Cryogenic Optical Microscope Faculty Advisor: Prof. Greg Kowalski Sponsors: Dale Larson, James...

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?