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Microscopes and microscopy


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Microscopes and microscopy

  2. 2. The Basics of the Microscope• A microscope is an optical instrument thatuses a lens or a combination of lenses tomagnify and resolve the fine details of anobject.• The earliest methods for examining physicalevidence in crime laboratories relied almostsolely on the microscope to study thestructure and composition of matter.
  3. 3. The Basics of the Microscope• The microscope is still one of the most usedtools in a crime laboratory for analyzingevidence, even with the advent of othermodern instrumentation.
  4. 4. The Basics of the Microscope• The earliest and simplest microscope was thesingle lens commonly referred to as amagnifying glass. The handheld magnifyingglass makes things appear larger than they arebecause of the way light rays are refracted, orbent, in passing from the air into the glass,and back to the air.• The magnified image is observed by lookingthrough the lens.
  5. 5. • Since the image appears to be on the same sideof the lens as the object, it cannot be projectedonto a screen. Such images are termed virtualimages and they appear upright, not inverted.Figure 1 presents an illustration of how a simplemagnifying lens operates. The object (in thiscase the subject is a rose) is being viewed with asimple bi-convex lens. Light reflected from therose enters the lens in straight lines asillustrated in Figure 1. This light is refracted andfocused by the lens to produce a virtual imageon the retina. The image of the rose is magnifiedbecause we perceive the actual size of theobject (the rose) to be at infinity because oureyes trace the light rays back in straight lines tothe virtual image (Figure 1). This is discussed ingreater detail below.Simple Lenses
  6. 6. • The ordinary magnifying glass can achieve amagnification of about 5 to 10 times. Highermagnifying power is obtainable only with acompound microscope, constructed of twolenses mounted at each end of a hollow tube.The object to be magnified is placed under thelower lens, called the objective lens, and themagnified image is viewed through the upperlens, called the eyepiece lens.History and Anatomy of theMicroscope
  7. 7. • As shown in the figure to theleft, the objective lens formsa real, inverted, magnifiedimage of the object. Theyeyepiece, acting just like amagnifying glass, furthermagnifies this image into avirtual image, which is seenby the eye. The combinedmagnifying power of bothlenses can produce an imagemagnified up to 1,500 times.History and Anatomy of theMicroscope
  8. 8. • Microscopes are instruments designed toproduce magnified visual or photographic imagesof small objects. The microscope mustaccomplish three tasks: produce a magnifiedimage of the specimen, separate the details inthe image, and render the details visible to thehuman eye or camera. This group of instrumentsincludes not only multiple-lens designs withobjectives and condensers, but also very simplesingle lens devices that are often hand-held, suchas a magnifying glass.History and Anatomy of theMicroscope
  9. 9. • The microscope illustrated in Figure 1 is asimple compound microscope inventedby British microscopist Robert Hookesometime in the 1660s. This beautifullycrafted microscope has an objective lensnear the specimen and is focused byturning the body of the microscope tomove the objective closer to or fartherfrom the specimen. An eyepiece lens isinserted at the top of the microscopeand, in many cases, there is an internal"field lens" within the barrel to increasethe size of the viewfield. The microscopein Figure 1 is illuminated through the oillamp and water-filled spherical reservoir,also illustrated in Figure 1. Light from thelamp is diffused when it passes throughthe reservoir and is then focused onto thespecimen with a lens attached to thereservoir. This early microscope sufferedfrom chromatic (and spherical)aberration, and all images viewed inwhite light contained "halos" that wereeither blue or red in color.History and Anatomy of theMicroscope
  10. 10. • Since so many microscope users rely upon directobservation, it is important to understand therelationship between the microscope and the eye.Our eyes are capable of distinguishing color in thevisible portion of the spectrum: from violet to blue togreen to yellow to orange to red; the eye cannotperceive ultraviolet or infrared rays. The eye also isable to sense differences in brightness or intensityranging from black to white and all the gray shades inbetween. Thus, for an image to be seen by theeye, the image must be presented to the eye in colorsof the visible spectrum and/or varying degrees oflight intensity.History and Anatomy of theMicroscope
