Module Director: Anita Hendrickson
206 685-2273
anitah@u.washington.edu
A. Description
The Morphological Imaging (MI) module provides NEI and other funded visual neuroscience investigators with a variety of morphological and morphometry services. These include 1) preparation and sectioning of tissue for electron microscopy (EM); 2) access to a well-maintained Phillips CM10 transmission EM; 3) regular and specialized EM techniques such as Vibratome tissue sectioning and embedding, and immunogold labeling, including training in these methods; 4) access to a Zeiss 510 multi-photon microscope and training in its use; 5) access to and training on a Nikon widefield microscope equipped with a 12 megapiixel Nikon CCD camera for high resolution bright field photography and an ultra-sensitive Hamamatsu CCD camera for imaging fluorescently-tagged probes; 6) digital image capture, processing and analysis using Adobe Photoshop, UIC MetaVue, AutoQuant, Autodeblur and NIH ImageJ; and 7) morphological image analysis and enhancement using SVI Huygens deconvolution software. These services offer investigators sophisticated image capture and analysis using equipment or software that would be difficult to fund in a single laboratory.
Daniel Possin, Research Scientist III, has overseen the MI module since 1982. He will continue to provide his unique blend of skills in morphologiy and computer graphics to MI users. Mr. Possin supervises two MI technicians. Edward Parker began work at the UW in July 2005 as a Research Scientist II. Mr. Parker is highly skilled in EM technology and was recently recruited from Univ. California Berkeley. He provides routine and specialized EM tissue processing and immunolabeling, as well as being responsible for training of students and faculty in EM methods and daily maintenance of the EM facility. A third FTE who is trained in computer graphics will be hired in summer 2005 to process digital images for manuscripts, posters and other scientific publications.
Location:
The MI module occupies three main areas. 1) RR 834 (390ft2). One side contains all wet lab facilities for EM tissue processing, EM tissue sectioning, glass knife making and Vibratome tissue sectioning. The other half contains Mr. Possin’s Apple MAC G5 computer, a new Microtek 2500F scanner and –an Epson 2200 printer used in record keeping and iimage processing. It also contains a MAC G5 computer and dual monitors which are used for image manipulation by the Huygens deconvolution software. Currently, additional space is being sought to separate the computer and wet lab facilities, but this will have to await the selection of a new Chair for Ophthalmology. 2) RR017 (296ft2) houses the Zeiss 510 multi-photon confocal microscope and associated equipment. This room is climate and air flow controlled to reduce fluctuations in the confocal lasers. 3)RR019 (373ft2) contains the Phillips CM10 transmission EM and an adjacent darkroom for EM negative developing. Both rooms have a filtered water supply and the darkroom has a temperature controlled water supply to insure correct and consistent film development.
Services:
EM tissue preparation and sectioning:. Tissue will be fixed, dehydrated and embedded appropriate for the desired EM techniques. Semi-thin and thin sections will be cut, stained, and supplied to investigators for light and EM examination. Alternatively, for less experienced investigators, thin sections will be examined and photographed in the EM by Mr. Parker with study prints provided to the investigator. Specialized EM techniques such as immunogold labeling will also be done by Mr. Parker or Mr. Possin as required by specific research groups. Training in tissue processing and EM usage for individual investigators, fellows and students will be the responsibility of Mr. Parker in consultation with Mr. Possin. Mr. Parker will also be responsible for aligning and maintaining the Phillips CD10 EM for daily use by investigators experienced in EM. 25% time of the FTE to be hired this summer will be used to print EM negatives for study prints, and then to scan and digitally manipulate selected EM negatives for publication. We have found this to be the most economical and time-efficient method of handling EM images.
Zeiss 510 Multiphoton Confocal Microscopy: Mr. Possin will schedule, maintain and provide routine service for this facility. He has received training by Zeiss personnel specifically for the 510, and has been able to troubleshoot all but the most serious problems with this new equipment. Mr. Possin attended the Imaging premeeting at ARVO 2005 and he will attend the International Confocal Workshop held at University of Briitish Columbia, Vancouver Canada in June 2006 for further exposure to modern imaging methodology and concepts. Mr. Possin will train individuals in confocal tissue preparation and the use of the microscope, and will assist users in obtaining satisfactory images on a daily basis. The Zeiss 510 currently is used 8-12 hrs/day, 6-7 days a week , so overseeing this instrument takes a major portion of Mr. Possin’s time.
