1. F ORENSIC MICROSCOPY

2. T YPES OF MICROSCOPES There are several different types of microscopes that are used in forensic analysis. What type of microscope used often depends on the sample itself, and the type of analysis that is being conducted.

3. Compound Light Microscopes: Most common microscope found in high school labs: Sample is illuminated from below with white light, and the light is then focused towards the eye piece. As the light passes through, different parts of sample will absorb/scatter/reflect light differently. This is what gives us the contrast in the sample. Usually used for objects 1 micrometer to 5 mm.

4. While compound light microscopes are useful in high school laboratories, there are several drawbacks or limitations for use in forensics lab. Most biological samples do not absorb light very well, so most of the light is reflected so samples appear very light with little to no contrast. To circumvent this, we can use stains to look for specific organelles. Other ways of adjusting contrast is adjusting the amount of light that passes through the sample, or using different colored light.

5. Staining is a way of adding chemical compounds to the sample that are better at absorbing light. Stains often bind to a specific structure or molecule, which allows us to select stains for particular organelles. Example: Crystal Violet is used to distinguish between Gram + and Gram – bacteria.

7. Gram Positive Gram Negative

8. D ARK FIELD MICROSCOPY Used to enhance contrast and works almost opposite of compound light or bright field microscopy.

10. Rather than light passing directly through a sample, the light is first redirected through a condenser. The condenser lens is a concave lens that causes the sample to be illuminated only from certain azimuths, directions, angles. The light is then scattered by the sample, and the scattered light is then focused to the eyepiece.

11. One of the drawbacks is that there is a high chance of a glare off of the specimen If the sample has different densities, it will scatter light differently and may cause inaccuracies within the image. Any air bubbles will cause the light to be refracted and scattered in a much different manner, causing distortion and inaccuracies.

12. P OLARIZED LIGHT MICROSCOPY

13. Light as we know it belongs to the electromagnetic spectrum, and the energy behaves as a wave. However, even though light is considered a wave, it is also composed of particles that are called photons. This is known as the dual nature of light, to be both a particle and a wave.

14. Light is categorized as a transverse wave, which means that the particles are moving up and down, as the wave moves left to right

15. Light can also be categorized as unpolarized and polarized. Unpolarized light is when the photons are oscillating in many different orientations. Light is considered to be polarized when it contains waves that only oscillate or vibrate in a certain direction. Polarized light is achieved by passing light through a “filter” with very narrow, vertical, rectangular slits. This results in light only oscillating in one direction and is called plane polarized light.

18. You can take polarized light and pass it through two filters, one after another. If you second filter aligns with your first, then the light will pass through. However, if your 2 nd filter is oriented in a different way, the light will not pass through the second filter and the light will be blocked.

20. Certain chemical compounds have an ability to refract and twist light into different orientations. Polarized light microscopy works on the following premise: Light is first polarized and then passed through the sample, so the light entering the sample has all already been aligned. Then depending on the chemicals in the substance, the light will be twisted. The second filter can be oriented to line up with the twisted light.

21. Polarized light microscopy is often used to present a clearer picture of the sample. Unpolarized light reflects, refracts and absorbs light in all different orientations, which can distort the image. One of the most important uses – to detect the presence of certain chemical compounds are present in the sample. Often used to test and identify the composition of minerals, fibers, soil.

22. P HASE C ONTRAST MICROSCOPY Phase contrast microscopes were developed to improve the amount of contrast seen with biological materials. Biological materials are often very similar in transparency and have little pigmentation. However, the different organelles have very different refractive indices Different organelles have a different ability to refract or bend light.

23. More background information on light as a wave: As a wave of light is slowed down by travelling through air, or bent by a sample: this causes light to be considered “out of phase” with the other light that bypassed the sample. This means that when the two waves – one in phase, one out of phase – are lined up with each other, the waves have the potential to cancel each other out.

24. Waves that are completely out of phase, totally cancel each other out, as shown below – so no light would be seen.

25. Constructive addition from in phase waves and Destructive addition from out of phase waves.

26. Most biological samples, when light passes through/over them, they bend and slow down light so that the resulting wave is about a quarter, or half a wavelength out of phase. Since it is only half a wavelength out of phase, it causes a reduction in the brightness – hence increases contrast.

29. F LUORESCENCE MICROSCOPY As light shines on a molecule, the electrons gain energy and become excited. For certain molecules, the light they emit - as they come back down to ground state – has a longer wavelength than the light they absorbed.

31. F LUORESCENCE IN THREE STEPS All fluorescence can be boiled down to three basic steps: 1. Absorption of photon (light) 2. Loss of some energy (vibration) 3. Emission of lower wavelength photon. You are able to filter out the “exciting light” and only see the emission light.

33. Certain biological materials contain chemicals that are capable of fluorescing when exposed to “exciting light” Semen and fingerprints are some of the more common materials that can be detected with fluorescent microscopy. The other option is to add a fluorescent dye or stain to the sample, which will bind to specific structure and allow for better visualization.

34. Example: sperm cells are often very difficult to identify from epithelial cells in rape investigations. In order to test DNA, you need to identify the sperm cells, and their nuclei. A fluorescent dye is added that binds to the nuclei in sperm cells, and then light is shown on the sample to excite the molecules. The original light is filtered out, and the only light visible is the fluorescent light given off by the dye (in the nuclei).

