1. Microscopy CHE 113 Forensic Science James T. Spencer, Copyright 2004-2008

2. 2 Microscopy and Stains Micro means small Skopein means to see Microscopes Preparation of specimens for light microscopy

3. 3 Sizes

4. 4 Brightfield Microscope Compound Comparison Stereoscopic Polarizing Microspectrophotometer Darkfield Microscope Phase-contrast Microscope Fluorescence Microscope Electron Microscope Transmission Scanning The Instruments

5. 5 How Microscopes Work

6. 6 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

7. 7 Other Early MicroscopesOther Early Microscopes Microscopy History Leeuwenhoek Microscope Hooke Microscope Late 1600’s 1670

8. 8 Later MicroscopesLater Microscopes Microscopy History Pacino, 1870 Olympus (modern)

9. 9 Microscopy History

10. 10 The most common microscope - compound light microscope (LM). Two sets of lenses: ocular and objective. Brightfield Mic. 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. Optical system comprised of condenser, objective lens, eyepiece lens and illuminator. The compound light microscope uses visible light. ( = 400 - 700). Virtual image is any specimen viewed through a lens. Compound Light Microscope

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12. 12 Electromagnetic Radiation Magnetic and Electronic Parts mutually perpendicular

13. 13 Electromagnetic Spectrum

14. 14 Darkfield Microscopy The darkfield microscope shows a light silhouette of an organism against a dark background. The light reaches the specimen from an angle with the help of an opaque disk. The specimen then appears "lit up" against the dark background. Most useful for extremely small living organisms that are invisible in the light microscopes. An example would Treponema pallidum, the causative agent for syphilis.

15. 15 Darkfield Microscopy Brightfield Darkfield

16. 16 Important tool for firearms examiner Two compound microscopes combined into one unit When viewer looks through the eyepiece, a field divided into two equal parts is observed Bullet comparisons- requires reflective light Hair & Fiber comparisons Questioned documents Comparison Microscope

17. 17 Comparison Microscope Split-image comparison of firing pin imprints in coaxial incident light

18. 18 Comparison Microscope Split-image comparison of banknotes: on the left the original, on the right the forgery

19. 19 Phase-Contrast Microscope A phase-contrast microscope splits a beam of light into 2 types of light, direct and refracted (reflected) and brings them together to form an image of the specimen. Where they are "in-phase" the image is brighter, where they are "out of phase" the image is darker, and by amplifying these differences in the light, it enhances contrast It allows the detailed observation of living organisms, especially the internal structures.

20. 20 Polarizing Microscope Based upon the rotation of polarized light.

21. 21 Light confined to a single plane of oscillation is said to be polarized. Many crystals are birefringent (light is refracted into 2 separate rays). Reduces glare by transmitting light in a vertical plane only. Polarizing Microscope

22. 22 Why do molecules rotate polarized light? A molecule possessing nonsupeposable mirror images is termed chiral. Naturally occurring substances are often found as just one mirror image (enantiomer). Enantiomers are identical in physical properties and identical chemical properties when they react with nonchiral reagents; only in a chiral environment will the differences show. Enantiomers rotate of polarized light in different directions.

23. 23 Optical Activity Non-superimposable mirror images Mirror Left Hand Right Hand

24. 24 Optical Isomers Amino Acids are Chiral Mirror Plane Enantiomers alanine

25. 25 Optical Isomerism Enantiomers have the same physical properties (i.e., b.p., m.p., viscosity, etc...). Enantiomers react identically toward other non-chiral molecules Enantiomers frequently react very differently toward chiral reagents (v. impt. biologically) Frequently, one enantiomer will be biol. active while the other is biol. inactive. Interaction of Chiral Reagents (L and R hands)

26. 26 In fluorescence microscopy, specimens are first stained with fluorochromes and then viewed through a compound microscope by using an ultraviolet (or near- ultraviolet) light source. The microorganisms appear as bright objects against a dark background. Fluorescence microscopy is used primarily in a diagnostic procedure called fluorescent- antibody (FA) technique, or immunofluorescence. This technique is especially useful in diagnosing syphilis & rabies. Fluorescence Microscope

