W13: MICROSCOPY

Units of measurement:

• 1 μm micrometer = ____ m

10^-6

Units of measurement:

• 10-6 m = ____ mm

10^-3

Units of measurement:

• 1 nm nanometer = ____ m

10^-9

Units of measurement:

• 10-9 m = ____ mm

10^-6

Units of measurement:

• 1000 nm = ___ μm

1

Units of measurement:

• 0.001 μm = __ nm

1

Microscope with only 1 lens

simple microscope

In this microscope the image from the objective lens is magnified again by the ocular lens.

Compound microscope

Total magnification of compound microscope

objective lens x ovular lens

Identify the objective:

Low power objective

• Obj. Lens magnification: 10

• Ocular lens magnification: 10

• Total magnification: 100x

Identify the objective:

• High power objective

• Obj. Lens magnification: 40

• Ocular lens magnification: 10

• Total magnification: 400x

Identify the objective:

• Oil immersion objective

• Obj. Lens magnification: 100

• Ocular lens magnification: 10

• Total magnification: 1000x

The ability of the lenses to distinguish two points.

Resolution

A microscope with a resolving power of ______ can distinguish between two points.

0.4 nm

_______ wavelengths of light provide greater resolution.

Shorter

The light-bending ability of a medium.

Refractive index

Used to keep the light from bending.

Immersion oil

• Utilized transmitted light

• Dark objects are visible against a bright background.

• Light reflected off the specimen does not enter the objective lens

Brightfield Illumination

• Utilized reflected light with a special condenser

• Light objects are visible against a dark background.

• Light reflected off the specimen enters the objective lens. Figure 3.4a, b

Darkfield Illumination

• Accentuates diffraction of the light that passes through a specimen.

• Direct and reflected light rays are combined at eye. Increasing contrast

• Binary fission, motility and endospore.

Phase-Contrast Microscopy

• Greater resolution

• Light reaches specimens only from the side

• Only the specimen itself is illuminated

Advantages of Dark field microscopy

• May be used to visualize live samples and avoid distortion from cell stain

• Image contrast is derived from the differential refractive index of cell structures.

Advantages of Phase contrast microscopy

• Uses Dual beam optical system (light)

• Accentuates diffraction of the light that passes through a specimen

• uses two beams of light adding color to provide better contrast.

Differential Interference Contrast Microscopy

• Uses UV light

• Fluorescent substances absorb UV light and emit visible light.

• Cells may be stained with fluorescent dyes (fluorochromes).

• Detect antigen and antibodies

Fluorescence microscopy

Fluorescence microscopy was usually used on Visualization of ____________________
(e.g., chlorophyll) or ____________

1.) auto fluorescent cell structures
2.) fluorescent stains

• Uses fluorochromes and a laser light.

• The laser illuminates each plane in a specimen to produce a 3-D image.

Confocal microscopy

Confocal microscopy is used to examine ________________ cross section.

Vertical and horizontal

• Uses electrons instead of light.

Electron microscopy

In electron microscopy, the _________ wavelength of electrons gives greater resolution.

shorter

• Specific requirement which is the preparation of ultrathin sections of
  specimens.

• Light passes through specimen, then an electromagnetic lens, to a screen or film.

• Specimens may be stained with heavy metal salts.

Transmission electron microscopy

What type of specimen Transmission electron microscopy requires?

Ultrathin sections of specimens

What is the resolution of transmission electron microscopy?

2.5 nm

• No requirement of ultrathin preparation sample

• An electron gun produces a beam of electrons that scans the surface of a whole specimen.

• Secondary electrons emitted from the specimen produce the image.

Scanning electron microscopy

Who are responsible for the image produced in scanning electron microscopy?

secondary electrons

What is the scanning resolution of scanning electron microscopy?

20 nm

Give 1 example of bacteria you can scan in TEM

Pseudomonas

Give 1 example of bacteria you can scan in SEM

Mycobacterium

• uses a metal probe to scan a specimen.

Scanning-Probe microscopy

What is the resolution of scanning-probe microscopy?

1/100 of an atom

• uses metal and diamond probe inserted into the specimen.

• produces 3d image

Atomic force microscopy