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Optical Microscopes

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Outline

Justification History Components of the

Optical Microscope Theory of operation

Basic Microscope Magnification

Refraction of light and Snell’s Law

The thin lens equation

Imaging and resolution Confocal Microscopy Vendors

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Why Optical Microscopes?

Still one of the most commonly used methods of inspection due to ease of use and sample preparation.

Often used to prepare samples for electron microscopy or scanning probe microscopy

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Labomed Stereo Zoom Scope

TopIlluminationPower

Eye pieceZoom

Focus

Top Lamp

Main power

Bottom LampWith removable glass cover

BottomIlluminationPower

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Nikon Optiphot

Lamp

Future camera

Coarse focus

Fine focus

Base

Eyepiece

Stage

Objective Lenses

NosepieceUse this to change the objectives never touch the actual objective lenses

Bright fieldDark field

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Nikon OptiphotBeamsplitter Apertures

Stage X, YCoarseFocus

FineFocus

LampIntensityControl

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Zeiss AxiotronEyepiecesyou look through these

BrightfieldNormal lightingDarkfieldIndirect lighting

CameraUsed to capture images

CoarseFocusFine

Focus

Light SourceBe sure to turn this off when you are finished using it

Fiber optics

Used to quickly lower the stage so the objectives do not become damaged

Objective lensesUsed to change magnificationNever touch these

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Zeiss Axiotron

Stage Rotation

Stage X and Y

Stage handle

Vacuum ChuckNot connected to vacuum

Beam splitterFor camera

Motorized objectiveAlways use these buttons to change objectives

Always use the front button to increase the magnification and the back button to decrease magnification. Otherwise you may damage the objective lens

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Zeiss Axiotron

Optional filtersNot used

ApertureAdjust so it is just beyond the field of view

FiltersYellow filter should be used for viewing photoresist

The other filter can be used to enhance contrast

PolarizerCan be used to enhance contrast

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Magnification

Objective lenses up to 100x or 150x Eyepieces 10x – 15x Magnification = OL x EP The limit of optical magnification is about

2000x.

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Refractive IndexWaves do not have the same speed in all materials.

The speed of light in vacuum is c=2.998 x 108 m/s.The speed of light in glass is slower, only about v=2.000 x 108 m/sThe refractive index is the ratio of these two speeds

cn =

vThis part slows down first

θi

θt

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Refraction and Snell’s Law

http://hyperphysics.phy-astr.gsu.edu/hbase/geoopt/refr.html

θi

θt

c v

Snell’s law describes refraction of sound ( a change in direction as it passes through a boundary) If the incident angle is not zero and the velocities are not equal.

i i

t t

sin θ θc

v sin θ θ

This part slows down first

θi

θt

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Snell’s LawA light wave traveling through a air with propagation speed 2.998 x 108 m/s passes into water with propagation speed 2.308 x 108 m/s with an incident angle of 50˚.

What is the angle of transmittance?

i i

t t

sin θ θc

v sin θ θ

θi

θt

c v

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A Convex Lens

The focal point of a lens

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Theory of Operation

f is focal point distanceo is object distancei is image distanceM is magnification

1 1 1

o i f

iM

o

o f

f i

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Thin Lens Calculations

1 1 1

o i f

iM

o

If a lens has a 4.5 cm focal length and a specimen is 5.3 cm from it where will the image appear to be and what will be the magnification?

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Imaging

Light field Dark field Resolution Depth of Field Polarizing filters, Nomarski Yellow Filters Apertures Beam splitter Digital image capture Limits of resolution

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Sample PreparationBiological sample may be placed on a glass slide with a cover slip. Various stains can be used to highlight different parts of the sample.

When gold particles are small enough they no longer appear as yellow shiny metal but can have a variety of colors which depends on their size. These are called quantum dots.

These can be combined with antibodies and used to stain different specific proteins different vibrant colors.

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-and-Tissue-Analysis/qdots-microspheres-nanospheres.html

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Inspection

The optical microscope is the most common method of inspection because they are relatively easy to use and easy to prepare samples for.

They are also commonly used when preparing samples for the electron and scanning probe microscopes.

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Confocal Microscope

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Optical Microscope Vendors

Olympus www.olympus.com Zeiss www.zeiss.com/micro Nikon www.nikonusa.com Leica www.leica-microsystems.com Microscope U

http://www.microscopyu.com/