1. Analytical Tools Microscopy Chapter 4 ©2010 Elsevier, Inc.

2. INTRODUCTION Microscopy is applicable to every area of forensic science – More than simply looking at small things ©2010 Elsevier, Inc.

3. MAGNIFICATION SYSTEMS A single lens used to form an enlarged image of an object is a simple magnification system ©2010 Elsevier, Inc.

4. MAGNIFICATION SYSTEMS A compound magnification system consists of two stages of magnification and the total magnification is the product of the magnification of the first and second lens ©2010 Elsevier, Inc.

5. MAGNIFICATION SYSTEMS – Observer looks at the first image with a lens that produces an enlarged image called a virtual image The image perceived by the eye; a real projectable image does not exist – I.e. your image in a mirror – A real image is one that could be projected onto a screen ©2010 Elsevier, Inc.

6. THE LENS – A lens is a translucent material that bends light in a known and predictable manner – Size and position of an image produced by a lens can be determined through geometry based on the focal length of the lens Focal length is the distance between two points of focus on either side of the lens – Determines much of the image quality Resolution is the minimum distance two objects can be separated and still be seen as two objects ©2010 Elsevier, Inc.

7. THE LENS ©2010 Elsevier, Inc.

8. THE LENS – Magnification with one lens is not indefinite – As magnification increases, lens diameter decreases to bend the light more to make a larger image Practical limit for simple lens is 10x to 15x ©2010 Elsevier, Inc.

9. COMPOUND MAGNIFYING SYSTEMS – Exceeds the limits imposed by a simple lens A second lens is placed in front of the first to further enlarge the image Total magnification of the microscope is the product of the two lenses – Lenses of up to 40x can be used in a compound microscope ©2010 Elsevier, Inc.

10. COMPOUND MAGNIFYING SYSTEMS Lenses in compound microscopes have resolution limits – Continued magnification of an image is possible, but the resolution is not improved » Called empty magnification ©2010 Elsevier, Inc.

11. THE MICROSCOPE ©2010 Elsevier, Inc.

12. THE MICROSCOPE – Eyepiece or ocular is the lens that one looks into when viewing an object microscopically A microscope having one eyepiece is monocular A microscope having two eyepieces is binocular – Area seen when looking through the eyepieces is called the field of view – Objective lens is closest to the – object or specimen being studied Most important part of microscope ©2010 Elsevier, Inc.

13. THE MICROSCOPE – Numerical aperture is an angular measure of the lens’ light-gathering ability and its resolving quality – Tube length is the distance from the lowest part of the objective to the upper edge of the eyepiece Standardized at 160mm – Coverslips are thin glass plates that are placed on top of mounted specimens Protects the specimen and objective from damage ©2010 Elsevier, Inc.

14. THE MICROSCOPE ©2010 Elsevier, Inc.

15. THE MICROSCOPE – The stage is the platform where the specimen sits during viewing Moved to focus the specimen – Portion of the specimen in the field of view is sitting in the same horizontal plane May be mechanical, rotating or both – Mechanical stages have knobs for control of movement – Rotating stages can spin 360° but not move back and forth ©2010 Elsevier, Inc.

16. THE MICROSCOPE – The condenser is used to obtain a bright, even field of view and improve image resolution Lenses located below the stage that focus or condense the light onto the specimen field of view – A condenser diaphragm is used to eliminate excess light and adjust for contrast in the image – Field diaphragm allows more or less light into the lens system of the microscope ©2010 Elsevier, Inc.

17. THE MICROSCOPE ©2010 Elsevier, Inc.

18. THE MICROSCOPE – Illumination is critical to a quality image Critical and Köhler are the two main types of illumination used in microscopy – Critical illumination concentrates light on the specimen with the condenser lens » Produces an intense lighting that highlights edges, but may be uneven – Köhler illumination sets the light rays parallel throughout the lens system, allowing them to evenly illuminate the specimen » Considered standard ©2010 Elsevier, Inc.

19. REFRACTIVE INDEX – Refraction, or bending of light, is one characteristic that allows lenses to focus a beam of light onto a single point Occurs when light passes from one medium to another when there is a difference in the index of refraction between the two materials ©2010 Elsevier, Inc.

20. REFRACTIVE INDEX Refractive index is defined as the relative speed at which light moves through a material with respect to its speed in a vacuum – N=C/v » N is index of refraction, C is speed of light, v is velocity of light in that material – Angle of refracted light is dependent on both the angle of incidence and the composition of the material into which it is entering ©2010 Elsevier, Inc.