27. 27 Hand section of sugarcane vascular bundle viewed with fluorescence microscope Sugarcane vascular bundle viewed with traditional staining and transmitted light (Bright Field) microscopy Hand-section of Sugarcane stem with a vascular bundle Stained with Toluidine Blue & Viewed with Bright Field microscopy

28. 28 An egg that was stained with a dye that appears yellow in a fluorescence microscope. An unfertilized egg stained with a dye that appears red in a fluorescence microscope. The line indicates size (10 micrometers = 1/100 of a millimeter). The egg is about 100 micrometers in diameter, so about ten eggs would fit side by side on a pencil line.

29. 29 Also called the dissecting microscope Working distance below objective lens & specimen. Image is 3D. Doesn’t reverse or invert the image, so examiner can manipulate the specimen Stereomicroscope

30. 30 Electron Microscope A beam of electrons, instead of light, is used with an electron microscope. Electron microscopes can magnify greater because the wavelengths of electrons are much smaller than those of visible light = 0.005nm as opposed to 500nm (one hundred thousand times smaller) The best compound light microscopes can magnify 2000x, electron microscopes can magnify up to 100,000x 2 types: TEM & SEM

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32. 32 Transmission electron microscopes (TEM): Thin sections (or layers) (2-D) can be seen in a transmission electron micrograph. Electrons pass directly through the specimen. Magnification: 10,000-100,000x Resolving power: 2.5 nm. The specimens must be fixed, dehydrated and viewed under a high vacuum. These treatments not only kill the specimens but may cause shrinkage and distortion. Transmission Elec. Microsc.

33. 33 Transmission Elec. Microsc.

34. 34 TEM Pictures Silver Nanoprisms Gold Nanoparticle

35. 35 Scanning electron microscopes (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: 1000-10,000x and Depth of Field very high. Can be used to identify the elements present in the specimen under examination. Scanning Elec. Microscopy

36. 36 Scanning Elec. Micros.

37. 37 SEM Images Human Hair (1100X) Diatom

38. 38 SEM Images Semiconductor Chip (600X)

39. 39 SEM Images Bread Mold (200X) Ni Boride (SU Chem)

40. 40 Definitions Types Simple Differential Special Stains (Microscopy)

41. 41 Staining Samples Staining simply means coloring the sample with a dye that emphasizes certain structures or components. “Fixing”- Before a sample can be stained, it must be attached or fixed, to the slide, otherwise the stain might wash it from the slide. “Smearing” - When a specimen is fixed, a thin film of material containing the sample is spread over the surface of the slide. This film (smear) is allowed to air dry or by flame.

42. 42 Stains are salts composed of a positive and a negative ion, one of which is colored and is known as the chromophore. The color of “basic dyes” originates in the positive ion. In “acidic dyes” it originates in the negative ion. Stains

43. 43 Dyes: Acids and Bases Basic dyes (cation) include crystal violet, methylene blue and safranin are more commonly used for bacterial cells. Acidic dyes (anion) are not attracted to most bacterial cells - the stain colors the background. This preparation of colorless bacteria against the colored background is called negative staining. It’s valuable for cell shapes, sizes & capsules. Distortions are kept to a minimum because heat fixing in not necessary. Examples of acidic dyes are eosin, nigrosin & India ink.

44. 44 Simple Stains Aqueous or alcohol solution of a single basic dye. Used to highlight microorganisms to determine cellular shapes and arrangements. Examples include, methylene blue, carbolfuchsin, crystal violet, & safranin.

45. 45 Differential Stains Differential Stains React differently with different kinds of bacteria in order to distinguish them. Gram Stain Divides bacteria into two large groups, gram-positive & gram-negative.

46. 46 Negative Staining for Capsules. Since most capsules do not accept stains, the capsules appear as halos around bacterial cells & stand out against a dark background. Endospore (Spore) Staining. Used to detect the presence of endospores in bacteria. When stain (malachite green) is applied to a heat-fixed smear of bacterial cells, the stain penetrates the endospores and stains them green. Flagella Staining Used to demonstrate the presence of flagella. Special Staining

47. 47 End Chapter 7

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