MODULE 2 | Forensic Microscopy

• Principal Purpose of Microscope

  The principal focus of any microscope is to form an enlarged image of a small object. In simple terms, it is for magnification purposes.
• Aspects of Forensic Science Where Microscopy is Applied

  • Firearm Examination
  • Trace Evidence (PLM)
  • Document Investigation aka QDE
  • Forensic Serology
• Sample preparation for microscopy is not required in the following fields:

  1. Firearms Examination
  2. Forensic Serology
  3. Questioned Document
• Sample preparation for microscopy is required in the field of trace evidence.
• Antonie Van Leeuwenhoek

  • 1632-1723
  • Father of Microbiology
  • Created a simple microscope that could magnify to about 275x and published drawings of microorganisms in 1683
  • Discovered bacteria, free-living and parasitic microscopic protists, sperm cells, blood cells, and microscopic nematodes
  • Incorrectly known as "the inventor of microscope"
• George Adams Sr.

  • Made many microscopes from about 1740-1772 but he was predominantly just a good manufacturer, not an inventor. It was actually believed that he was a copier.
• Simple microscopes could attain around 2-micron resolution, while the best compound microscopes were limited to around 5 microns because of chromatic aberration.
• Chester Moore Hall

  • He designed a system that used a concaved lens next to a convex lens which could realign all the colors. This was the first achromatic lens.
  • George Bass was the lens-maker who actually made the lenses, but he did not divulge the secret until over 20 years later to John Dolland who copied the idea in 1759 and patented the achromatic lens.
• Giovanni Battista Amici


  In 1827, he built high quality microscopes and introduced the first matched achromatic microscope. He had previously (1813) designed "reflecting microscopes" using curved mirrors rather than lenses. He recognized the importance of coverslip thickness and developed the concept of "water immersion".
• Speed of Light

  Light travels at 300,000 km/s in space and it goes down to 197,000 km/s in glass.
• How big are light waves?

  They range from about 400nm to about 700nm in length.
• Absorption


  When light passes through an object the intensity is reduced depending upon the color absorbed. Thus, the selective absorption of white light produces colored light.
• Refraction


  Direction change of a ray of light passing from one transparent medium to another with different optical density.

  
• Reflection


  It is the phenomenon in which light travelling in one medium, incident on the surface of another returns to the first medium. This is also known as the bouncing back of light.
• Transmission


  It occurs when light passes through the surface of a medium.
• Diffraction


  Light rays bend around edges—new wavefronts are generated at sharp edges—the smaller the aperture the lower the definition.

  A) Small diffraction effect

  B) Large diffraction effect

  
• Dispersion


  Separation of light into its constituent wavelengths when entering a transparent medium—the change of refractive index with wavelength, such as the spectrum produced by a prism or a rainbow. This is also known as the separation of light.
• Magnification of Human Eye

  • An object can be focused generally no closer than 250mm from the eye (depending on how old you are)
  • This is considered to be the normal viewing distance from 1x magnification
  • Young people may be able to focus as close as 125mm so they can magnify as much as 2x because the image covers a larger part of the retina — i.e., it is magnified at the place where the image is formed.
• Opaque


  These are objects that do not allow light to pass through; they absorb or reflect all light.
• Translucent


  These are objects that can be seen through, but not clearly; they absorb, reflect, and transmit light.
• Transparent


  These are objects that allow almost all of the light to pass through, so they can be seen through clearly.
• Primary Colors


  Colors:

  1. Red
  2. Green
  3. Blue

  Light:

  1. Cyan
  2. Magenta
  3. Yellow
• Subtractive Colors


  It works by partially or entirely masking colors on a lighter background, usually white. The ink reduces the light that would otherwise be reflected.
• Additive Colors


  It is a property of a color model that predicts the appearance of colors made by coincident component lights.
• Light and Refraction


  Convex (or positive) lenses converge (or focus) light and can form images. Concave (or negative) lenses diverge (or spread out) light rays.

