Light microscopy uses visible light and magnification lenses to examine stained or unstained specimens. It has limitations in resolution but is simple to use. Electron microscopy uses electron beams instead of light for higher magnification and resolution down to individual atoms or molecules, but requires cutting or coating specimens. Probe microscopy magnifies over 100 million times using scanning tunneling or atomic force probes, achieving resolution of 1/100th of an atom.
Light microscopy uses visible light and magnification lenses to examine stained or unstained specimens. It has limitations in resolution but is simple to use. Electron microscopy uses electron beams instead of light for higher magnification and resolution down to individual atoms or molecules, but requires cutting or coating specimens. Probe microscopy magnifies over 100 million times using scanning tunneling or atomic force probes, achieving resolution of 1/100th of an atom.
Light microscopy uses visible light and magnification lenses to examine stained or unstained specimens. It has limitations in resolution but is simple to use. Electron microscopy uses electron beams instead of light for higher magnification and resolution down to individual atoms or molecules, but requires cutting or coating specimens. Probe microscopy magnifies over 100 million times using scanning tunneling or atomic force probes, achieving resolution of 1/100th of an atom.
a. Bright-field microscopes i. Shows colored or darkened organisms or specimens (achieved through staining) against a light background ii. Has several objective lenses (parfocal microscopes remain in focus when objectives are changed) iii. Light reflected off the specimen does not enter the objective lens iv. The simplest and most common v. Uses white light to illuminate specimen vi. Disadvantages: 1.
Limited resolving power
2. Not ideal for observing colourless organisms (unless
staining will be performed) vii. Two types 1. Simple a. Contain a single magnifying lens b. Similar to magnifying glass c. Leeuwenhoek used simple microscope to observe microorganisms 2. Compound a. Precision instrument composed of a series of lenses for magnification b. Uses light (natural & artificial) as source of illumination c. Light passes through specimen into objective lens d. Oil immersion lens increases resolution e. Have one or two ocular lenses
f. Total magnification = magnification of objective
lens X magnification of ocular lens g. Most have condenser lens (direct light through specimen) b. Dark-field microscopes i. Limits the entry of light creating a dark background against the illuminated specimen ii. Improves the contrast of unstained, transparent specimens iii. Excludes the unscattered beam from the image iv. Best for observing pale objects v. Only light rays scattered by specimen enter objective lens vi. Increases contrast and enables observation of more details vii. Disadvantage: Sample must be very strongly illuminated, which can cause damage to the sample c. Phase microscopes i. Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining ii. Light rays in phase produce brighter image, while light rays out of phase produce darker image iii. Contrast is created because light waves are out of phase iv. Two types 1. Phase-contrast microscope a. Employs special condensers that allow light to pass through the specimen at different speeds allowing for an effective contrast between the different structures in the organism b. Accentuates diffraction of the light that passes through a specimen c. Difference in densities and composition within the imaged objects however often give rise to changes in the phase of light passing through them, hence they are sometimes called "phase objects". d. Makes cell structures visible
e. Allows their study with the specimen still alive,
thus an excellent way to observe living cells f. Enhances the contrast between intracellular structures having slight differences in refractive index 2. Differential interference contrast microscope a. Accentuates diffraction of the light that passes through a specimen; uses two beams of light b. Creates image by detecting differences in refractive indices and thickness of different parts of specimen c. Excellent way to observe living cells d. Fluorescent microscopes i. Direct UV light source at specimen ii. Makes use of fluorescent dyes - May have affinity to different molecules Cellular Immunological Molecular iii. Makes use of high energy light (eg. UV) - Fluorochromes will emit lower frequency lights iv. Specimen radiates energy back as a longer, visible wavelength v. UV light increases resolution and contrast vi. Some cells are naturally fluorescent; others must be stained vii. Specimens usually stained with fluorochromes viii. Shows a bright image of the object resulting from the fluorescent light emitted by the specimen ix. Used in immunofluorescence to identify pathogens and to make visible a variety of proteins e. Confocal microscopes i. Use fluorescent dyes ii. Use UV lasers to illuminate fluorescent chemicals in a single plane
iii. Resolution increased because emitted light passes through
pinhole aperture iv. Computer constructs 3-D image from digitized images b.) Electron Microscopy a. Light microscopes cannot resolve structures closer than 200 nm b. Beams of electrons are used to produce images instead of light c. Wavelength of electron beam is much shorter than light, resulting in much higher resolution d. Electron microscopes have greater resolving power and magnification making very small structures and details visible e. Magnifies objects 10,000X to 100,000X f. Detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms g. Needs preparation of samples h. Two types i. Transmission electron microscopes 1. Internal structure Need slicing 2. Uses electrons instead of light 3. The shorter wavelength of electrons gives greater resolution 4. Ultrathin sections of specimens. 5. Beam passes through specimen, then an electromagnetic lens, to a screen or film. 6. Specimens may be stained with heavy metal salts. 7. 10,000-100,000x; resolution 2.5 nm ii. Scanning electron microscopes 1. Surface of the specimen 2. An electron gun produces a beam of electrons that scans the surface of a whole specimen 3. Secondary electrons emitted from the specimen produce the image 4. 1000-10,000x; resolution 20 nm
c.) Probe Microscopy
a. Magnifies more than 100,000,000 times b. Two types i. Scanning tunneling microscopes 1. Scanning tunneling microscopy uses a metal probe to scan a specimen 2. Resolution 1/100 of an atom ii. Atomic force microscopes 1. Atomic force microscopy uses a metal and diamond probe inserted into the specimen 2. Produces 3-D images 3. Metal coat not needed