Nikon Wide-field Microscope: Mr. Possin will be responsible for training individuals in the use of this new microscope facility. This is particularly important so that users maximize the capability of its dual CCD cameras, each of which has very specific photographic perimeters. He also will train users in MetaView, the image capture and analysis software used by the Nikon computer system. He will advise users on the advisability and methodology for the best capture of images for deconvolution processing.
Image analysis: Mr. Possin will consult with users of the Nikon and multiphoton microscopes about methodology to obtain the best images for deconvolution. He currently is training in the use of the Huygens propriatory software and will remain the chief interface for this facility. A FTE will be hired who is experienced in the use of programs such as Adobe Photoshop to manipulate and enhance digital images. Over the past three years digital image processing for publications and posters has become a major task for Mr. Possin and the MI module. The addition of an additional graphics person will provide 75% time for an ongoing and enhanced service to MI users for image processing for manuscripts, posters and other scientific publication.
B. History of the MI Module
The MI module has been part of the Univ. Washington Vision CORE grant since its inception in 1976, although it has gone through several revisions as histological, morphometry, and imaging techniques have changed, or the needs of UW users have altered. During the current grant period this module was called Morphology and Morphometry, but in 2005, the name was changed to Morphological Imaging (MI). In part this reflects that, over the current grant period, the need for film based photographic services has dropped dramatically while the demand for digital imaging and its processing has skyrocketed. It also reflects a decrease in user demand for morphometry services like whole neuron reconstruction. Those vision researchers who need sophisticated morphometry methodology have limited access to the Child Health and Development Center morphology module on campus. As part of this change, in 2005 the Research Imaging module of the current grant period was integrated into MI, including one FTE. This new person will be trained in graphics and will be primarily responsible for digital image processing and manipulation using Adobe Photoshop for manuscripts, posters and other scientific publication. A smaller part of their job will be to print EM negatives for study prints and to scan and manipulate EM images for publication. John Clark, Ph.D., professor and chair of Biological Structure was appointed in 2005 to serve with Anita Hendrickson as joint module director.
As mentioned above, over the current grant period there has been a major revolution in digital imaging technology for biological microscopy. This prompted the purchase of 1) a Zeiss 510 multiphoton confocal microscope in 2000, funded in large part by the Murdock Foundation; 2) a Nikon E1000 light microscope equipped with the latest Nikon wide field lenses, a 12 megapixel CCD camera to optimize brightfield photography, and an ultrasensitive Hamamatsu CCD camera for fluorescent probe imaging; and 3) SVI Huygens deconvolution software to further enhance information extraction from images obtained by these two new microscopes.
EM has and will continue to be a major service offered by MI. Although we have investigated converting to digital imaging for our Phillips CM10 EM, we believe that film still provides the highest resolution images at the lowest cost. Retrofitting our older EM for a CCD camera would be difficult and expensive. Although film availability is not an issue at present, we have purchased a 2 year supply to give us time to find alternative sources, if an availability problem does arise. MI will continue to offer EM negative printing for study prints, but we now utilize EM negative scanning and further processing by Adobe Photoshop for publication prints. The cooperative purchase of a modern transmission EM is being discussed by several groups at the UW, including the Vision CORE MI module.
