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
- 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)
- 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
- 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
- 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
- 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
- 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
- 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
- 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)
- 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.