Phase Contrast Microscopy

Expand allAdvantages 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) 

Expand allAdvantages of differential interference microscopy:    

• 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: 

• 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 

Expand allAdvantages 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

Expand allAdvantages 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

Expand allAdvantages 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.

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Image Analysis