MICROSCOPY

• Microscopy is an essential instrument to analyze samples and detect tissue elements and microorganisms that help in the diagnosis and management of diseases
• Parfocal


  Objectives that can be changed with minimal or no refocusing
• Types of viewing heads

  • Monocular
  • Binocular
  • Trinocular
• Monocular heads


  • One eyepiece when viewing the specimen
  • Using an LCD camera would occupy the eyepiece
  • Are light weight and are inexpensive
• Binocular heads

  • Have two eyepieces
  • More convenient and comfortable to use; most common choice
• Trinocular heads


  • Have a third eyepiece tube that can be used by another person simultaneously or by an LCD camera
  • More expensive than the other two types
• Types of microscope based on application

  • Biological microscope
  • Stereoscopic microscope

  
• Biological microscope


  • Magnification: 4x (scanner), 10x (LPO), 40x (HPO), 100x (Oil)
  • Light comes from beneath the stage
  • For blood cells, bacteria and protozoa, etc.
• Stereoscopic microscope


  • Magnification: 7x to 46x
  • Light comes from above and beneath the stage
  • For flowers, rocks, insects, etc.

  
• Types of microscope based on structure

  • Inverted microscope
  • Upright microscope

  
• Inverted microscope


  • Observe target from below
  • Specimens observed are cells soaked in culture dishes
• Upright microscope


  • Observe target from above
  • Specimens observed are placed on slides

  
• Types of microscope based on material used for visualization

  • Light/Optical microscopy
  • Electron microscopy
  • X-ray microscopy
  • Scanning probe microscopy

  
• Total magnification


  Product of the magnifying power of the objective and eyepiece
• Proper handling of microscope

  • Carry the microscope with two hands, supporting the base with one hand and holding the arm with the other hand
  • Always hold the microscope in a vertical position
  • Only clean optical surfaces with good quality lens tissue and commercial lens cleaner
  • Do not use Oil for 10x and 40x objectives
  • Clean the oil immersion lens after use
  • Always remove the slides with the LPO/Scanner raised
  • Store the microscope with the LPO/Scanner position and the stage control
  • The top lens of the eyepiece should be polished to remove dust or finger marks, and microscope should be checked for critical illumination
  • Rotation of the eyepiece should be polished to remove dust or finger marks, and microscope should be checked for critical illumination
  • Dust removal should be done with an air bulb
  • If dust is present on the condenser, the object being viewed will come in and out when racked up and down
• Proper use of a microscope

  • Do not adjust the objectives by holding the objectives itself, but by the revolving nosepiece
  • Adjust brightness according to your preference using the rheostat and condenser
  • Rheostat - It is a dial that controls the intensity of the light produced
  • Brush off dust and debris on the lenses
  • Disinfect Microscope using 70% Ethyl Alcohol before and after using
  • Storage Position: Lowered stage, Scanner position, oculars at the bottom, wire unattached and coiled, rheostat turned to minimum, and switch is off. Oil or debris must be removed prior to storage. No slides should be on the stage. Cover when not in use

  
• Types of light microscopes

  • Bright-Field microscope
  • Dark-Field microscope
  • Phase-Contrast microscope
  • Differential Interference Contrast Microscopy
  • Confocal Microscopy
  • Fluorescence Microscopy
  • Scanning Electron Microscope
  • Transmission Electron Microscope
  • Polarizing Microscopy

  
• Bright-Field microscope


  • Most common type of light microscope
  • A dark sample on a bright background
  • Also known as a compound microscope because it uses 2 sets of lenses to magnify an image
  • First Lens: Objectives
  • Second Lens: Ocular Lens (Eyepiece)
  • Objects or cells appear dark against a light background
  • Ocular → 10x
  • Disadvantage: Light dust is easily observed

  
• Dark-Field microscope


  • Used for unstained specimens that cannot be easily viewed with a bright-field microscope
  • Contrast comes from the sample scattering the light
  • Bright specimens behind a dark background
  • Uses an opaque condenser to block majority of the light coming from the light source
  • Objects or cells appear light against a dark background
• Phase-Contrast microscope


  • Adaptation of bright-field microscope
  • Uses 2 phase rings attached to the objective and condenser
  • Enhances visualization and detail of specimens
  • Used for living or unstained cells and tissue sections that are usually transparent and colorless (unstained); also for living cultured cells
  • Creates a good contrast between the specimen and the background to improve visualization
  • Principle: Light diffraction

  
• Differential Interference Contrast Microscopy


  • A modified version of phase-contrast microscopy that produced 3D because of the optics used
  • Nomarski optics - Allows the production of 3D images of living cells - Used to observe cells of tissue cultures

