Cryogenic optical microscope
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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. Problem Statement.

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Cryogenic Optical Microscope

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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


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)


Problem Statement (cont.)

  • 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

(Source: webphysics.davidson.edu)


Design Requirements

  • 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)


Impact Statement

  • 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


Why below -140oC?

  • 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

(Source: www.nims.go.jp)


Optical Microscope

  • Numerical Aperture (NA)

    • Indicates the resolving power of the lens

    • Larger NA = better resolution

    • Inversely related: WD NA

(Source: www.microscopyu.com)


Optical Microscope

  • Numerical Aperture (NA)

    • Indicates the resolving power of the lens

    • Larger NA = better resolution

    • Inversely related: WD NA

(Source: www.microscopyu.com)


Design #1

Preliminary Designs

Design #2

Design #3


Thermal Modeling

TGU=16oC

Design #3


Cold Temperature Objective Lens

  • 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


Design Evolution

Window

(Image Path)

Cold Lens

Position

LN2 Level

Vacuum Chamber

Inner Skirt to

Hold N2 Gas

Post to Hold Sample

for Imaging

Outer Housing


TEM Cryo-Transfer Apparatus

  • Cold Finger

  • LN2 Workstation


Current Design

Upper Assembly

(Contains Objective Lens)

Cold Finger

Work Station


Current Design


Current Design


Current Design

LN2 Reservoir


Current Design

Sample

Position


Prototype


External Optical Microscope


Boiling

CHF

(Source: www.alamthermal.com)

(Source: www.spaceflight.esa.int)

(Source: www.nuc.berkeley.edu)


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

Complete Assembly in

Imaging Position

External Nitrogen

Flush

Thermocouples in

Key Locations


Testing

Test #1

Test #2

Pre-Cooled

Copper

=Tsample

Test #3

Test #4


Testing

Test #1

Test #2

Pre-Cooled

Copper

=Tsample

Test #3

Test #4


Testing

Test 1

Test 2

Test 3

Test 4

Imaging

Cutoff


Microscope

Aberrations

Partial Focus

Focused: 2.7µ

10µ Line Spacing

(filters omitted)

Special Thanks to:

Antoine van Oijen

Resolution Target


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


Recommendations

  • 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


Questions?


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