Microscopy, Histometry

Question 1

Refraction

Answer 1

• Light being deflected in passing obliquely through the interface between one medium and another or through a medium of varying density

Question 2

Refractive index (RI)

Answer 2

• ratio of sine values of angle of incidence/angle of
  refraction
• sin i / sin r = refractive index (n)

Question 3

Wavelength

Answer 3

• detected by the human eye as the colour of light

Question 4

Amplitude

Answer 4

• detected by the human eye as the intensity or brightness of the light

Question 5

Frequency

Answer 5

• the number of vibrations of the beam per second

Question 6

Simple microscopes

Answer 6

• e.g. reading lenses, watchmaker’s eye loupes (no more than 20x)

Question 7

Compound microscopes

Answer 7

• combination of objective lens and eyepiece (20x-1000x/2000x)

Question 8

Where is there no point of magnifying much beyond 2000x?

Answer 8

• there are other limits on the

microscope image that make very high magnifications useless (e.g. the resolution)

Question 9

Resolution

Answer 9

• ability to see two structures that are very close together as separate structures

Question 10

Numerical aperture (NA)

Answer 10

• measure of the resolving power of a lens (always engraved on barrel of a lens)
• Resolution = (0.61 x wavelength)/NA
• NA dry lens: <0.9
• NA oil-immersion lens <1.4 for high power lens

Question 11

Brightfield microscopy

Answer 11

• image is seen against a bright background.
• Objects are usually stained and therefore they absorb some wavelengths of light
• Many of the other special forms of microscopy make use of our ability to pick out
  very small amounts of light against a dark background.

Question 12

Darkfield microscopy

Answer 12

• uses darkfield illumination.
• ideal for viewing unstained objects, transparent and absorb little or no light
• No direct light enters the objective due to use of an annulus (or patch stop) in substage condenser or a special background illuminator.
• Only if something in the specimen bends the light will any light enter the objective and be seen (diffracting objects).

Question 13

Polarising microscopy

Answer 13

• Birefringent materials
• use of polarised light (light vibrating in a single plane).
• White light - polarising filter - specimen - analyser filter
• Crossed polarising filters – no light seen (back background)
• No specimen (aligned - bright)
• Specimen with birefringent property (crossed - bright on dark background)

Question 14

Polarising light

Answer 14

• Crystal of calcite (Nicol prisms) – older microscopes

- Polarising filters with precisely aligned crystals – more commonly nowadays

Question 15

Fluorescent microscopy

Answer 15

• Strong U.V. lamp (U.V + visible light)
• Collector
• Exciter filter (U.V only)
• Deflecting mirror
• Condenser
• Specimen (U.V + visible light)
• Objective
• Barrier filter (visible light only)
• Ocular
• Eye

Question 16

Phase contrast microscopy

Answer 16

• Related to the darkground microscopy
• Annulus in substage condenser is arranged to let
  light enter the objective
• Light is focused on a phase plate in the objective
• illuminated light: passes straight through specimen
• refracted light: diffracted or refracted through specimen

Question 17

Production of contrast in phase contrast microscopy

Answer 17

• deviated and undeviated rays cancel each other out

- image of specimen is dark against brighter background

Question 18

Interference microscopy

Answer 18

• Interference can be used as a method of converting changes in the phase of light, which are invisible to the human eye, into intensity
  changes that are visible.
• Constructive interference (increased amplitude or brightness)
• Destructive interference (lower amplitude or dimmer)

Question 19

Wollaston prism (beam splitter) in interference microscopy

Answer 19

• Polarised light is directed through two-layered modified beam splitter, splits the beam into two beams
• path of one beam is directed through the specimen and the other isn’t - upper Wollaston prism (beam combiner) combines the 2 beams
• Different parts have different refractive indices, causes amplitude variations that are visualized as differences in brightness

Question 20

Transmission electron microscopy

Answer 20

• Use electrons (higher resolution, up till about 10 nm)
• Magnetic lenses not glass
• magnification is altered by changing electrical current
• Vacuum microscope tube (air absorbs and deflects electrons)
• Special air locks
• Specimens must be very thin and dry
• Electron beam will damage the specimen overtime
• Specimen mounted on copper grid to disperse electric charge
• phosphorescent screen, makes electrons visible in screen
• Images with shades of grey
• Heavy metals to enhance contrast between structures

Question 21

Histometry

Answer 21

• Measurement of histological images
• Simple measurement techniques include methods of determining size (length, area and volume), number and optical density of
  microscopic features.
• Mostly research (as compared to diagnostic)
• Used for statistical analysis
• Allows 3D reconstitution from 2D images of sections
• Allows for standardisation (e.g. between different centres)

Question 22

Application of histometry

Answer 22

• Improved assessment of certain histological changes
• dependent on recognition of shapes – direct visual
  appraisal covers this
• Sometimes the eye cannot discriminate consistently on a quantitative basis of cellularity – needs histometry
• staining intensity is difficult to assess by eye - needs
  computer-controlled image analysis

Question 23

Muller-Lyer illusion

Answer 23

• Both horizontal lines are equal in length but one line appears longer than the other line due to presence of angled fins.

Question 24

Delbeouf illusion

Answer 24

• The inner circle on one appears to be larger than the outer circle on the other due to the effect of outer circles present
• An outer circle apparently enlarges the inner enclosed circle and an inner circle shrinks the other circle.

Question 25

Titchener illusion

Answer 25

• inner circles are equal but the one surrounded with smaller circles seems larger than the one surrounded by larger circles

Question 26

Poggendorff illusion

Answer 26

• Line A appears to connect with line a and line B with line b
• However, the only straight connection is the line B-a

Question 27

Kanitsza figure

Answer 27

• 3 dark circles with missing segments give a strong impression of a triangle

Question 28

Measurement in histometry

Answer 28

• size: area, length, volume
• estimating size by comparison with a known object
• Using a graticule placed in the eyepiece (calibrated with stage micrometer)
• Taking a photograph and measuring the photograph
• Digital image analysis
• Number
• Density (colour intensity, grey value)

Question 29

Use of Eyepiece Micrometer and Stage Micrometer

Answer 29

• Eyepiece micrometer (graticule) is calibrated by
  comparison with a standard stage micrometer (a slide
  engraved with a 1 or 2 mm etching (sub divided)). This slide is the fixed standard
• Eyepiece micrometer can now be used as a simple ruler

Question 30

Examination of 2D structure

Answer 30

• microstructure is measured by sampling it w/stereological probes
• most common stereological probes are points, lines, surfaces and volumes

Question 31

Examination of 3D structure

Answer 31

• tissue architecture disturbance is ideally suited to Stereology
• Parameters looked at are: surface area per volume, volume density of specialised cytoplasm, number of cells per volume
• Not possible without measurement
• Many 3D parameters are relevant to tissue functions

Question 32

Volume measurement

Answer 32

• Provided the following conditions are met:
• objects are homogeneous (evenly arranged) in tissues (=isotropic)
• the sections are thin compared with the objects
• More than one level (i.e. section) is measured

Question 33

Theorem of Delesse

Answer 33

• Area proportion = volume proportion