard, D.A. (1994). Cryo automated electron tomography: towards high resolution reconstructions of plastic embedded structures. J. Microscopy 174: 75-84. (pdf).

Kam, Z, Jones, M.O., Chen, H., Agard, D.A., and Sedat, J.W. (1993). Design and construction of an optimal illumination system for quantitative wide-field multi-dimensional microscopy. J. Bioimaging 1: 71-81. (pdf).

Koster, A.J., Braunfeld, M.B., Fung, J.C., Abbey, C.K., Han, K.F., Liu, W., Chen, H., Sedat, J.W., and Agard, D.A. (1993). Towards automatic three-dimensional imaging of large biological structures using intermediate voltage electron microscopy. EMSA Bulletin 23: 176-188. (pdf).

Swedlow, J.R., Sedat, J.W., and Agard, D.A. (1993). Multiple chromosomal populations of topoisomerase II detected in vivo by time-lapse, three-dimensional wide field microscopy. Cell 73: 97-108. (pdf).

Koster, A.J., Braunfeld, M.B., Sedat, J.W., and Agard, D.A. (1992). Automated TEM control for electron tomography. Electron Microscopy 1: 119-123. (pdf).

Chen, H., Clyborne, W., Sedat, J.W., and Agard, D. A. (1992). PRISM: An integrated system for display and analysis of three-dimensional microscope images. SPIE Biomedical Image Processing and Three-Dimensional Microscopy 1660: 784-790. (pdf).

Koster, A.J., Chen, H., Sedat, J.W., and Agard, D.A. (1992). Automated microscopy for electron tomography. Ultramicroscopy 46: 207-227. (pdf).

Holmes, T.J., Liu, Y-H., Khosla, D., and Agard, D.A. (1991). Increased depth-of-field and stereo pairs of fluorescence micrographs via inverse filtering and maximum likelihood estimation. J. Microscopy 164 (3): 217-237. (pdf).

Kam Z; Minden J S; Agard D A; Sedat J W; Leptin M Drosophila gastrulation: analysis of cell shape changes in living embryos by three-dimensional fluorescence microscopy. Development (Cambridge, England) (1991), 112(2), 365-70.

Kam, Z., Minden, J.S., Agard, D.A., Sedat, J.W., and Leptin, M. (1991). Drosophila gastrulation: analysis of cell shape changes in living embryos by three-dimensional fluorescence microscopy. Development 112: 365-370. (pdf).

Minden, Jonathan; Kam, Zvi; Agard, David; Sedat, John; Alberts, Bruce. Embryonic lineage analysis using three-dimensional, time lapse in vivo fluorescent microscopy. Proceedings of SPIE-The International Society for Optical Engineering (1990), 1205(Bioimaging Two-Dimens. Spectrosc.), 29-42. (pdf).

Hiraoka, Y., Sedat, J.W., and Agard, D.A. (1990). Determination of the three-dimensional imaging properties of an optical microscope system: partial confocal behavior in epi-fluorescence microscopy. Biophys. J., 57: 325-333.

Chen, H., Sedat, J.W., and Agard, D.A. (1989). Software and hardware for 3-D gray-level image analysis and quantization. SPIE New Methods in Microscopy and Low Light Imaging 1161: 31-41. (pdf).

Agard, D.A., Hiraoka, Y., and Sedat, J.W. (1989). Three-dimensional microscopy: image processing for high-resolution subcellular imaging. SPIE New Methods in Microscopy and Low Light Imaging 1161: 24-30. (pdf).

Minden, J.S., Agard, D.A., Sedat, J.W., and Alberts, M.B. (1989). Direct cell lineage in Drosophila melanogaster by time-lapse, three-dimensional optical microscopy of living embryos. J. Cell Biol. 109: 505-516.

Shaw, P.J., Agard, D.A., Hiraoka, Y., and Sedat, J.W. (1989). Tilted view reconstruction in optical microscopy. Three-dimensional reconstruction of Drosophila melanogaster embryo nuclei. Biophys. J. 55: 101-110. (pdf).

Hiraoka, Y., Sedat, J.W., and Agard, D.A. (1987). The use of a charge-coupled device for quantitative optical microscopy of biological structures. Science 238: 36-41.(pdf).

Agard, D.A. (1983). A least-squares method for determining structure factor in three-dimensional tilted-view reconstruction. J. Mol. Biol. 167: 849-852. (pdf).

Agard, D.A., Steinberg, R.A., and Stroud, R.M. (1980). Quantitative analysis of electrophoretograms: a mathematical approach to super-resolution. Analyt. Biochem. 111: 257-268. (pdf).

Agard, D.A. and Sedat, J.W. (1980). Progress in the three-dimensional analysis of biological specimens utilizing image processing techniques. J. SPIE 264: 110-117.

Stroud, R.M. and Agard, D.A. (1979). Structure determination of asymmetric membrane profiles using an iterative fourier method. Biophys. J. 25(3): 495-512. (pdf).