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Shared Instrument Grant S10 application

Shared Instrument Grant S10 application. Cryo -EM upgrade of U Maryland EM Core Facility Submission deadline March 23 rd , 2009 I will need your write up by March 13 th , 2009. Why do we need cryo -EM upgrade?.

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Shared Instrument Grant S10 application

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  1. Shared Instrument Grant S10 application Cryo-EM upgrade of U Maryland EM Core Facility Submission deadline March 23rd, 2009 I will need your write up by March 13th, 2009

  2. Why do we need cryo-EM upgrade? • Traditional EM sample preparation relies on chemical fixation, dehydration and resin embedding • Chemical fixation is a very slow process, whereas cryo fixation can fix entire sample in seconds. • Chemical fixation with glutaraldehdye or praraformaldedhye may modify antigen or ultrastructure due to cross-linking • Resin embedding require dehydration, proteins may be lost during lengthy dehydration procedures • Resin may mask protein antigens and make them unaccessable by antibody detection • CryoEM techniques maintain cells in frozen hydrated states that are most representative of live cells

  3. Principal Steps of Cryopreparation Suspension Tissue Chemical Fixation Cryoprotection CRYO FIXATION Freeze Fracture Right now, we do not have any capability of cryo EM sample preparation or observation in UMB campus CryoUltramicrotomy Freeze Substitution Cryo Transfer Thin film replication Low Temp embedding Freeze Drying Room Temp Embedding Immuno labelling Ultramicrotomy Cryo TEM Microanalysis Amorphous Frozen studies Room Temp TEM Immunocytochemistry Morphology

  4. Principal Steps of Cryopreparation Suspension Tissue Chemical Fixation Cryoprotection CRYO FIXATION Freeze Fracture We will be able to perform all these experiments if we acquire cryo EM prep equipment and cryo EM upgrade listed in the next page CryoUltramicrotomy Freeze Substitution Cryo Transfer Thin film replication Low Temp embedding Freeze Drying Room Temp Embedding Immuno labelling Ultramicrotomy Cryo TEM Microanalysis Amorphous Frozen studies Room Temp TEM Immunocytochemistry Morphology

  5. Equipment requested in this S10 application • Tecnai T12 Cryo-upgrade • Low dose exposure techniques and performance test 13,406 • Cryo transfer holder and pumping station 69,947 • Cryo EM sample prep equipment • Cryo-ultramictrotome (Leica UC6 and EMFC 6 cryo chamber) 109,337 • Freeze-substitution system 40,588 • High pressure freezer (EMPACT2)and Rapid transfer system (RTS) 249,009 • Plunge freeze device (vitrobot) 85,000 Total $566,858

  6. High pressure FreezerLeica EMPACT2 • Rapid freezing of cells, tissues (including biopsy samples) under high pressure. • Thickness of tissue (200mm) can be 10-20 X greater than with plunge freezing • High pressures lowers the freezing point, increases the freezing rate, prevent the expansion of water and inhibit crystalline ice formation. • Cryoprotectants may not be required, thus eliminating the possibility of induced artifacts. • High pressure frozen samples can then be processed for viewing in the frozen hydrated state or at room temperature in the SEM or TEM • The rapid transfer system (RTS) accessory is designed for correlative LM/EM, i.e., for time resolution studies in conjunction with confocal microscopy. • A cell can be viewed under the confocal microscope, then quickly frozen with the EM PACT2/EM RTS, allowing the same cell to be viewed in the electron microscope. The EM RTS can also be used to enhance the speed of taking a biopsy in conjunction with the Leica’s new Microbiopsy Transfer System.

  7. Advantages of high pressure freezing Brain tissue Conventionally aldehyde fixed (CAF) Conventionally aldehyde fixed and HPF (CAF+HPF) J Struct Biol, 2008 March;161(3): 359-371 G. E. Sosinsky et. al.

  8. Advantages of high pressure freezing J Struct Biol, 2008 March;161(3): 359-371. G. E. Sosinsky et. al.

  9. Accessory kit requested with HPF • Rapid transfer system(RTS) • Flat specimen holder system for • Specimen tube system for particulate sample • Microbiopsy transfer system • Freeze fracture system • Live cell kit • Optical workstation

  10. Journal of Microscopy, Vol. 23, Pt 2 2008, pp. 317-328, P. Verkade

  11. Live cell kit For LM-EM correlative studies Journal of Microscopy, Vol. 23, Pt 2 2008, pp. 317-328, P. Verkade

  12. Leica EM AFS2 Freezer substitution • What is Freeze substitution? Low temperature substitution of dehydrating agents and fixatives into rapidly frozen samples allow for crosslinking of cellular components and the removal of water at temperatures low enough to avoid damaging effects of ambient-temperature dehydration • Cryo-fixation followed by freeze substitution demonstrated superior preservation of fine structure over chemical fixation method. • Low temperature embedding by using acrylic resin further enhance the efficiency in antigen localization. • Go to Leica website for more information about each instrument: • http://www.leica-microsystems.com/pdfs.nsf/(ALLIDs)/449FB6C60ECB0F57C125703C0032008D/$FILE/Leica_EMProductOverview-Brochure_EN.pdf

  13. Cryo-ultramicrotome • Cryo-ultramicrotome will allow us to section unfixed (or lightly fixed ) sample processed in high pressure freezer. • Frozen thin section an be immunogold labeled without dehydration and resin embedding • Frozen thin sections can also be transferred directly to the cryoTEM for observation • See next page for correlative microscopy studies by using ultrathin frozen sections generated from cryo-ultramicrotome

  14. Collodial gold-4 nm Fluoronanoprobe +silver enhancement

  15. If you are interested to be a user for this SIG, Please contact me (rhsia@umaryland.edu) as soon as possible I will need the followings from you by Friday March 13th: • Title of project: • Name • Department • Current support: including name of grants, grant type, grant number and dates, does not have to be NIH or RO1 • Research interests/activities • Project description describing how cryo sample preparation and cryo EM can be used in your research.  Please refer to as many equipment listed in the S10 as possible. • Include any previous EM data/images you have. • Enhancement of research by access to the cryo EM equipment • Any other references you need to cite in your write-up • I have put a few related pdf references in the EM facility website. (www.dental.umaryland.edu/Core-imaging/cryoEMref). Please feel free to download those files if they can be of help for your writing

  16. Thank you for your support • if you have any question please feel free to contact me (rhsia@umaryland.edu, X67992) • You could generate comparison and support data by trying out the cryo EM equipment during the our workshop on March 16-17. For more details, go to www.dental.umaryland.edu\Core-imaging\cryoworkshop Registration deadline is March 6th , 2009

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