37. There are certain drawbacks to using fluorescence microscopy: In order to excite materials, and see the emission – the sample has to be relatively thin. To get around this: they are developing new technique called “two photon fluorescence microscopy” – which uses two photons rather than one to excite molecules Relatively low resolution.

38. IR MICROSCOPY Infrared light, with wavelengths between 700- 1400 nm is not visible to our eyes. However, many compounds that are of particular interest in forensic cases, readily absorb light in this region. The absorption spectrums of dyes, drugs, fibers, pigments are very specific to the type of molecule.

39. When a compound is subjected to IR light, they produce an absorption spectrum. Within the spectrum, there is a region called the “fingerprint” that is specific to the type of molecule you are working with. This spectrum is then compared to standards and used to identify the compounds in the sample. Using IR in this way is known as IR microspectrophotometry

40. As you shine IR light on the sample, chemicals will absorb it. However, we cannot detect IR. A material that is sensitive to IR is put in place, such as film or an electronic detector. Essentially, each part of the field of view is mapped, and depending on the spectrum that is produced – the chemical composition can be determined. Often use for small samples of ink, gunshot residue (GSR) drugs, explosives, fingerprint oils.

41. S TEREO MICROSCOPY What do we do when the sample is too thick to allow light to be shown through it? Similar to the dark field microscope, the stereo microscope also depends on light bouncing off the surface. Also known as the dissecting microscope. Gives a low magnification, 3D image of the surface, with a focus on surface structures.

42. C OMPARISON MICROSCOPY Incredibly popular in firearm investigations. Typically used to compare the fine details of evidence at the crime scene with a standard or reference. Two separate compound microscopes joined together at the eyepiece. Allows you to move each sample independently until you can get the best comparison.

43. Comparison microscopy is used to compare the striations on a bullet from a crime scene to a test bullet fired with the recovered gun from the crime scene. If the striations match up, it is very likely that the crime scene bullet came from the gun that they recovered. This can also be done with bullet casings, forged documents and analysis for hair and fibers.

45. One major drawback to all of these types of microscopes is the resolution. Each type relies on shining or scattering light through or on a sample to get a projected image. The resolution of the image our eyes can see is limited to about half the wavelength projected. Because most microscopy uses visible light, our eyes are limited to about 300 nm for the resolution. So as we magnify 1000x, 2000x – we lose a lot of the fine details because we are limited to a “large” wavelength of light

46. If we want to visualize smaller objects and finer details, we need to use wavelengths that are much smaller so that when we magnify them we can keep our resolution. For this, scientists have developed techniques that use electrons to illuminate the sample rather than visible light.

47. How do electrons give us smaller wavelengths? A brief intro to quantum physics: very very brief! We already know that light has a “dual nature” – an ability to behave both as particle and a wave. We can say this about all particles. If we assume that every particle can also behave as a wave, we can calculate the wavelength for a particle – the particle in this case being the electron.

48. The equation: Wavelength = h / mv Where: H = Planck’s constant: 6.63x10 -34 m 2 kg/s m = mass v = velocity This equation demonstrates that the larger the object, the smaller the wavelength. Conversely, the smaller the object the larger the wavelength.

49. For an electron, the wavelength is about 0.8nm which is much smaller than the wavelength of visible light. This allows us to get a much better resolution. We can more dramatically increase the resolution by playing around with the velocity of the particle. Based on the equation, the higher the velocity, the smaller the wavelength. By accelerating an electron to high velocities, we can shrink the wavelength and therefore get much better resolution for incredibly tiny structures.

50. A beam of high velocity electrons are aimed at a sample.. In order to focus this sample, electrical and magnetic fields are used rather than optical lenses like in traditional microscopy. The beam of electrons must be passed through a vacuum because air molecules can also readily absorb electrons – so they would never make to the sample.

51. Scanning Electron Microscope: Works similar to a stereoscope: uses scattered electrons, rather than electrons passed through the sample. As the beam hits the surface, both electrons and x- rays are bounced back. The SEM detector collects the electrons and converts them into a magnified image. This is done continuously over the entire sample, and all of the images are fused together into one picture.

52. Some road bumps: Because electrons are being shot at the surface, excess negative charge can build up: If the surface is conductive: the excess negative charges are able to flow right off the surface and not build up. If surface is non-conductive: a thin coating of a conductive material must be applied to channel away excess charge

53. Because of the incredibly small wavelength of the electrons, the pictures we are able to get are incredibly detailed and we are able to see several different surface features in focus at once. In addition, the x-rays given off are also analyzed. The x-rays given off are unique to what element(s) are in a given sample. By analyzing the x-rays given off, we can get an idea of the chemical composition of different parts of the sample. This is known as Energy Dispersive X-ray Analysis (EDXA)

54. T RANSMISSIVE ELECTRON MICROSCOPE Similar to bright field microscope – electrons pass through the sample: Some of the electrons are scattered and don’t make it up to viewing, this creates a darkening in the image. Because of different densities and layers, shadows are created as the electrons are absorbed and scattered differently. Used to study very small features, down to a couple of angstroms. (10 atoms lined up = 1 angstrom). Study pathogens, fine minerals, unknown materials to determine chemical composition.