  11. 11. • The eye receptors of theretina used for sensing colorare the cone cells; the cellsfor distinguishing levels ofintensity, not in color, arethe rod cells. These cells arelocated on the retina at theback of the inside of the eye.The front of the eye (seeFigure 2), including the iris,the curved cornea, and thelens are respectively themechanisms for admittinglight and focusing it on theretina.History and Anatomy of theMicroscope
  12. 12. • More than five hundredyears ago, simple glassmagnifiers were developed.These were convex lenses(thicker in the center thanthe periphery). Thespecimen or object couldthen be focused by use ofthe magnifier placedbetween the object and theeye. These "simplemicroscopes" could spreadthe image on the retina bymagnification throughincreasing the visual angleon the retina.History and Anatomy of theMicroscope
  13. 13. History and Anatomy of theMicroscope• The "simple microscope" ormagnifying glass reached itshighest state of perfection, in the1600s, in the work of Anton vonLeeuwenhoek who was able tosee single-celled animals (whichhe called "animalcules") and evensome larger bacteria with asimple microscope similar to theone illustrated in Figure 3. Theimage produced by such amagnifier, held close to theobservers eye, appears as if itwere on the same side of the lensas the object itself. Such animage, seen as if it were teninches from the eye, is known as avirtual image and cannot becaptured on film.Anton Von Leeuwenhoek (1632-1723)Father of Microbiology
  14. 14. • Around the beginning of the 1600s,through work attributed to theJanssen brothers (see themicroscope in Figure 4) in theNetherlands and Galileo in Italy, thecompound microscope wasdeveloped. In its simplest form, itconsisted of two convex lensesaligned in series: an object glass(objective) closer to the object orspecimen; and an eyepiece (ocular)closer to the observers eye (withmeans of adjusting the position ofthe specimen and the microscopelenses). The compound microscopeachieves a two-stage magnification.The objective projects a magnifiedimage into the body tube of themicroscope and the eyepiecefurther magnifies the imageprojected by the objective.History and Anatomy of theMicroscopeZacharias JanssenMicroscopeGalileo’s Microscope byZacharias Janssen(circa 1590)
  15. 15. • The eighteenth and nineteenth centurieswitnessed a great improvement in themechanical and optical quality ofcompound microscopes. Advances inmachine tools allowed moresophisticated parts to be fabricatedand, by the mid 1800s, brass was thealloy of choice for the production of high-quality microscopes. A number of Britishand German microscope manufacturersflourished during this time period. Theirmicroscopes varied widely in design andproduction quality, but the overallprinciples defining their optical propertiesremained relatively constant. Themicroscope illustrated in Figure 5 wasmanufactured by Hugh Powell and PeterLealand about 1850. The tripod baseprovided a sturdy support for themicroscope, which many people considerthe most advanced of its period.History and Anatomy of theMicroscope
  16. 16. • During the first quarter of the twentiethcentury, many microscope manufacturershad begun substituting cast iron for brassin microscope frames and stages. Ironwas much cheaper and could be not bedistinguished from brass when paintedblack. They also started to electroplatemany of the critical brass componentssuch as knobs, objectivebarrels, nosepieces, eyepieces, andmechanical stage assemblies (illustratedin Figure 6). These early twentiethcentury microscopes still subscribed to acommon design motif. They weremonocular with a substage mirror thatwas used with an external lamp toilluminate the specimen. A typicalmicroscope of the period is the ZeissLaboratory microscope pictured in Figure6. This type of microscope is veryfunctional and many are still in use today.History and Anatomy of theMicroscope
  17. 17. • Modern microscopes far exceed the design specifications ofthose made prior to the mid 1900s. Glass formulations arevastly improved allowing greater correction for opticalaberration than ever before, and synthetic anti-glare lenscoatings are now very advanced. Integrated circuittechnology has allowed manufacturers to produce "smart"microscopes that incorporate microprocessors into themicroscope stand. Photomicrography in the late twentiethcentury is easier than ever before with auxiliaryattachments that monitor light intensity, calculate exposurebased on film speed, and automatically performcomplicated tasks such as bracketing, multiple exposure,and time-lapse photography.History and Anatomy of theMicroscope
