Advanced Microscopies

Invention of a light microscope with 7-10X improved axial resolution

Light microscopy, especially fluorescence microscopy, has become the cornerstone of modern cell biology research. Previously, Dr. John Sedat (UCSF) and I had developed wide-field deconvolution microscopes that provided significantly enhanced resolution compared to conventional microscopes. Our design and software are available commercially through a licensing agreement with Applied Precision, Inc. of Seattle. Because of their high resolution and superb sensitivity (10-100x that of confocal microscopes), these microscopes have become the instrument of choice for in vivo studies such as those using Green Fluorescent Protein. While we continue to develop and improve on the data collection, deconvolution, and analysis software to facilitate our own studies on cellular structure, we have long sought to further improve resolution, especially in the difficult focus (Z axis) direction.

A brilliant postdoc (Dr. Mats Gustafsson) working with Sedat and myself has invented a new class of interference-based microscopies that provide dramatic resolution enhancements. The basic idea is to position the sample between two objective lenses. Emitted fluorescent light from the two lenses is then coherently combined and imaged by a CCD detector. Because constructive interference is critically-dependent on the precise matching of the paths lengths between the sample and the detector, the resolution in the Z direction is improved. Alternatively, one can use the same system to coherently interfere the emission light reaching the sample. As previously shown for the standing wave microscope, this can also provide higher resolution Z information. While both of these approaches provide higher axial resolution than possible with either conventional or confocal microscopes, they suffer because some Z information is systematically absent. This can be remedied and the resolution further extended by combining the illumination interference with the image interference. The result is a spectacular improvement in Z resolution to 7-10x that possible with conventional or confocal microscopes. In fact, the resolution is now greater by a factor of 2 in the axial (Z) direction than in the XY plane. The validity of this approach has been demonstrated by an initial prototype - all of the imaging properties expected from theory have been achieved. The full-width at half height in the axial direction is 70nm! Further, this resolution can be obtained on real biological samples. This pushes the light microscope into resolution regimes previously only attainable with electron microscopy.

Development of Automated Electron Tomography

A necessary aspect of our analysis of cellular structures such as centrosomes and chromosomes has been the development of EM-Tomography. This work is in close collaboration with Dr. John Sedat and his lab at UCSF. This technique, uniquely is suited for examining the highly individual supramolecular assemblies that typify cell biology. While single particle reconstruction methods are clearly the best method for the analysis of large well-behaved complexes such as ribosomes, still larger structures are generally not sufficiently homogeneous to average. For these samples (organelles, chromosomes, cytoskeletal components, etc.) all data needs to be collected from a single specimen. Efficient data collection (typically 100-150 different tilted images) under low dose conditions is critical to optimal reconstructions.

While EM Tomography is extremely powerful, and has the potential to dramatically impact cell biology, its technical difficulty has limited its general use. For our own work as well as for the cell biology community, we wish to sufficiently automate all aspects of EM Tomography to make it routine. While we are probably the world leaders in EM Tomography, there is much to do - both for automating the whole process but also to improve the quality of the reconstructions. The central problems are: i) lack of stage eucentricity which leads to changes in position and focus after each tilt, ii) sensitivity to beam damage, iii) determining the relationship between electron intensity and specimen mass, iv) post-data collection image alignment, and v) optimizing the reconstruction process to correct for limited number of tilts and finite detector size.

During the past 5 years we have made significant progress in this area. Our automated tomography data collection has been routinely used for collection of high quality data. This solves the problem of tracking and correcting problems due to changes in focus and translation during data collection. We have carried out an in-depth analysis of the mechanism of image formation for the rather thick samples (0.2 - 0.7µm) typically used in cellular tomography. Using an analysis method previously only used in material science at atomic resolutions (exit wavefront reconstruction), we determined that the observed images are a combination of both phase contrast as well as amplitude contrast (they were expected to be pure amplitude contrast) and are contaminated by multiple elastic and inelastic scatter. Exit wavefront reconstruction images were used to assess the performance of multi-focal filter methods for removing the unwanted effects of multiple elastic and multiple inelastic scatter. We have also worked out cryo low dose data collection methods to optimize data resolution and high pressure freezing methods for improving specimen preservation. Current efforts are also directed at improved 3D reconstruction methodologies.

Relevant Publications

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).