21. REFRACTIVE INDEX – The normal is a line perpendicular to the boundary between two substances – Snell’s Law: N1 x sin(q1) = N2 x sin(q2) N is refractive indices of material 1 and material 2, q is angle of light traveling through the material with respect to the normal Mounting media or mountants are used so samples can be viewed in transmitted light – Samples must be in a material with a refractive index that is close to their own ©2010 Elsevier, Inc.

22. REFRACTIVE INDEX ©2010 Elsevier, Inc.

23. POLARIZED LIGHT MICROSCOPY Exploits optical properties of materials to discover details about the structure and composition of materials – Isotropic materials demonstrate the same optical properties in all directions Gases, liquids, certain glasses and crystals – Anisotropic materials have optical properties that vary with the orientation of the incoming light and the optical structure of the material ©2010 Elsevier, Inc.

24. POLARIZED LIGHT MICROSCOPY ©2010 Elsevier, Inc.

25. POLARIZED LIGHT MICROSCOPY ©2010 Elsevier, Inc.

26. POLARIZED LIGHT MICROSCOPY PLM uses the fact that anisotropic materials divide light rays into two parts to yield information about the material – A polarizer is a special filter that lets light pass through only in a “preferred” direction Light that vibrates only in one direction is called polarized light – The analyzer is a polarizing filter that is aligned opposite of the polarizer Located above the objectives; can manually be slid into or out of the light path ©2010 Elsevier, Inc.

27. POLARIZED LIGHT MICROSCOPY – Birefringence is the result of the division of light into at least two rays when it passes through certain types of material Δn= n e - n o – n o is the refractive index for the ordinary ray, ne is the refractive index for the extraordinary ray ©2010 Elsevier, Inc.

28. POLARIZED LIGHT MICROSCOPY ©2010 Elsevier, Inc.

29. POLARIZED LIGHT MICROSCOPY – Retardation is difference in velocity of the ordinary and extraordinary rays R=t(n2 – n1) – r is retardation, t is thickness and n2 – n1 is birefringence ©2010 Elsevier, Inc.

30. POLARIZED LIGHT MICROSCOPY – In the PLM, the out-of-phase waves of light strike the analyzer and are diffracted into various colors depending on the wavelengths being added and subtracted, called interference colors Colors are indicative of the fiber’s polymer type and molecular organization Birefringence of a fiber can be determined with PLM – Numerical difference between the maximum and minimum refractive indices ©2010 Elsevier, Inc.

31. POLARIZED LIGHT MICROSCOPY Michel-Levy Chart gives diameter, birefringence and retardation ©2010 Elsevier, Inc.

32. FLUORESCENCE MICROSCOPY – Fluorescence is the luminescence of a substance excited by radiation Emission stops when excitation stops Emitted wavelength is longer than excitation radiation – Luminescence is subdivided into phosphorescence, which is characterized by long-lived emission, and fluorescence ©2010 Elsevier, Inc.

33. FLUORESCENCE MICROSCOPY – In a fluorescence microscope, the specimen is illuminated with light of a short wavelength Part of light is absorbed by specimen and reemitted as fluorescence Fluorescence has less energy that exciting radiation Fluorophores, or fluorescent components, can be excited by near UV invisible radiation and seen in the visible range ©2010 Elsevier, Inc.

34. FLUORESCENCE MICROSCOPY – Fluorescence microscope has special light source and a pair of complementary filters Lamp should be rich in short wavelengths – High-pressure mercury arc lamps Primary or excitation filter is placed between lamp and specimen Secondary, barrier, or suppression filter prevents excitation light from reaching the observer’s eye ©2010 Elsevier, Inc.

35. Electron Microscopy – Electron microscopes employ a particle beam of electrons focused by magnetic lenses Much higher resolving power and greater depth of field than light microscopes Either transmission (TEM) or scanning (SEM) ©2010 Elsevier, Inc.

36. Electron Microscopy In TEM, the electron beam passes through a very thinly sliced specimen In SEM, a beam of electrons rasters across a specimen to provide a non-colored image of its surface ©2010 Elsevier, Inc.

37. Electron Microscopy » SEM is used to analyze paint, particles, fractures, toolmarks, and gunshot residue ©2010 Elsevier, Inc.

38. CHAPTER SUMMARY Microscopy provides fast, low-cost, and definitive results to the trained scientist ©2010 Elsevier, Inc.