  18. 18. • The microscope illustrated in Figure7 is an Olympus Provis AX70research microscope. Thismicroscope represents the lateststate-of-the-art design thatincorporates multiple illuminators(episcopic and diascopic), analyzersand polarizers, DICprisms, fluorescenceattachments, and phase contrastcapabilities. The photomicrographysystem is the ultimate insophistication and performancefeaturing spotmeasurement, automatic exposurecontrol, and zoom magnification forflexible, easy framing. The Y-shapedframe is designed to be user-friendly by offering the maximum inoperator comfort and ease of use.History and Anatomy of theMicroscope
  19. 19. • The optical principles of the compoundmicroscope are incorporated into the basic designof different types of light microscopes. Themicroscopes most applicable for examiningforensic specimens are as follows:– The compound microscope– The comparison microscope– The stereoscopic microscope– The polarizing microscope– The microspectrophotometerHistory and Anatomy of theMicroscope
  20. 20. • The parts of the compound microscope areillustrated at the left. This microscope consists of amechanical system, which supports themicroscope, and an optical system.• The mechanical system of the compoundmicroscope is composed of six main parts:– BASE: the support on which the instrument rests– ARM: a C-shaped upright structure, hinged to thebase, that supports the microscope and acts as a handlefor carrying– STAGE: The horizontal plate on which the specimens areplaced for study. The specimens are normally mountedon glass slides that are held firmly in place on the stageby stage clips.– BODY TUBE: A cylindrical hollow tube on which theobjective and eyepiece lenses are mounted at oppositeends. This tube is merely a corridor through which lightpasses from one lens to another.– COARSE ADJUSTMENT: This knob focuses the microscopelenses on the specimen by raising and lowering the stage.– FINE ADJUSTMENT: The movements effected by thisknob are similar to those of the coarse adjustment butare of a much smaller magnitude.The Compound Microscope
  21. 21. • The optical system is made up offour parts:– ILLUMINATOR: Most modernmicroscopes use artificial lightsupplied by a lightbulb to illuminatethe specimen being examined. If thespecimen is transparent, the light isdirected up toward and through thespecimen stage from an illuminatorbuilt into the base of the microscope.This is known as transmittedillumination. When the object isopaque – that is, not transparent – thelight source must be placed above thespecimen so that it can reflect off thespecimen’s surface and into the lenssystem of the microscope. This type ofillumination is known as vertical orreflected illumination.The Compound Microscope
  22. 22. – CONDENSER: The condenser is a lens systemunder the microscope stage that collects lightrays from the base illuminator and focusesthem on the specimen. The simplestcondenser is known as the Abbé condenser.It consists of two lenses held together in ametal mount. The condenser also includes aniris diaphragm that can be opened or closedto control the amount of light passing intothe condenser.– OBJECTIVE LENS: This is the lens positionedclosest to the specimen. To facilitatechanging from one objective lens toanother, several objectives are mounted on arevolving nosepiece or turret located aboutthe specimen. Most microscopes areparfocal, meaning that when the microscopeis focused with one objective in position, theother objective can be rotated into place byrevolving the nosepiece while the specimenremains very nearly in correct focus.– EYEPIECE or OCULAR LENS: This is the lensclosest to the eye. A microscope with onlyone eyepiece is monocular; one constructedwith two eyepieces is binocular.The Compound Microscope
  23. 23. Each microscope lens is inscribed with a numbersignifying its magnifying power. The image viewed bythe microscopist will have a total magnification equalto the product of the magnifying power of theobjective and eyepiece lenses. For example, aneyepiece lens with a magnification of 10 times (10x)used in combination with an objective lens of 10x has atotal magnification power of 100x. Most forensic workrequires a 10x eyepiece in combination with either a4x, 10x, 20x, or 45x objective. The respectivemagnifications will be 400x, 100x, 200x, and 450x.The Compound MicroscopeMagnification and Resolution
  24. 24. • In addition, each objective lens is inscribed with itsnumerical aperture. (N.A.). The ability of an objective lensto resolve details into separate images instead of oneblurred image is directly proportional to the numericalaperture value of the objective lens. For example, anobjective lens of N.A. 1.30 can separate details at half thedistance of a lens with an N.A. of 0.65. The maximumuseful magnification of a compound microscope isapproximately 1,000 times the N.A. of the objective beingused. This magnification is sufficient to permit the eye tosee all the detail that can be resolved. Any effort toincrease the total magnification beyond this figure will yieldno additional detail and is referred to as emptymagnification.The Compound MicroscopeMagnification and Resolution
