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Introduction to the light microscope

front 1

Light microscopy

back 1

Light microscopy refers to the use of any kind of microscope that use visible light to observe specimens

The light microscope, so called because it employs visible light to detect small objects, is probably the most well-known and well-used research tool in biology.

front 2

Types of light microscopy

back 2

–Bright-field

–Dark-field

–Fluorescence

–Phase contrast

front 3

Microscope parts and care

back 3

Always carry with 2 hands, one on the arm and one on the base

Only use lens paper for cleaning the lens

Keep liquids away!

front 4

Ocular lens (eyepiece)

back 4

Remagnifies the image formed by the objective lens

front 5

Body tube

back 5

Transmits the image from the objective lens to the ocular lens

front 6

Objective lenses

back 6

Lens that is closest to the slide and provide initial magnification on a specimen (Primary lenses that magnify the specimen)

front 7

Mechanical Stage

back 7

Holds the microscope slide in position

front 8

Condenser

back 8

Focuses light through specimen

front 9

Iris Diaphragm

back 9

Controls the amount of light entering the condenser and the light that reach specimen

front 10

Illuminator

back 10

Light source

front 11

Fine focusing knob

back 11

Used after initial focusing, to sharpen the image

front 12

Coarse focusing knob

back 12

Used or initial focusing, should never be used when the high power or oil immersion lens

front 13

Total Magnification

back 13

Magnification by the objective lens X (multiply) Magnification by the Ocular lens

front 14

Objective Scanning

4 X

back 14

locate specimen

front 15

Objective Low Power

10 X

back 15

view the whole or large portions of specimen

front 16

Objective High Power

40 X

back 16

see small, detailed parts of specimen

front 17

Objective Oil Immersion

100 X

back 17

see very small specimens (e.g., bacteria)

is used to keep light from bending

front 18

Bright-field Microscopy

back 18

  • It is is the simplest of all the optical microscopy illumination techniques.
  • It produces an image that it is transmitted through a specimen. The specimen restricts light transmission and appears “shadowy” against a bright background.
  • Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light and contrast in the sample is caused by absorbance of some of the transmitted light in dense areas of the sample.
  • Most of the biological specimens are colorless or transparent, therefore staining of the sample is used in order to improved the contrast between the specimen and the background.

front 19

HOW do the image is formed?

back 19

  • The light comes from the bottom in this case
  • It passes through the condenser lens (it concentrates the light (making illumination of the specimen more uniform)
  • The light is refracted (bend) as it passes through the objective lens producing a magnifying real image
  • The image is magnified again at it passes through the ocular lens to produce a virtual image that appears below or within the microscope.

front 20

Resolution

back 20

Magnification is due to the RESOLUTION of light as it passes through the lens

the clarity of an image

front 21

Bright-field Microscopy
Resolution and Refractive Index

back 21

  • Resolution is the ability of the lenses to distinguish two points
  • A microscope with a resolving power of 0.4 nm
    can distinguish between two points ≥ 0.4 nm
  • Shorter wavelengths of light provide greater resolution
  • The refractive index is a measure of the
    light-bending ability of a medium
  • The light may bend in air so much that it misses
    the small high-magnification lens
  • Immersion oil is used to keep light from bending

front 22

Bright-field Microscopy
Oil immersion

back 22

  • Light bends when it passes from glass to air or from air to glass because air and glass have different refractive indices. The bending of light as it passes through the glass slide to the air and then to the glass lens decreases the resolving power. At high magnification (100X) it can prevent a clear image from being viewed. This decrease in resolution can be prevented by putting immersion oilbetween the slide and the lens because immersion has the same refractive index as glass

front 23

Light of shorter wavelength produce a clearer image than light of longer wavelength

back 23

True

front 24

Best limit o esolution

back 24

The best limit of reso;ution (resolving power) for a light microscope is 0.2 Nm ( 200nm)

front 25

What position should the stage be when you store the microscope?

back 25

at the lowest position

front 26

If the resolving power of your microscopy is 250 nm, will you be able to distinguish two point that are 260 nm apart ? why?

back 26

Yes because the have a distance apart and it would possible to see it