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• WavelengthWavelength

– shorter wavelength greater resolution

(450-500nm)

• Numerical apertureNumerical aperture

0.61 0.61 λλ

n sinn sinθθ

• 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 MicroscopyPhase Contrast Microscopy

• In phase contrast a phase plate is placed in In phase contrast a phase plate is placed in the light path. the light path.

• In a phase-contrast microscope, the annular In a phase-contrast microscope, the annular rings in the objective lens and the condenser rings in the objective lens and the condenser separate the light.separate the light.

• Barely refracted light passes through the Barely refracted light passes through the center of the plate and is not retarded.center of the plate and is not retarded.

Phase Contrast MicroscopyPhase Contrast Microscopy

• Highly refracted light passes through the Highly refracted light passes through the plate farther from center. plate farther from center.

• The interference produced by these two The interference produced by these two paths produces images in which the dense paths produces images in which the dense structures appear darker than the structures appear darker than the background. background.

The Phase-Contrast Microscope

Phase Contrast MicroscopyPhase 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 Differential Interference Contrast MicroscopyMicroscopy

Differential Interference Contrast Differential Interference Contrast MicroscopyMicroscopy

• DIC works by separating a polarised light DIC works by separating a polarised light source into two beams which take slightly source into two beams which take slightly different paths through the sample. different paths through the sample.

• The beams interfere when they are The beams interfere when they are recombined. recombined.

Differential Interference Contrast Differential Interference Contrast MicroscopyMicroscopy

• This gives the appearance of a three-This gives the appearance of a three-dimensional physical relief corresponding dimensional physical relief corresponding to the variation of optical density of the to the variation of optical density of the sample, emphasizing lines and edges sample, emphasizing lines and edges though not providing a topographically though not providing a topographically accurate image.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 MicroscopyDark Phase Microscopy

Dark Phase MicroscopyDark Phase Microscopy

• Opaque disc is placed underneath the condenser lensOpaque disc is placed underneath the condenser lens

• Only light that is scattered by objects on the slide can Only light that is scattered by objects on the slide can reach the eye.reach the eye.

• Light is reflected by specimen on the slide.Light is reflected by specimen on the slide.

• Bright white against a dark background. Bright white against a dark background.

• Pigmented objects-false colorsPigmented objects-false colors

Electron MicroscopyElectron Microscopy

• Use of electrons (short wavelength)Use of electrons (short wavelength)• 100,000 time smaller wavelenght than light100,000 time smaller wavelenght than light

• 0.0037nm (light 400-700nm)0.0037nm (light 400-700nm)

• 1000 time better resolution1000 time better resolution

• Magnetic field direct the path of electronMagnetic field direct the path of electron• Increase in electron velocityIncrease in electron velocity

• Decrease in wavelengthDecrease in wavelength

• Resolution 0.1nm (100x more than light Resolution 0.1nm (100x more than light microscope)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 microscopeTransmission 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