Microscopy

• Define Resolution

  The measure of the minimum distance of two distinguishable points
• Define Contrast

  Visible differences in brightness or colour between parts of the sample
• Why must the specimen for a Conventional bright field light microscope samples be partially transparent

  • Light passes through the specimen
• Describe the uses of Confocal microscopy


  • Generates 3D images
  • Removes out-of-focus images
  • Can look inside thick specimens
  • Specimens are fluorescently stained
• Describe super-resolution


  • Gathers light from fluorescent molecules are records their position
• Describe the features of a Scanning electron microscope


  • The electron beam is scanned across the surface of the specimen
  • Electrons are reflected from the surface and collected by a detector
  • Electrons are detected and converted into an electronic signal
  • The image represents the topology of the sample
• Describe the Sample preparation for a light microscope


  
  

  1. Whole mounts

  2. Tissue sections

  3. Fixation [Chemicals to prevent autolysis and preserve structures]

  4. Dehydration and clearing [Remove water for wax impregnation]

  5. Embedding [Infiltrate with molten wax and transfer to a mould]

  6. Sectioning [Microtome produces thin sections collected onto glass slides]

  7. Staining [Wax is removed]
• Sample preparation for transmission electron microscopy


  1. Whole mounts

  2. Tissue sections

  3. Fixation [GLutaraldehyde and osmium tetroxide]

  4. Dehydration [Ethanol]

  5. Embedding [Plastic resins]

  6. Sectioning [Ultramicrotome]

  7. Staining [Heavy metal stains]
• Sample preparation for scanning electron microscopy


  1. Fixation [Glutaraldehyde and osmium tetroxide]

  2. Dehydration [Ethanol]

  3. Critical point drying [Ethanol removed]

  4. Coating [Gold]
• What is cell fractionation.

  Allows you to separate organelles for studying in isolation.
• Cell fractionationprocesses

  1. Homogenisation

  2. centrifugation
• Define Magnification

  The ratio of an object's image size to its real size
• What is the main similarity between a TEM and a light microscope

  The arrangement of lenses.
• Why are heavy metal stains used

  Tissues have little contrast under electron beams.
• Why is the TEM able to give a 3-D appearance.

  It has a great depth of focus.
• What type of structure is an electron microscope able to view

  Ultrastructure
• What are the four types of microscopy

  • Light
  • Fluorescence
  • Confocal
  • Electron
• What are the two types of electron microscopes

  Transmission and Scanning.
• What are the three parameters for microscopy

  Magnification, Resolution and Contrast.
• What are the two types of light microscopes

  Dissecting, Fluorescence, Contrast, Confocal and compound bright field.
• What is the main difference between a dissecting and compound brightfield microscope.

  Compound brightfield microscopes allow for higher magnification.

  Dissecting microscopes allow for 3D visualisation of the specimen.

  Compound brightfield microscopes have 4 objective lenses, whereas the dissecting microscope only has one.
• What are the objective lens magnifications for a compound brightfield

  4, 10, 40, 100.
• What name is given to the *100 magnification lens.

  Oil immersion.
• Label the compound brightfield microscope

  A) Eye piece

  B) Objective lens

  C) Stage Clip

  D) Stage

  E) Coarse adjustment

  F) Fine adjustment

  G) Stage controls

  H) Light adjustment

  I) Light switch

  J) Light source

  K) Condensor

  L) Diaphragm

  
• What are the types of contrast microscopes

  Phase and Differential.
• What is the advantage of light microscopes

  It can visualise living cells.
• What is the disadvantage of a light microscope.

  It's resolution is limited.
• What is the relationship between resolution and wavelength of radiation

  Inversely proportional.
• Why are stained specimens used.

  Increase contrast.
• What is the disadvantage of staining a specimen.

  Cells must be fixed, meaning they are preserved so cannot be living.
• How does phase contrast microscopy work

  Enhances contrast in unstained cells by amplifying differences in density within the specimen.
• What is the main difference between phase and differential contrast

  Differential contrast uses optical modifications.
• What is the general term used for contrast microscopy

  Advanced light microscopy
• Briefly describe advanced light microscopy

  Allows for observation of transparent specimens. Light phase shifts are caused by the specimen and cause contrast.
• Describe how fluorescence microscopy works

  Shows the locations of specific molecules within a cell. Molecules are tagged with flourcescent substances. These abosrb short-wavelengths and emit long-wavelengths [visible light].
• What is the removal of out-of-focus images called

  Optical sectioning
• Describe how a confocal microscope works

  Uses lasers for optical sectioning. Only regions within a narrow depth of focus are imaged. Regions outside the plane appear black.
• Describe deconvolution microscopy

  Algorithms remove out of focus light to improve resolution
• What are the two types of microscopy that break the resolution limit of a light microscope

  Deconvolution and super-resolution microscopy.
• What does it mean to break the resolution limit of a light microscope.

  Additional modificaitons that help to increase the resolution of a microscope.