  25. 25. • The experienced microscopist weighs a number ofimportant factors before choosing a magnifying power:– FIELD OF VIEW: A first consideration must be the size of thespecimen area, or the field of view, that the examiner wishesto study. As magnifying power increases, the field of viewdecreases. Thus, it is best to first select a low magnification inwhich a good general overall view of the specimen is seen, andto switch later to a higher power in which a smaller portion ofthe specimen can be viewed in more detail.– DEPTH OF FOCUS: The depth of focus is also a function ofmagnifying power. After a focus has been achieved on aspecimen, the depth of focus defines the thickness of thatspecimen. Areas above and below this region will be blurredand can be viewed only when the focus is readjusted. Depthof focus decreases as magnifying power increases.The Compound MicroscopeMagnification and Resolution
  26. 26. • Forensic microscopy oftenrequires a side-by-sidecomparison of specimens. Thiskind of examination can be bestperformed with a comparisonmicroscope, such as the onepictured at left.• Basically, the comparisonmicroscope is two compoundmicroscopes combined into oneunit. The unique feature of itsdesign is that it uses a bridgeincorporating a series of mirrorsand lenses to join twoindependent objective lensesinto a singular binocular unit.The Comparison Microscope
  27. 27. • A viewer looking through theeyepiece lenses of the comparisonmicroscope observes a circularfield, equally divided into two partsby a fine line. The specimenmounted under the left-handobjective appears in the left half ofthe field, and the specimenmounted under the right-handobjective appears in the left half ofthe field.• Comparison microscopes designedto compare opaque objects, suchas bullets and cartridge casings, areequipped with vertical or reflectedillumination.• Comparison microscopes used tocompare hairs or fibers usetransmitted illumination.The Comparison Microscope
  28. 28. The Stereoscopic Microscope• The details that characterize thestructures of many types of physicalevidence do not always requireexamination under very highmagnifications. For such specimens, thestereoscopic microscope has proven quiteadequate, providing magnifying powersfrom 10x to 125x. This microscope has theadvantage of presenting a distinctivethree-dimensional image of an object.Also, whereas the image formed by thecompound microscope is inverted andreversed, the stereoscopic microscope ismore convenient because prisms in itslight path create a right-side-up image.• The stereoscopic microscope shown isactually two monocular compoundmicroscopes properly spaced and alignedto present a three dimensional image of aspecimen to the viewer, who looksthrough both eyepiece lenses.
  29. 29. • The stereoscopic microscope is undoubtedly the mostfrequently used and versatile microscope found in thecrime laboratory. Its wide field of view and greatdepth of focus make it an ideal instrument forlocating trace evidence in debris, garments, and tools.Furthermore, its potentially large working distance(the distance between the objective lens and thespecimen) makes it ideal for microscopic examinationof big, bulky items. When fitted with verticalillumination, the stereoscopic microscope becomesthe primary tool for characterizing physical evidenceas diverse as paint, soil, gunpowder residues, andmarijuana.The Stereoscopic Microscope
  30. 30. • Before we can discuss the polarizing lightmicroscope, we must first talk about the theory oflight. As with all matter, knowledge of the nature andbehavior of light is fundamental to understandingphysical properties important to the examination offorensic evidence. Two simple models explain light’sbehavior. The first model describes light as acontinuous wave; the second depicts it as a stream ofdiscrete energy particles. Together, these two verydifferent descriptions explain all of the observedproperties of light, but by itself, no one model canexplain all the facets of behavior of light.The Polarizing MicroscopeThe Theory of Light
  31. 31. • The wave concept depicts light as having anup-and-down motion of a continuouswave, as shown below. Such a wave can becharacterized by two distinct properties:wavelength and frequency.Light as a Wave
  32. 32. Wavelength and Wave Motion• The distance between two consecutive crests (high points)or troughs (low points) of a wave is called the wavelength;it is designated by the Greek letter lambda (λ) and istypically measured in nanometers (nm), or millionths of ameter. The number of crests (or troughs) passing any onegiven point in time is defined as the frequency of thewave. Frequency is normally designated by the letter ƒand is expressed in cycles per second (cps). Frequencyand wavelength are inversely proportional to oneanother, as shown in the relationshipF = c/λwhere cequals the speed of light.
