Download The Bright-Field Microscope

Microscopy
Scale
Lenses and the Bending of Light
• light is refracted (bent) when passing from one
medium to another
• refractive index
– a measure of how greatly a substance slows the velocity
of light
• direction and magnitude of bending is determined
by the refractive indexes of the two media forming
the interface
Lenses
• focus light rays at a specific place called the
focal point
• distance between center of lens and focal
point is the focal length
• strength of lens related to focal length
– short focal length more magnification
The Light Microscope
• Types
–
–
–
–
Bright-field microscope
Dark-field microscope
Phase-contrast microscope
Fluorescence microscopes
• Modern
– Compound microscopes
– Image formed by action of 2 lenses
Image Quality
• Resolution
– Ability of a lens to separate or distinguish small objects that are
close together
– Factors
• Wavelength
– shorter wavelength  greater resolution
(450-500nm)
• Numerical aperture
0.61 λ
n sinθ
• Working distance
– distance between the front surface of lens and surface of cover
glass or specimen
• Brightness - How light or dark is the image?
• Focus - Is the image blurry or well-defined?
• Resolution - How close can two points in the image be before they are
no longer seen as two separate points?
• Contrast - What is the difference in lighting between adjacent areas of
the specimen?
The Bright-Field Microscope
• Dark image against a brighter background
• Stained Specimen
• Several objective lenses (3-5)
– Parfocal microscopes
• Total magnification
– Product of the magnifications of the ocular
lens and the objective lens
– 45x (objectice) X 10x (eye piece)= 450x
Figure 2.4
Phase Contrast Microscopy
• In phase contrast a phase plate is placed in
the light path.
• In a phase-contrast microscope, the annular
rings in the objective lens and the condenser
separate the light.
• Barely refracted light passes through the
center of the plate and is not retarded.
Phase Contrast Microscopy
• Highly refracted light passes through the
plate farther from center.
• The interference produced by these two
paths produces images in which the dense
structures appear darker than the
background.
The Phase-Contrast Microscope
Orlando Science Center
Phase Contrast Microscopy
The Differential Interference
Contrast Microscope
• creates image by detecting differences in
refractive indices and thickness of different
parts of specimen
Differential Interference Contrast
Microscopy
Differential Interference Contrast
Microscopy
• DIC works by separating a polarised light
source into two beams which take slightly
different paths through the sample.
• The beams interfere when they are
recombined.
Differential Interference Contrast
Microscopy
• This gives the appearance of a threedimensional physical relief corresponding
to the variation of optical density of the
sample, emphasizing lines and edges though
not providing a topographically accurate
image.
The Fluorescence Microscope
• exposes specimen to ultraviolet, violet, or blue light
• specimens usually stained with fluorochromes
• shows a bright image of the object resulting from the fluorescent light
emitted by the specimen
Dark Phase Microscopy
Dark Phase Microscopy
• Opaque disc is placed underneath the condenser lens
• Only light that is scattered by objects on the slide can reach
the eye.
• Light is reflected by specimen on the slide.
• Bright white against a dark background.
• Pigmented objects-false colors
Electron Microscopy
• Use of electrons (short wavelength)
• 100,000 time smaller wavelenght than light
• 0.0037nm (light 400-700nm)
• 1000 time better resolution
• Magnetic field direct the path of electron
• Increase in electron velocity
• Decrease in wavelength
• Resolution 0.1nm (100x more than light
microscope)
• denser regions in specimen, scatter more electrons
and appear darker
Types
• Transmission electron microscope
– internal structure
– electrons pass through thin specimens (50-1000
nm).
• scanning electron microscope
– 3d structure
– In scanning electron microscopy signals
emitted from the surface of thick specimens.
Transmission electron microscope
Electron micrograph of a cell in a root tip
Specimen preparation
• Vaccum to prevent colliding of electron
– dead specimen
– insufficient electron density
– electron dense salt (gold, uranium)
• Embedded in a polymer for thin sections
– microtome cut slices (micrometer thick)
• Sprayed onto copper grid
– viruses and macromolecules
• Flash frozen (for cryo EM)
• Artifact
Specimen preparation