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Cryogenic Optical Microscope

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Cryogenic Optical Microscope Cryogenic Optical Microscope MIM U 701-702 Technical Design Report Cryogenic Optical Microscope Project # 8 Final Report Design Advisor: Prof. Gregory Kowalski Design Team Mohammad Ali, John Delcore Sarah Kaufmann, David Rezac April 17, 2007 Department of Mechanical, Industrial and Manufacturing Engineering College of Engineering, Northeastern University Boston, MA 02115 1 Cryogenic Optical Microscope Design Team Mohammad Ali, John Delcore Sarah Kaufmann, David Rezac Design Advisor Sponsor Prof. Gregory Kowalski Harvard Medical School Abstract The transmission electron microscope (TEM) is extensively used in medical research because of its high magnification and resolution capabilities. One drawback of the TEM is that as the magnification increases, its field of view decreases, and more time is required to identify points of interest on a biological sample. This longer exposure can degrade the sample integrity. A solution is to first map the sample with fluorescent nano- particles, flash freeze it in vitreous ice at cryogenic temperatures, then study the sample under the optical microscope (OM). The OM provides a much larger field of view, allowing the identification of the key points of interest, before transferring it to the TEM. The described solution is comprised of a custom optical microscope with a built-in cooling system that is compatible with current TEM equipment. 2 TABLE OF CONTENTS Acknowledgements..............................................................................................................7 Copyright .............................................................................................................................7 1.0 PROBLEM STATEMENT............................................................................................8 2.0 IMPACT STATEMENT................................................................................................8 3.0 BACKGROUND ...........................................................................................................8 3.1 Purpose...............................................................................................................8 3.2 Patent Research..................................................................................................9 3.2.1 Cryogenic sample stage for an ion microscope .............................10 3.2.2 High resolution electron microscope cold stage ............................10 3.2.3 Freezing/perfusion microscope stage.............................................11 3.3 Current Sample Preparation Method ...............................................................12 3.3.1 Vitrobot..........................................................................................13 3.3.2 Blotting of Specimen .....................................................................13 3.3.3 Vitreous Ice Formulation ...............................................................15 3.3.4 Properties of Liquid Nitrogen (LN2) and Liquid Ethane ...............15 3.3.5 Intermediate Container...................................................................15 3.3.6 Transfer to Cold Finger..................................................................16 3.4 Transmission Electron Microscope (TEM) .....................................................17 3.4.1 3-Dimensional Modeling in TEM..................................................18 3.4.2 Electron versus Optical Microscopy..............................................18 3.5 Optical Microscopy..........................................................................................19 3.5.1 Defining Optical Microscopes.......................................................19 3.5.2 Resolution......................................................................................20 3.5.3 Objective Lens Properties..............................................................21 3.5.4 Use of Air Lenses...........................................................................23 4.0 MAPPING....................................................................................................................23 5.0 VITREOUS ICE ..........................................................................................................24 6.0 INITIAL DESIGN CONCEPTS..................................................................................25 6.1 Design Concept # 1..........................................................................................25 6.2 Design Concept # 2..........................................................................................26 6.3 Design Concept # 3..........................................................................................28 6.4 Design Concept # 4..........................................................................................29 7.0 PROGRESSION OF DESIGN ....................................................................................29 7.1 Thermal Modeling ...........................................................................................30 7.2 Introduction of Cold Lens................................................................................33 7.3 Microscope Construction.................................................................................34 7.4 Intermediate Design.........................................................................................35 8.0 CURRENT DESIGN ...................................................................................................37 8.1 Incorporating Microscopists’ Opinion.............................................................37 8.2 Translation of the Workstation Assembly .......................................................41 8.3 Guide Rails/Vertical Motion of Objective Lens ..............................................44 8.4 External Optical Microscope ...........................................................................45 8.4.1 Light Source...................................................................................46 8.4.2 Excitation Filter .............................................................................46 3 8.4.3 Emission Filter...............................................................................47 8.4.4 Dichroic Beam Splitter..................................................................47 9.0 INTERMEDIATE MODIFICATIONS .......................................................................47 9.1 Attachment of X-Y Translator.........................................................................47 9.2 Attachment of Upper Chamber and Collar ......................................................48 9.3 Dry Zone Installment .......................................................................................49 9.4 Opening Skirt Notch ........................................................................................50 10.0 BOILING ...................................................................................................................50 10.1 Boiling Curve.................................................................................................51 10.1.1 Free Convective Boiling................................................................51 10.1.2 Nucleate Boiling ............................................................................51 10.1.3 Critical Heat Flux...........................................................................52 10.1.4 Transition and Film Boiling...........................................................52 10.2 Cryo-Transfer Apparatus ...............................................................................53 10.3 Thermal Modeling .........................................................................................53 10.4 Initial Testing.................................................................................................57 10.5 Part Modifications and Further Modeling......................................................58 10.5.1 Initial Modification Concept..........................................................58 10.5.2 Final Modification Concept...........................................................60 10.6 Further Testing...............................................................................................62 11.0 CONTINUED TESTING...........................................................................................63 11.1 Test #1…........................................................................................................63 11.2 Test #2…........................................................................................................67 11.3 Test #3…........................................................................................................68 11.4 Test #4…........................................................................................................70 11.5 Test #5…........................................................................................................71 12.0 IMAGING..................................................................................................................72 12.1 Optical Mirror Images…................................................................................73 12.2 Resolution Target Images…. .........................................................................75 13.0 CONCLUSION..........................................................................................................75 14.0 RECCOMENDATIONS............................................................................................75 14.1 Thermal
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