1. Cryogenic Optical Microscope Faculty Advisor: Prof. Greg Kowalski Sponsors: Dale Larson, James Hogle, Ph.D. (Harvard Medical School) Design Team: Mohammad Ali John Delcore Sarah Kaufmann David Rezac

2. Problem Statement Infected, frozen sample to be analyzed in TEM  Very high resolution  Required to better understand cell behavior Imaging complications  Small field of view leads to lots of time searching  Electron bombardment  Congested area around cell (Source: www.brockhouse.mcmaster.ca)

3. Problem Statement Infected, frozen sample to be analyzed in TEM  Very high resolution  Required to better understand cell behavior Imaging complications  Small field of view leads to lots of time searching  Electron bombardment  Congested area around cell` (Source: www.lifesci.ucsb.edu)

4. It is necessary to first image the sample in an optical microscope (OM) and identify areas of interest Currently, frozen samples cannot be viewed in the OM Problem Statement (cont.) (Source: webphysics.davidson.edu)

5. Maintain specimen below -140°C Provide means to image the sample (microscope) Isolate sample from significant vibrations Protect sample grid from stresses that may cause deformation Prevent contamination by water contact (condensation) Design Requirements

6. Aid research and development for improved therapeutic advancements Improve quality and quantity of TEM images Enable microscopists  Label molecular components in OM  Analyze with high resolution of TEM Requires no additional expertise  Familiar operations for microscopists Impact Statement

7. The sample is embedded in vitreous ice  Stable below -140 o C Vitreous ice is the “glassy” amorphous solid form of water  Does not scatter electrons  Low vapor pressure Why below -140 o C? (Source: www.nims.go.jp)

8. Numerical Aperture (NA)  Indicates the resolving power of the lens  Larger NA = better resolution  Inversely related: WD NA Optical Microscope (Source: www.microscopyu.com)

9. Numerical Aperture (NA)  Indicates the resolving power of the lens  Larger NA = better resolution  Inversely related: WD NA Optical Microscope (Source: www.microscopyu.com)

10. Design #1 Preliminary Designs Design #2 Design #3

11. Thermal Modeling Design #3 T GU =16 o C

12. Able to function at LN 2 temperature Manufactured by Microthek Corp. in Germany 80x magnification 0.8 Numerical Aperture.96 mm working distance Withstands cyclic testing to liquid helium temperatures Cold Temperature Objective Lens

13. Design Evolution Cold Lens Position Vacuum Chamber Inner Skirt to Hold N 2 Gas Post to Hold Sample for Imaging Outer Housing Window (Image Path) LN 2 Level

14. Cold Finger LN 2 Workstation TEM Cryo-Transfer Apparatus

15. Current Design Cold Finger Upper Assembly (Contains Objective Lens) Work Station

16. Current Design

18. LN 2 Reservoir

19. Current Design Sample Position

20. Prototype

21. External Optical Microscope

22. Boiling CHF (Source: www.alamthermal.com) (Source: www.nuc.berkeley.edu)(Source: www.spaceflight.esa.int)

23. Boiling q in =9.47W SS in 116 minutes 1.98kg LN 2 q in =3.08W SS in 26 minutes 0.075kg LN 2

24. Boiling q in =9.47W SS in 116 minutes 1.98kg LN 2 q in =3.08W SS in 26 minutes 0.075kg LN 2

25. Testing Thermocouples in Key Locations Complete Assembly in Imaging Position External Nitrogen Flush

26. Testing Test #1 Test #4Test #3 Test #2 Pre-Cooled Copper =T sample

27. Testing Test #1 Test #4Test #3 Test #2 Pre-Cooled Copper =T sample

28. Testing Test 1 Test 2 Test 3 Test 4 Imaging Cutoff

29. Microscope Focused: 2.7µPartial FocusAberrations Resolution Target 10µ Line Spacing (filters omitted) Special Thanks to: Antoine van Oijen

30. Current Prototype Allows for imaging in the optical microscope  Stable cold environment maintains specimen below -140°C for 10-15 minutes Provides imaging resolution down to 2.7µ Incorporates TEM cold finger into the design  Protects sample from additional stresses or possible water contamination  Saves time and effort Whole system is on a 1,000 lb optical table  Reduces vibrations

31. Continue design stage with thermal mass concept Refine microscope operation Reduce footprint and isolate optical components Function specific improvements:  Automate XY and Z stages  Purchase camera suited to application Recommendations

32. Questions?