Micro lab

• Light microscopy techniques include bright-field, fluorescence, phase-contrast, confocal, and polarizing microscopy

  
• Brightfield microscopy:

  • Simple light microscope producing images against a white background
  • Advantages: simple to use, adaptable with new technology, can view some specimens without staining
  • Disadvantages: can't observe living specimens, very low contrast, intense lighting can kill microorganisms, limited resolution due to visible light
• Darkfield microscopy:

  • A brightfield microscope with a modification using an opaque disk to create a hollow cone of light focused on the specimen
  • Advantages: viewing live specimens, more detailed view of external features, adding a fluorescent dye increases visibility
  • Disadvantages: can mistake "dust" as an organism, requires strong illumination that can damage the specimen
• Phase contrast microscopy:

  • Useful for unstained cells and transparent tissue sections
  • Advantages: makes unstained cells visible, living things can be observed in their natural state without preparation of fixation and staining
  • Disadvantages: phase-contrast condensers and objective lenses add considerable cost to a microscope, requires alignment of light path
• Differential contrast microscopy:

  • A modification technique of phase-contrast microscopy, also known as "Nomarski" optics
  • Advantages: provides high-contrast images of living organisms with a three-dimensional appearance, useful in distinguishing structures within live, unstained specimens
• Fluorescence microscopy:

  • Uses fluorophores to create a luminiscent specimen with bright objects against a dark background
  • Advantages: clear examination for thick specimens, possible to make 3D images, non-invasive method
  • Disadvantages: uses expensive laser, requires fluorescent sample, high-intensity laser irradiation can be harmful to living cells and tissues
• Electron microscopy:

  • Scanning electron microscope uses beams of electrons to visualize specimens and generate highly-magnified three-dimensional images
  • Transmission electron microscope has a powerful magnification of about 2 million times that of the light microscope, produces high-quality images with high clarity
• A microscope is an essential tool to see microorganisms that are too small to be seen by the naked eye
• To use a microscope effectively, it is necessary to become familiar with its parts
• Ocular lens: what an observer looks through & is present in the upper portion of the microscope
• Eye Piece Tube clasps the eyepieces which are positioned above the objective lens
• Body Tube holds the objective and ocular lens at the two ends, including the head and the nosepiece
• Course Adjustment knob: rapid control allowing quick focusing by moving the objective lens/stage up & down (initial focusing)
• Fine Adjustment knob: slow but precise control used to fine-tune the image when viewing at higher magnifications
• Arm connects and supports the base & head of the microscope, used to carry the microscope
• Revolving Nosepiece holds 2 or more objective lenses & can be rotated to easily change power
• Objective Lens: main optical lenses ranging in various magnifications from 4x to 100x, generally including 3-5 lenses on a single microscope
• Stage Clip: operated to hold the slide in place
• Aperture: tiny hole in the stage through which transmitted light enters
• Stage: where the specimen to be viewed is placed
• Condenser: used to gather and focus the illuminator's light onto the specimen, located beneath the stage
• Illuminator: light source located at the base of the microscope
• Iris diaphragm: regulates the light that will go to the condenser
• Base: bottom part of the microscope
• Parfocal means there is little to no need for adjustment when switching between objectives
• Working Distance: the distance between the objective lens and the stage; inversely related to the size of the objective (smaller objective, larger working distance; larger objective, smaller working distance)
• Magnification: indicates how many times the object is enlarged
• Resolution: the ability to distinguish between two adjacent points
• Focal Point: the region where the image is formed
• Depth of Focus: the thickness of the specimen that is in focus
• Short wavelength and high numerical aperture lead to high resolution
• Numerical aperture: a measure of the ability of a lens to gather light; higher numerical aperture values indicate better resolution
• Low magnification results in a larger field of view, while high magnification results in a smaller field of view (and vice versa)
• The purpose of the microscope is to magnify objects that are too small to be seen with the naked eye.
• Resolution refers to the ability of a microscope to distinguish between two closely spaced points on an image, measured by the distance at which two point sources can no longer be distinguished as separate points.
• Confocal microscopy:

  • Uses light from a laser through the objective of a standard light microscope to excite a specimen within a narrow plane of focus
  • Fluorescent stains are generally used to increase contrast and resolution
  • Non-invasive method
  • Specimen is stained with a fluorescent dye and illuminated one plane at a time

  Advantages:

  • Useful for examining thick specimens like biofilms, which can be examined alive and unfixed
  • Possible to make 3D images
  • Provides clear examination for thick specimens

  Disadvantages:

  • Uses laser (expensive)
  • Requires fluorescent sample
  • High-intensity laser irradiation can be harmful to living cells and tissues
• Scanning Electron Microscope:

  • Uses beams of electrons instead of light, reflected back to the specimen, producing a three-dimensional image
  • Studies the surface features of cells and viruses
  • Involves coating the sample with a thin layer of conductive material like gold, silver, platinum, or cadmium (sputter coating)
• Transmission Electron Microscope:

  • Uses a particle beam of electrons to visualize specimens and generate a highly-magnified image, magnifying objects up to 2 million times
  • Techniques include adding heavy metal compounds to the fixative and ultrathin sectioning of embedded tissue with a glass knife
• Confocal microscopy uses a laser to scan multiple z-planes successively, producing high-resolution images at various depths that can be constructed into a 3D image by a computer, useful for examining thick specimens like biofilms
• Two-photon microscopy uses a scanning technique, fluorochromes, and long-wavelength light to penetrate deep into thick specimens like biofilms