Cryogenic Optical Microscope
Faculty Advisor: Prof. Greg Kowalski
Sponsors: Dale Larson , James Hogle, Ph.D. (Harvard Medical School)
Design Team:
Mohammad AliJohn DelcoreSarah KaufmannDavid Rezac
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)
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)
• 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)
• 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
• 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
• The sample is embedded in vitreous ice Stable below -140oC
• Vitreous ice is the “glassy” amorphous solid form of water Does not scatter electrons Low vapor pressure
Why below -140oC?
(Source: www.nims.go.jp)
• Numerical Aperture (NA)
Indicates the resolving power of the lens Larger NA = better resolution Inversely related: WD NA
Optical Microscope
(Source: www.microscopyu.com)
• Numerical Aperture (NA)
Indicates the resolving power of the lens Larger NA = better resolution Inversely related: WD NA
Optical Microscope
(Source: www.microscopyu.com)
Design #1
Preliminary Designs
Design #2
Design #3
Thermal Modeling
Design #3
TGU=16oC
• Able to function at LN2 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
Design Evolution
Cold LensPosition
Vacuum Chamber
Inner Skirt to Hold N2 Gas
Post to Hold Samplefor Imaging
Outer Housing
Window(Image Path)
LN2 Level
• Cold Finger
• LN2 Workstation
TEM Cryo-Transfer Apparatus
Current Design
Cold Finger
Upper Assembly(Contains Objective Lens)
Work Station
Current Design
Current Design
Current Design
LN2 Reservoir
Current Design
SamplePosition
Prototype
External Optical Microscope
BoilingCHF
(Source: www.alamthermal.com)
(Source: www.nuc.berkeley.edu)(Source: www.spaceflight.esa.int)
Boiling
•qin=9.47W•SS in 116 minutes•1.98kg LN2
•qin=3.08W•SS in 26 minutes•0.075kg LN2
Boiling
•qin=9.47W•SS in 116 minutes•1.98kg LN2
•qin=3.08W•SS in 26 minutes•0.075kg LN2
Testing
Thermocouples inKey Locations
Complete Assembly in Imaging Position
External NitrogenFlush
Testing
Test #1
Test #4Test #3
Test #2
Pre-CooledCopper
=Tsample
Testing
Test #1
Test #4Test #3
Test #2
Pre-CooledCopper
=Tsample
-200
-180
-160
-140
-120
-100
-80
-60
-40
-20
0
0 5 10 15 20 25 30 35
Time (min)
Tem
per
atu
re (
C)
Testing
Test 1
Test 2
Test 3
Test 4
ImagingCutoff
Microscope
Focused: 2.7µPartial FocusAberrations
Resolution Target
10µ Line Spacing
(filters omitted)
Special Thanks to:Antoine van Oijen
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
• 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
Questions?