Lecture 12 Light And Phase Microscopy

Question 1

3 basic light microscope types

Answer 1

Bright field - standard use, best for stained samples

Phase contrast - light interference shows contrast, useful for live study

Fluorescence - fluorescent tags used to observe specific aspects

Question 2

Lenses work by refraction

Answer 2

Light moves slower in dense transparent material, if it hits the interface between two materials at an angle it will be bent ( refracted)

Amount of light depends on angle of incidence

Question 3

Convex lens

Answer 3

Focal length depends on curvature of the lens. Long focal length lenses are less magnifying than short focal length lenses

Long focal length lens less curved - imagine thin slice/scoop from surface of a large sphere

Short focal length lens highly curved - closer to a small sphere itself

Question 4

Concave lens

Answer 4

Diverges parallel beams, focal point is the point they converge from.
Trace rays back behind lens to find focal point

Question 5

How does a lens magnify?

Answer 5

Magnifying glass - single convex lens produces an enlarged “virtual image” (image seen through the lens) if object is closer than focal point

More highly curved the lens (closer to a sphere) the shorter the focal length and higher the magnification.

If object is further away than the focal length then a “real image” is formed

Question 6

A microscope has 2 lenses

Answer 6

1) an objective lens to produce a magnified real image - near sample, short and fat with short focal length ( high power)

2) an eye piece lens to produce a magnified virtual image of the real image - this is what you see with your eye - secondary magnification, a convex lens of longer focal length (lower power)

Question 7

Bright field microscope

Answer 7

Light is passed through the specimen

Scattering of light/interference produced image

Image is magnified and focused on retina or detector (e.g. camera)

Used in histology for cross sectional tissue structure study at cellular level

Question 8

Resolution

Answer 8

Diffraction limit - limits how much detail is visible

Resolving power of optical microscope limited by:

Wavelength of visible light

Refraction index of material between lens and sample (air/oil/water)

Diameter of objective lens

Question 9

Resolution calculation

Answer 9

D= 0.61 gamma/N sin alpha

D= Resolution in nm
Gamma = wavelength of light in nm
N= refractive index (of medium lens & cover slip)
Alpha= half the angle of light collection

Question 10

Phase contrast microscope

Answer 10

Can amplify refractive index difference in cell components improving contrast - excellent for live cells

Process:

1) annular diaphragm creates cones of light
2) this illuminates the specimen
3) undiffracted light passes through the periphery of the lens, diffracted light is bent into the centre
4) peripheral light passes through thinned annules that retard light 1/4 wavelength less than the thicker central region - phase is shifted relative to the diffracted light
5) interference creates contrast

Question 11

Fluorescent microscopy

Answer 11

Different parts of the cell can be stained differently or antibodies attached to fluorescent molecules can be used to bind to specific proteins and locate them.