Microscopy, Histometry Flashcards
1
Q
Refraction
A
- Light being deflected in passing obliquely through the interface between one medium and another or through a medium of varying density
2
Q
Refractive index (RI)
A
- ratio of sine values of angle of incidence/angle of
refraction - sin i / sin r = refractive index (n)
3
Q
Wavelength
A
- detected by the human eye as the colour of light
4
Q
Amplitude
A
- detected by the human eye as the intensity or brightness of the light
5
Q
Frequency
A
- the number of vibrations of the beam per second
6
Q
Simple microscopes
A
- e.g. reading lenses, watchmaker’s eye loupes (no more than 20x)
7
Q
Compound microscopes
A
- combination of objective lens and eyepiece (20x-1000x/2000x)
8
Q
Where is there no point of magnifying much beyond 2000x?
A
- there are other limits on the
microscope image that make very high magnifications useless (e.g. the resolution)
9
Q
Resolution
A
- ability to see two structures that are very close together as separate structures
10
Q
Numerical aperture (NA)
A
- 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
11
Q
Brightfield microscopy
A
- 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.
12
Q
Darkfield microscopy
A
- 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).
13
Q
Polarising microscopy
A
- 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)
14
Q
Polarising light
A
- Crystal of calcite (Nicol prisms) – older microscopes
- Polarising filters with precisely aligned crystals – more commonly nowadays
15
Q
Fluorescent microscopy
A
- 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
16
Q
Phase contrast microscopy
A
- 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
17
Q
Production of contrast in phase contrast microscopy
A
- deviated and undeviated rays cancel each other out
- image of specimen is dark against brighter background
18
Q
Interference microscopy
A
- 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)
19
Q
Wollaston prism (beam splitter) in interference microscopy
A
- 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
20
Q
Transmission electron microscopy
A
- 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
21
Q
Histometry
A
- 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)
22
Q
Application of histometry
A
- 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
23
Q
Muller-Lyer illusion
A
- Both horizontal lines are equal in length but one line appears longer than the other line due to presence of angled fins.
24
Q
Delbeouf illusion
A
- 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.
25
Titchener illusion
- inner circles are equal but the one surrounded with smaller circles seems larger than the one surrounded by larger circles
26
Poggendorff illusion
- Line A appears to connect with line a and line B with line b
- However, the only straight connection is the line B-a
27
Kanitsza figure
- 3 dark circles with missing segments give a strong impression of a triangle
28
Measurement in histometry
- 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)
29
Use of Eyepiece Micrometer and Stage Micrometer
- 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
30
Examination of 2D structure
- microstructure is measured by sampling it w/stereological probes
- most common stereological probes are points, lines, surfaces and volumes
31
Examination of 3D structure
- 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
32
Volume measurement
- 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
33
Theorem of Delesse
- Area proportion = volume proportion