  
• 2 Important Parameters of Microscopy

  1. Magnification - It is a measure of how much larger a microscope causes an object to appear.
  2. Resolution - It is the smallest distance by which two points can be separated and still be distinguished as separate object. Moreover, it is the ability to see increasingly fine details as the magnification is increased. Remember, the smaller the resolution means better quality of the image.
• How to calculate microscope magnification?

  Multiply the magnification of the eyepiece by the magnification of the objective lenses.

  Formula:

  Magnification of Microscope = (eyepiece magnification) x (objective lenses magnification)
• Magnification of objective lenses

  1. Scanning objective - 4x
  2. Low Power Objective - 10x
  3. High Power Objective - 40x
  4. Oil Immersion - 100x
• Classification of Microscopes

  1. Light Microscope - It produces an image of a specimen by using visible light (photons). It can often be performed on living cells.
  2. Electron Microscope - It produces an image of a specimen by using a beam of electrons. The samples must be placed under a vacuum.
• Binocular (Stereo) Microscope


  It is an optical microscope that provides two optical paths for each eye resulting in a three-dimensional view. It is also known as dissecting microscope and stereo zoom microscope. It uses low magnification (10x-50x or 2x-100x) and the image it produces is upright. It is commonly used in document examination, as in trying to determine whether one pen stroke passes over another.
• Compound Microscope


  It uses multiple lenses to enlarge image of a sample. It view samples at high magnification (40-1000x), which is achieved by the combined effect of two sets of lenses: the ocular lens (in the eyepiece) and the objective lenses (close to the sample). It provides a single optical path resulting in the same image to both the left and right eye. It also uses high magnification and shows an upside down image.
• Scanning Electron Microscope (SEM)


  This powerful imaging tool allows you to see extremely small details of materials at high magnifications with excellent clarity and depth of field. It uses electrons to create image rather than light thus, it is classified as an electron microscope. It has the ability to conduct chemical and morphological analyses using spectroscopic methods. It applies the reflection property of light.
• Transmission Electron Microscope (TEM)


  It is an analytical technique used to visualize the smallest structures in matter. It can reveal stunning details at the atomic scale by magnifying nanometer structures up to 50 million times. It uses a beam of electrons to illuminate specimens, rather than photons used in traditional optical microscopy. It is used for pathogen analysis and for examination of paint pigments. It applies the transmission property of light.
• Infrared (IR) Microscope


  It uses a source that transmits infrared wavelengths of light to view an image of the sample. It allows for the measurement of IR spectra in addition to visualization of samples. It is used in drug identifications.
• Phase Contrast Microscope


  It is used to enhance the contrast of images of transparent and colorless specimens. It enables visualization of cells and cell components that would be difficult to see using an ordinary light microscope.
• Polarizing Light Microscope (PLM)


  It enhances image contrast and improves image quality in comparison to other microscopy methods. It uses both an analyzer and a polarizer to cross-polarize the light and pick up differences in the colors in the optical path of the specimen being examined.
• Polarized Light

  Light is an electromagnetic wave that vibrates in all directions. Any light which vibrates in more than one direction is called "unpolarized light"; whereas, a light wave that vibrates in a single direction is called "polarized light."
• Refractive Index


  MEASUREMENT: Immersion Method. Specimen placed in index of refraction oils until it disappears.

  DETERMINATIVE VALUE: Identification of unknown isotropic crystals. Comparison of glass particles.
• Refractive Indices


  MEASUREMENT: Orientation of principal vibration direction with polarizer microscope. Matching of two (uniaxial crystals) or three (biaxial crystals) with the index of refraction oils.

  DETERMINATIVE VALUE: Crystallographic identification of chemical crystals and minerals. Identification and comparison of artificial fibers.
• Birefringence


  MEASUREMENT: Numerical difference between two principal refractive indices. By subtracting larger refractive index from smaller or by measurement of retardation with a compensator and measurement of thickness.

  DETERMINATIVE VALUE: Identification of crystalline and semi-crystalline materials. Comparison of certain artificial fibers.