  
• Confocal Microscopy


  • Uses a high intensity light (usually layer) and a plate with a pinhole in front of the image detector to only capture the focused light only
  • Purpose of the pinhole: only allow focused light to pass through and be detected to sharpen the image
  • It needs a high intensity light source because only a few light rays can pass through the pinhole
  • Computer-driven mirror system is used to create digitally reconstructed 3D by capturing different spots of the specimen

  
• Fluorescence Microscope


  • Used to detect bacteria and viruses within cells and tissues through immunofluorescence
  • Fluorescence - Property where atoms absorb light at a particular wavelength and emit light at a longer wavelength
  • Used to view naturally fluorescent substances or those that have been stained with fluorochromes
  • Acridine orange - binds to DNA & RNA
  • DAPI & Hoeschst - binds to DNA and nuclei; blue fluorescence under UV

  
• Diagnosis of Syphilis


  • Fluorescent Treponemal antibody-absorption test (FTA-ABS) - For the diagnosis of syphilis - Fluorochrome used: Fluorescein isothiocyanate (FITC) → color green
• Scanning Electron Microscope


  • Electron Microscopy - Uses electrons instead of light to magnify images
  • Useful for providing 3-dimensional images of the surface of microscopic objects
  • Electrons are focused into the specimen causing the release of radiation
  • Radiation is then captured, detected, amplified, and then images on a screen

  
• Transmission Electron Microscope


  • A beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through
  • Electrons that pass through are gathered and focused by an electromagnetic lens to present an image
  • Provides a 2-D images of objects
  • Higher resolving power than SEM
  • Best for visualizing viruses and ultrastructure of cell organelles
  • Heavy metal ions are added as fixatives or dehydrating solutions to increase their electron density and visibility
  • Common Fixatives: Osmium tetroxide, lead citrate, and uranyl compounds
  • Cryofracture and freezing etching - techniques that allow TEM study of cells without fixation

  
• Polarizing Microscopy


  • For the recognition of stained and unstained structures
  • Utilizes two polarizing lenses, where the specimen is between the lenses
  • Birefringence - Ability to rotate the direction of vibration of polarized light
  • A feature of crystalline substances or substances containing highly oriented and repetitive molecules (cellulose, collagen, microtubules, and actin filaments) are only structures are seen through the polarizing microscope

  
• Other types of microscope

  • Scanning Probe Microscope
  • Stereomicroscope
  • Lattice-Light Sheet Microscope
  • Autoradiography

  
• Scanning Probe Microscope


  • The tip is moved across the sample many times. This is why these are called "scanning" microscopes
  • Used to make images of nanoscale surfaces and structures, including atoms
  • Use a physical probe to scan back and forth over the surface of a sample
  • A computer gathers data that are used to generate an image of the surface
  • Resolution of <1nm
  • The distance of the deflection is measured by a laser that is reflected off the top of the cantilever and into an array of photodiodes
  • SPMs can detect differences in height that are a fraction of a nanometer about the diameter of a single atom

  
• Stereomicroscope


  • A stereo, or dissecting microscope provides a 3D view of the specimen
  • Two separate viewing angles (separate objective lenses and eyepiece for each eye) yield a 3D image
  • Ideal with dealing with thick or opaque samples
  • Utilizes light that is naturally reflected from the object

  
• Lattice-Light Sheet Microscope


  • 3D imaging or large samples
  • The beams are thinner and longer than any other microscopes
  • With much thinner light sheets, smaller samples can be imaged at higher resolutions
  • The sample is illuminated with a light sheet from the side, exciting fluorophores close to the focal plane

  
• Autoradiography


  • Cells or tissues are radioactively labeled with radioactive metabolites which causes them to produce weak radiation in certain areas as to where the metabolites are located
  • The radiation released is detected by microdetectors to produce an image
  • Radioactive metabolites used: nucleotides, amino acids, & sugars
• Disadvantages of microscopes

  • Optical Microscopy
  • Electron Microscopy
  • Scanning Probe Microscopy
  • X-Ray
• Optical Microscopy


  • Resolution of approximately 0.2 micrometers
  • Limits the practical magnification to -1,500x
  • Out-of-focus light from points outside the focal plan reduces image clarity
  • Internal structure difficult to observe
• Electron Microscopy

  • Large
  • Expensive equipment
  • Extremely sensitive to vibration and external magnetic fields
• Scanning Probe Microscopy


  • Slower in acquiring the image
  • Maximum image size is smaller
• 1. Ray

  • Due to the high energy can destroy the sample and cause the damage of cells
  • Possible mutations