M ICROSCOPY 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

H OW M ICROSCOPES W ORK

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

Later MicroscopesLater Microscopes Microscopy History Pacino, 1870 Olympus (modern)

C OMPOUND L IGHT M ICROSCOPE The most common microscope - compound light microscope (LM). Two sets of lenses: ocular and objective. The total magnification: multiply magnification of the objective lens with the ocular lens. e.g., ocular is 10x and the objectives is 100, total mag. will be 1000x. e.g., ocular is 10x and the objectives is 100, total mag. will be 1000x. Optical system comprised of condenser, objective lens, eyepiece lens and illuminator. The compound light microscope uses visible light. ( = 400 – 700 nm). Virtual image is any specimen viewed through a lens. 6

C OMPOUND MICROSCOPE 1. Base 2. Arm 3. Stage 4. Body tube 5. Coarse adjust 6. Fine adjust 7. Illuminator 8. Condenser 9. Objective lens 10. Occular lens

E LECTROMAGNETIC S PECTRUM

C OMPARISON M ICROSCOPE Two compound microscopes combined into one unit When viewer looks through the eyepiece, a field divided into two equal parts is observed Firearms Identification: Bullet comparisons Hair & Fiber comparisons Questioned documents 9

C OMPARISON M ICROSCOPE 10 Split-image comparison of firing pin imprints in coaxial incident light

C OMPARISON M ICROSCOPE 11 Split-image comparison of banknotes: on the left the original, on the right the forgery

S TEREOMICROSCOPE Also called the dissecting microscope Working distance from objective lens to specimen. Examiner can manipulate the specimen 12

13 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 X

I LLUMINATION Vertical 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 view Depth 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.”

16 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

E LECTRON M ICROSCOPE 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 = 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 18

S CANNING E LECTRON M ICROSCOPY (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: ,000x and Depth of Field very high. Can be used to identify the elements present in the specimen under examination.

S CANNING E LECTRON M ICROSCOPE (SEM): S AMPLE C HARACTERISTICS 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 I MAGES Human Hair (1100X) Diatom

SEM I MAGES Semiconductor Chip (600X)

SEM I MAGES Bread Mold (200X)