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Imaging Services

 

 Phase contrast microscopy

Advantages of phase contrast microscopy:
  • It is possible to visualize certain cell organelles and structures that are invisible with bright-field
  • Suitable for living cells (long time lapses can be acquired)
  • High-contrast, high-resolution images
  • Good for studying and interpreting thin specimens
  • Can be used in conjunction with fluorescence microscopy
Disadvantages of phase contrast microscopy:
  • Not good for thick specimens (can appear distorted)
  • Shade-off (a steady reduction of contrast moving from the center of the larger objects toward its edges)
  • Halo effect (surrounding by bright areas, which obscure details along the perimeter of the specimen)
  • Modern techniques provide solutions (apodized phase contrast minimizes halo effect) but these limits can never be eliminated completely

Differential Interference Contrast Microscopy (DIC) 

Advantages of Differential Interference Microscopy (DIC):    
  • It is possible to visualize certain cell organelles and structures that are invisible with bright-field (transparent objects to be seen by using the difference in light’s refraction)
  • The specimen will appear bright in contrast to the dark background
  • Able to use a full width condenser aperture setting resulting in a brighter image
  • No halo effect occurs with differential interference contrast
  • Gives a greater depth of focus - can produce very clear images of thick specimens
  • Can be used in conjunction with fluorescence microscopy
  • Suitable for living cells (long time lapses can be acquired
Disadvantages of differential interference microscopy (DIC): 
  • The three-dimensional image of a specimen may not be accurate.
  • The enhanced areas of light and shadow might add distortion to the appearance of the image

Widefield Fluorescence Microscopy 

Advantages of fluorescence microscopy:
  • Allows labelling of organelles, molecules and other features of interest
  • Allows tracking the dynamics of processes involving labeled features in real-time and in vivo
  • The technique is highly sensitive: can detect a few molecules per cubic micrometer
  • Location of structures too small to be visible in a light microscope
  • Possibility to use different colors to track distinct molecules
  • Multicolor fluorescence microscopy allows to address possible interactions between molecules by observing colocalization
  • Quantitative imaging
Disadvantages of fluorescence microscopy: 
  • Photobleaching - dyes become nonfluorescent due to its molecular structure being altered as a result of exposure to excitation light)
  • Phototoxicity - cells become damaged due to interaction between fluorescent dye and excitation light
  • Inability to show morphology of surrounding structures.
  • Chromatic and spherical aberration
  • The availability of target specific antibodies
  • Limited specificity of the antibody
  • Limited ability of the antibody to diffuse to the target

 Confocal microscopy

Advantages of confocal microscopy:
  • Better vertical resolution
  • The ability to serially produce thin optical sections through fluorescent specimens that have a thickness ranging up to 50 micrometers or more
  • Better horizontal resolution
  • Image information is restricted to a well-defined plane, rather than being complicated by signals arising from remote locations in the specimen
  • More efficient use of light (requires less intense light, minimize photodamage)
  • Reduction in background fluorescence
  • Improved signal-to-noise
  • Optical sectioning of both living and fixed specimens
  • The ability to adjust magnification electronically by varying the area scanned by the laser without having to change objectives
  • Improved quantitative imaging
Disadvantages of confocal microscopy: 
  • The limited number of excitation wavelengths available with common lasers
  • Harmful nature of high-intensity laser irradiation to living cells and tissues
  • High quality images may require significant acquisition times

 Bright-field microscopy

Advantages of bright-field microscopy:
  • The optics do not change the color of the observed structures.
  • Stains are used to make certain structures visible.
  • Bright-field microscopy requires fewer adjustments before one can observe the specimens.
  • Can be used to view fixed specimens or live cells.
  • Frees fluorescent channels in microscopes
  • Eliminates distortions caused by the overlapping of the color emissions of the stains and the excitation of the fluorescing materials.
  • There are relatively cheap, fast and simple staining protocols to visualize:
  •      The nuclei and cytoplasm (Haematoxylin and Eosin Staining, Methylene Blue       Neutral/Toluylene Red, Nile Blue)
  •      Types of cells (Papanicolaou staining)
  •      Cell walls (Crystal Violet with Mordant)
  •      Bacteria (Giemsa stain, Gimenez stain)
  •      Spores (Malachite Green)
  •      Intracellular lipid globules (Nile Red)
  •      Lipids (Osmium Tetroxide)
  •      Collagen (Fuchsin, Safranin)
  •      Starch (Iodine)
  •      Mitochondria (Fuchsin)
  •      Proteins (Coomassie Blue)
  •      Glycogen (Carmine)
  •      Mucins (Bismarck Brown)
Disadvantages of bright-field microscopy:
  • low contrast
  • most cells must be stained to be seen
  • staining may introduce extraneous details
  • intense light used for high magnification applications can damage specimens or kill living cells

Time lapse imaging

 Time-lapse imaging (serial images taken at regular time points to capture the dynamics process) can be performed using phase contrast, DIC, fluorescence, and confocal microcopy modes.