Chapter 1 - The Compound Light Microscope

Question 1

What are the main two factors that influence resolution?

Answer 1

1. Wavelength

2. Numerical Aperture (NA)

Question 2

What is resolution (a.k.a. resolving power)?

Answer 2

The ability of an optical system to render two or more closely spaced object points as distinct images.

Question 3

How does wavelength influence resolution?

Answer 3

It affects it in two ways:

a. Resolution is improved if shorter wavelength light is used. e.g. using blue light instead of red light.
b. Intake angle of objective lens - a large intake angle offers better resolution than one with a small intake angle (good diagram on page 22 of book)

Question 4

State Abbe’s equation. State what each part stands for.

Answer 4

d min = λ/2NA

d min = the minimum distance between two objects in which they can both still be differentiated as two separate points.
λ = the wavelength of the light used (average is ~500nm for white light)
NA = n sin α where NA = numerical aperture, n = refractive index of the medium between the slide & the objective lens (usually air or oil), α = half the intake angle of the objective lens being used.

Question 5

State one way in which NA can be increased.

Answer 5

By using emersion oil in between the slide and the objective lens instead of it just being air. Air has a refractive index of 1 however oil has one of 1.5.

Question 6

A substance with a low refractive index (n) will bend light ___ than one with a higher n.

Answer 6

Less

Question 7

The light microscope anatomy can be subcategorised into three sections. What are they and what parts of the microscope are found in each?

Answer 7

1. Illumination system: coarse and fine focus knobs, field iris, light source, transformer, filter holder, collector lens, condenser lens and condenser iris
2. Specimen: stage, specimen
3. Image forming system: oculars, objective lens, ocular focusing and adjusting knobs

Question 8

What are the two types of light sources you can use and which is better? (____ illumination)

Answer 8

1. Critical illumination - simpler, potential for the light to look uneven
2. Köhler illumination - requires extra piece of equipment (collector lens), more expensive, evens out irregularities though
   Therefore they are both as good as each other!

Question 9

What is the difference between the Condenser Iris and Field Iris?

Answer 9

FI = controls area of illumination. If open too much it creates glare (decreased contrast and image quality). If it is not open enough it creates a shadow.
CI = controls angle/aperture of illumination. Firstly it focuses the light onto the specimen and secondly it matched the NA of the condenser to the NA of the objective lens being used.
Both need to be set correctly to prevent glare or shade or loss of resolution.
Good diagram on page 27 of notes on how to properly set the CI.

Question 10

How does altering the Condenser Iris affect: resolution, intensity, contrast and depth of field?

Answer 10

R: it improves until condenser iris is opened enough to fill the objective lens with light (when it is perfectly matched)
I: decreases with smaller NA as the opening is smaller in the condenser iris allowing fewer light rays through
C: increases with smaller NA. The diffraction effects are more pronounced with smaller NA and diffraction halos around each image point become more obvious.
D of F: increases as NA decreases (as you close the condenser iris more). Good diagram on page 29.