MICROSCOPY

Micro means small

Skopein means to see

Microscopes

Preparation of specimens for light microscopy

Bright-field Microscope Compound Comparison Stereoscopic Microspectrophotometer Scanning Electron Microscope

The Instruments

HOW MICROSCOPES WORK

•Dutch spectacle-makers (1590), Janssens, Dutch spectacle-makers (1590), Janssens, discovered that nearby objects appeared discovered that nearby objects appeared greatly enlarged with lenses. greatly enlarged with lenses. •Galileo (late 1600s), based on the Janssens Galileo (late 1600s), based on the Janssens experiments, worked out a experiments, worked out a much better instrument much better instrument with a focusing device.with a focusing device.

Microscopy History

Janssen

Galileo

•Later MicroscopesLater Microscopes

Microscopy History

Pacino, 1870

Olympus (modern)

COMPOUND LIGHT MICROSCOPE

The most common microscope - The most common microscope - compound light microscope compound light microscope (LM).(LM).

Two sets of lenses: ocular and Two sets of lenses: ocular and objective. objective.

The total magnification: The total magnification: multiply multiply magnification of the objective lens magnification of the objective lens with the ocular lens. with the ocular lens. e.g., ocular is 10x and the objectives e.g., ocular is 10x and the objectives

is 100, total mag. will be 1000x.is 100, total mag. will be 1000x. Optical system comprised of Optical system comprised of

condenser, objective lens, eyepiece condenser, objective lens, eyepiece lens and illuminator.lens and illuminator.

The compound light microscope uses The compound light microscope uses visible light. visible light. (( = 400 – 700 nm). = 400 – 700 nm).

Virtual image is any specimen Virtual image is any specimen viewed through a lens.viewed through a lens.

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COMPOUND MICROSCOPE

1. Base2. Arm3. Stage4. Body tube5. Coarse adjust6. Fine adjust7. Illuminator8. Condenser9. Objective lens10. Occular lens

ELECTROMAGNETIC SPECTRUM

COMPARISON MICROSCOPE Two compound

microscopes combined into one unit

When viewer looks through the eyepiece, a field divided into two equal parts isobserved

Firearms Identification: Bullet comparisons

Hair & Fiber comparisons Questioned documents

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COMPARISON MICROSCOPE

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Split-image comparison of firing pinimprints in coaxial incident light

COMPARISON MICROSCOPE

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Split-image comparison of banknotes:on the left the original, on the right the forgery

STEREOMICROSCOPE Also called the dissecting microscope Working distance from objective lens to

specimen. Examiner can manipulate the specimen

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Stereoscopic Microscope• most widely used microscope in crime laboratories• most versatile of the microscopes used• A binocular microscope.

Advantages:• large working distance for bulky samples• three-dimensional image• image that is not inverted or reversed

Disadvantage:• magnifying power of only 10-125X

ILLUMINATIONVertical Illumination (ex. stereomicroscope)

• Illumination from above the specimen• Used when studying opaque samples• Light is reflected off the specimen’s surface into the lens

Transmitted Illumination (ex. compound microscope)• Used only with transparent specimen• Light is directed up through the specimen from the base.

Microscope Characteristics

Field of View = the span of the area in viewDepth of Focus = the thickness of the region in focus

Field of view and depth of focus both decrease as magnifying power increases.

• High powers of magnification “sacrifice” both the size of the area in view and the thickness of the region in focus• Low powers of magnification “sacrifice” detail within the region under examination and “in focus.”

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MICROSPECTROPHOTOMETERS

• Available as visible or infrared spectrometers.• Details of the sample may be viewed directly.• The visible or infrared spectrum of the specimen being viewed may be obtained at the same time.

One genuine and one counterfeit $50 bill

Inked line on bill

Visible spectrum of each

ELECTRON MICROSCOPE A beam of electrons,

instead of light, is used Can magnify greater

because the wavelengths of electrons are much smaller than those of visible light = 0.005 nm as opposed to 500 nm

The best compound light microscopes can magnify 2000x, electron microscopes can magnify up to 100,000x

2 types: TEM & SEM

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SCANNING ELECTRON MICROSCOPY (SEM)

3-D views of the surfaces by aiming a beam of electrons onto the specimen. Electrons are bounced off the surface of the

specimen and form a 3D image that is stereoscopic in appearance.

Magnification: 1000-100,000x and Depth of Field very high.

Can be used to identify the elements present in the specimen under examination.

SCANNING ELECTRON MICROSCOPE (SEM):SAMPLE CHARACTERISTICS

The sample must not be “soft.” “Soft” samples are destroyed by the electron beam.

The sample will emit X-rays characteristic of the elements present at the surface of the sample. The sample composition may be analyzed while the sample is being “viewed.”

SEM IMAGES

Human Hair (1100X)

Diatom

SEM IMAGES

Semiconductor Chip (600X)

SEM IMAGES

Bread Mold (200X)