  33. 33. Wavelength and Wave MotionWave length PeakAmplitudeTrough
  34. 34. • Many of us have held a glass prism up toward thatsunlight and watched it transform light into the colorsof the rainbow. The process of separating light into itscomponent colors is called dispersion. Visible lightusually travels at a constant velocity of nearly 300million meters per second. However, on passingthrough a prism, each color component of light isslowed to a speed slightly different from those of theothers, causing each component to bend at a differentangle as it emerges from the prism. The bending oflight waves because of the change in velocity if calledrefraction.Wavelength and Wave Motion
  35. 35. Wavelength and Wave MotionThe observation that a substance has a coloris consistent with this description of whitelight. For example, when light passes througha red glass, the glass absorbs all thecomponent colors of light except red, whichpasses through or is transmitted by the glass.Like wise, one can determine the color of anopaque object by observing its ability toabsorb some of the component colors of lightwhile reflecting others back to the eye. Coloris thus a visual indication that objects absorbcertain portions of visible light and transmitor reflect others. Scientists have longrecognized this phenomenon and havelearned to characterize different chemicalsubstances by the type and quantity of lightthey absorb. This has important applicationsfor the identification and classification offorensic evidence.
  36. 36. The Multispectral SunThis animation shows views of the Sun at variousfrequencies across the electromagnetic spectrum. Notehow different features and regions of the Sun are visiblein the different views. The visible light view shows thephotosphere, including several sunspots. The infraredview shows the lower chromosphere immediately abovethe photosphere, where temperatures are still relativelycool. Most of the high energy photons that produce theUV and X-ray views come from higher up in the Suns hotatmosphere. Notice how the areas of the atmosphereabove sunspots tend to be especially bright in the X-rayand UV views. Sunspots are visible indicators of magneticdisturbances on the Sun that high energy phenomenasuch as solar flares and coronal mass ejections.Most of the individual views portray the Sun as seenthrough a very narrow range of wavelengths; forexample, the IR view is just a narrow band of infrared"light" with a wavelength around 1,083 nanometers (asopposed to the entire IR portion of the spectrum, whichranges across wavelengths from 750 nm to 1 mm), whilethe first UV image is centered around a wavelength of30.4 nanometers. These narrow wavelength "windows"in the EM spectrum are actually the "fingerprints" ofspecific elements at specific temperatures.
  37. 37. • The observation that a substance or objecthas color is consistent with the description ofwhite light. For example, when light passesthrough red glass, the glass absorbs all thecomponent colors of light except red, whichpasses through or is transmitted by the glass.Likewise, one can determine the color of anopaque object by observing its ability toabsorb some of the component colors of lightwhile reflecting others back to the eye.Wavelength and Wave Motion
  38. 38. • Color is thus a visual indicationthat objects absorb certainportions of visible light andtransmit or reflect others.Scientists have long recognizedthis phenomenon and havelearned to characterizedifferent chemical substancesby the type and quantity oflight they absorb. This hasimportant applications for theidentification and classificationof forensic evidence.Perception of COLOR
  39. 39. The Electromagnetic Spectrum• Similarly, the range of colors that make up thevisible spectrum can be correlated withfrequency. For instance, the lowest frequenciesof visible light are red; waves with a lowerfrequency fall into the invisible infrared (IR)region. The highest frequencies of visible lightare violet, waves with a higher frequency extendinto the invisible, ultraviolet (UV) region. Nodefinite boundaries exist between any colors orregions of the electromagnetic spectrum; instead,each region is composed of a continuous range offrequencies, each blending into the other.