C. Publications Using the MI Mod
La Spada AR, Fu Y-H, Sopher BL, Libby RA, Wang X, Li LY, Einum DD, Huang J, Possin D, Smith AC, Martinez RA, Koszdin KL, Treuting PM, Ware CB, Hurley JB, Ptacek LJ, Chen S. (2001). Polyglutamine-expanded ataxin-7 antagonizes CRX function and induces cone-rod dystrophy in a mouse model of SCA7. Neuron 31: 913-927 (Cover story)
Garden GA, Libby RT, Fu Y-H, Kinoshita Y, Huang J, Possin DE, Smith AC, Martinez RA, Fine GC, Grote SK, Ware CB, Einum DD, Morrison RS, Ptacek LJ, Sopher BL, La Spada AR. (2002). Polyglutamine-expanded ataxin-7 promotes non-cell autonomous Purkinje cell degeneration and displays proteolytic cleavage in ataxic transgenic mice. J Neurosci 22: 4897-4905 (Cover story)
La Spada AR & Taylor JP. (2003). Polyglutamines placed into context. Neuron 38: 681-684
Chen S, Peng G-H, Wang X, Smith AC, Grote SK, Sopher BL & La Spada AR. (2004). Interference of CRX-dependent transcription by ataxin-7 involves interaction between the glutamine regions and requires the ataxin-7 carboxy-terminal region for nuclear localization. Hum Mol Genet 13: 53-67 (Cover story)
Palhan VB, Chen S, Peng G, Tjernberg A, Chait BT, Gamper AM, Fan Y, La Spada AR^, Roeder RG^. (2005). Polyglutamine-expanded ataxin-7 inhibits STAGA histone acetyltransferase activity to produce retinal degeneration in a dominant negative manner. Proc Natl Acad Sci USA 102: 8472-8477
Brockerhoff, S. E., F. Rieke, H. R. Matthews, M. R. Taylor, B. Kennedy, I. Ankoudinova, G. A. Niemi , C. L. Tucker, M. Xiao, M. C. Cilluffo, G. L. Fain, and J. B. Hurley. (2003). Light stimulates a transducin-independent increase of cytoplasmic Ca2+ and suppression of current in cones from the zebrafish mutant nof. J. of Neurosci. 23 (2), 470 – 480.
Kennedy, B. N., G. W. Stearns, V. Ramamurthy, I. Ankoudinova, D.W. Raible, J. B. Hurley, and S. E. Brockerhoff. (2004) Zebrafish rx3 and mab21l2 are required for eye morphogenesis. Dev. Biol. 270, 336 – 49.
Taylor, M. R., H. A. Van Epps, M. J.B. Hurley and S. E. Brockerhoff (2004). A zebrafish model for pyruvate dehydrogenase deficiency: rescue of neurological dysfunction and embryonic lethality using a ketogenic diet. Proc. Natl. Acad. Sci. USA 101 (13), 4584-4589.
Van Epps H. A. , M. Hayashi, L. Lucast, G. W. Stearns, J. B. Hurley, P. De Camilli, S. E. Brockerhoff (2004). The zebrafish nrc mutant reveals a role for the polyphosphoinositide phosphatase Synaptojanin 1 in cone photoreceptor ribbon anchoring. J. of Neuroscience, 24:8641-50
Taylor MR, Kikkawa S, Diez-Juan A, Ramamurthy V, Kawakami K, Carmeliet P, Brockerhoff S. E. (2005). The Zebrafish pob Gene Encodes a Novel Protein Required for Survival of Red Cone Photoreceptor Cells. Genetics; Feb 16;
Nawrot M, West K, Huang J, Possin DE, Bretscher A, Crabb JW, Saari JC. Cellular retinaldehyde-binding protein interacts with ERM-binding phosphoprotein 50 in retinal pigment epithelium. Invest Ophthalmol Vis Sci 2004;45:393-401.
Saari JC, Nawrot M, Kennedy MN, Garwin GG, Hurley JB, Huang J, Possin DE, Crabb JW. Visual cycle impairment in cellular retinaldehyde binding protein (CRALBP) knockout mice results in delayed dark adaptation. Neuron 2001;29:739-748 (B/I; M/M).
Pendergrass,W., Wolf, N, Poot, M. Efficacy of MitoTracker Green and CMXRosamine to measure changes in mitochondrial membrane potentials in living cells and tissues. Cytometry Part A.61A:162-9 , 2004.
Wolf, N., Penn, P., Pendergrass, W., Van Remmen, H., Bartke, A., Rabinovitch, P., Martin, G.M. Age-related cataract progression in five mouse models for anti-oxidant protection or hormonal influence. Exp Eye Res. 2005
Schriner, S., Linford, N., Martin, G., Treuting,P., Ogburn, C., Emond, M., Coskun, P., Ladiges, W., Wolf,N., V an Remmen, H., Wallace, D., Rabinovitch, P. Extension of murine lifespan by overexpression of catalase targeted to mitochondria. Science,.E pub, 2005.