  40. 40. • Ordinarily, light in any region ofthe ES is a collection of wavespossessing a range of wavelengths.Under normal circumstances, thislight comprises waves that are allout of step with each other(incoherent light).However, scientists can producelight that has all its wavespulsating in unison. This is called alaser (light amplification bystimulated emission of radiation).Light in this form is very intenseand can be focused on a very smallarea. Laser beams can be focusedto pinpoints that are so intensethat they can zap microscopicholes in a diamond.Lasers
  41. 41. • The light’s wavelike motion in space can beinvoked to explain many facets of its behavior.The waves that compose a beam of light can bepictured as vibrating in all directionsperpendicular to the direction in which the lightis traveling. However, when a beam of lightpasses through certain types of speciallyfabricated crystalline substances, it emergesvibrating in only one plane. Light that is confinedto a single plane of vibration is said to be plane-polarized.The Polarizing Microscope
  42. 42. • The device that creates polarized lightis called a polarizer.• Because polarized light appears nodifferent to the eye from ordinarylight, special means must be devisedfor detecting it. This is accomplishedsimply placing a second polarizingcrystal, called an analyzer, in the pathof the polarized beam. If the polarizerand analyzer are aligned parallel toeach other, the polarized light passesthrough and is seen by the eye. If, onthe other hand, the polarizer andanalyzer are set perpendicular to oneanother, or are “crossed”, no lightpenetrates, and the result is totaldarkness or extinction.The Polarizing Microscope
  43. 43. • Essentially, the polarizer is placedbetween the light source and thesample stage to polarize the lightbefore it passes through the specimen.The polarized light penetrating thespecimen must then pass through ananalyzer before it reaches the eyepieceand finally the eye. Normally thepolarizer and analyzer are “crossed” sothat when no specimen is in place, thefield appears dark. However,introducing a specimen that polarizeslight reorients the polarized light,allowing it to pass through the analyzer.This result produces vivid colors andintensity contrasts that make thespecimen readily distinguishable.The Polarizing Microscope
  44. 44. • Few instruments in a crime laboratory can match theversatility of the microscope. The microscope’s magnifyingpower is indispensable for finding minute traces ofphysical evidence. Many items of physical evidence can becharacterized by a microscopic examination of theirmorphological features. Likewise, the microscope can beused to study how light interacts with the material underinvestigation, or it can be used to observe the effects thatother chemical substances have on such evidence. Each ofthese features allows an examiner to better characterizeand identify physical evidence. Recently, linking themicroscope to a computerized spectrophotometer hasadded a new dimension to its capability. This combinationhas given rise to a new instrument called themicrospectrophotometer.The Microspectrophotometer
  45. 45. The MicrospectrophotometerWith the development of themicrospectrophotometer, a forensicanalyst can view a particle under amicroscope while a beam of light isdirected at the particle to obtain itsabsorption spectrum. An absorptionspectrum is the characteristic patternof dark lines or bands that occurswhen electromagnetic radiation ispassed through an absorbing mediuminto a spectroscope. An equivalentpattern occurs as colored lines orbands in the emission spectrum ofthat medium. The range offrequencies of electromagneticradiation readily absorbed by asubstance by virtue of its chemicalcomposition is unique to thatsubstance and creates a “fingerprint”.
  46. 46. • Depending on the type of light employed, anexaminer can acquire either a visible or an IRspectral pattern of the substance being viewedunder the microscope. The obvious advantageof this approach is to provide added informationto characterize trace quantities of evidence. Amicrospectrophotometer designed to measurethe uptake of visible light by materials is shownon the previous slide.The MicrospectrophotometerAbsorption spectrum of the planet Gliese581g to analyze the possibility of thepresence of life, or the capability of theplanet to support life (similar to ours).
  47. 47. • Visual comparison of color is usually one of the first steps in examining paint, fiberand ink evidence. Such comparisons are easily obtained using a comparisonmicroscope. A forensic scientist can use the microspectrophotometer to comparethe color of materials visually while plotting an absorption spectrum for each itemunder examination. This displays theExact wavelengths at which each itemabsorbs light in the visible-light spectrum.Another emerging technique in forensicscience is the use of the infrared spectro-photometer to examine fibers and paints.The “fingerprint” IR spectrum is uniquefor each chemical substance.The Microspectrophotometer
  48. 48. • All the microscopesdescribed thus far use lightcoming off the specimen toproduce a magnifiedimage. The scanningelectron microscope (SEM)is, however, a special casein the family ofmicroscopes . The image isformed by aiming a beamof electrons onto thespecimen and studyingelectron emissions on aclosed TV circuit.The Scanning Electron Microscope(SEM)
  49. 49. • Palynology: The branch of scienceconcerned with the study of pollen, spores ,and similar palynomorphs, living and fossil.• Forensic Palynology: the use of pollen andspore evidence in legal cases (Mildenhall,1982). In its broader application, the field offorensic palynology also includes legalinformation derived from the analysis of abroad range of microscopic organisms--suchas dinoflagellates, acritarchs, andchitinozoans--that can be found in bothfresh and marine environments (Faegri etal., 1989). However, in most samplingsituations forensic palynologists rarelyencounter these other types of organismsbecause most are restricted to fossildeposits. Studies of palynomorphs trappedin materials associated with criminal or civilinvestigations are slowly gaining recognitionas valuable forensic techniques.Introduction to ForensicPalynology
  50. 50. • Of the many plant species onearth, more than half a million producepollen or spores. The pollen or sporesproduced by each species has a uniquetype of ornamentation and morphology.This means that pollen or spores can beidentified and used to provide linksbetween a crime scene and a person orobject if examined by a trained analyst.This technique is called forensicpalynology and includes the collectionand examination of pollen and sporesconnected with crime scenes, illegalactivities, or terrorism. Microscopy isthe principal tool used in the field offorensic palynology.Forensic Palynology: Pollen andSpores as Evidence
  51. 51. • In nature, pollen grains are thesingle-celled male gametophytes ofseed-bearing plants. The pollengrain wall (exine) is durable becauseit protects and carries the spermcells needed for plant reproduction.Spores consist of both male andfemale gametes of plants such asalgae, fungi, mosses, and ferns.Pollen-producing plants are eitheranemophilous (their pollen isdispersed by wind) orentomophilous (their pollen iscarried and dispersed by insects orsmall animals).Forensic Palynology: Characteristics ofSpores and Pollen
  52. 52. • Fairly precise geographicallocations can often beidentified by the presenceof different mixtures ofairborne pollens producedby anemophilous plants.For example, it may bepossible to identify ageographical origin by aprofile of the pollensamples retrieved from asuspect’s clothing simply bythe type and percentages ofairborne pollen grains.Forensic Palynology: Characteristics ofSpores and Pollen
  53. 53. • Entomophilous plants usuallyproduce a small amount ofpollen that is very sticky innature. Therefore, this type ofpollen is very rarely deposited onclothing or other objects exceptby direct contact with the plant.This information is useful whenreconstructing the events of acrime because it indicates thatthe clothing, a vehicle, or otherobjects may have come intodirect contact with plant typesfound at a crime scene.Forensic Palynology: Characteristics ofSpores and Pollen
  54. 54. • Both spores and pollen are microscopic in size and areproduced by the adult plants and dispersed by themillions, and both can be analyzed using similarmethods that use a variety of microscopic techniques.Using a compound light microscope with magnificationcapabilities up to 1,000x, analysts usually can identifypollen and spores as coming from a specific plantfamily or genus, and at times even a unique species.However, often the pollen or spores of related speciesmay appear so similar that identification of the speciesis possible only with careful analysis using an SEM.Forensic Palynology: Analysis ofSpores and Pollen
  55. 55. • Unique shapes, aperture type, and surfaceornamentation are typically used toidentify spore samples. Useful features forcharacterizing pollen grains include shape,apertures, and wall and surfacesculpturing. Shapes of pollen grainsinclude spheres, triangles, ellipses,hexagons, pentagons, and many othergeometric variations. Apertures are theopenings on pollen grains from which thepollen tube grows and carries the sperm tothe egg to complete fertilization.Forensic Palynology: Analysis ofSpores and Pollen
  56. 56. • To avoid destruction orcontamination, early collection offorensic pollen samples foranalysis is important and shouldbe completed as soon as possibleat a crime scene by a trainedpalynologist. This kind of expert’sfirst task is to calculate theestimated production anddispersal patterns of spores andpollen (called the pollen rain) forthe crime scene or area ofinterest, and then using thatinformation to produce a kind of“pollen fingerprint” of thatlocation.Forensic Palynology: Analysis ofSpores and Pollen