Microscopy Flashcards
Describe, in principle, what a microscope does. (F)
An instrument which enables you to magnify an object
Name 4 different types of microscope. (F)
- Light Microscope
- Transmitting Electron Microscope (TEM)
- Scanning Electron Microscope (SEM)
- Laser Scanning Confocal Microscope (LSCM)
State what “SEM” and “TEM” are abbreviations for. (F)
- Scanning Electron Microscope
- Transmitting Electron Microscope
Outline how an SEM works.
- beam of electrons sent across surface of specimen
- reflected electrons are collected
Outline how a TEM works.
- beam of electrons transmitted through a specimen
- focused to produce an image
Outline how a laser scanning confocal microscope works.
- single spot of focused light (laser) moved across a specimen (point illumination)
- causes fluorescence from components labelled with “dye”
- emitted light from specimen filtered through pinhole aperture
Describe the use and properties of light microscopy. (F)
Use:
- easily available
- can be used out in field
Properties:
- relatively cheap
- can view living/dead specimens
Describe the use and properties of SEM. (F)
Use:
- can produce images with high magnifications and clear resolution
Properties:
- dead specimens (vacuum)
- can see more detail of cell ultrastructure
Describe the use and properties of TEM. (F)
Use:
- can produce images with high magnifications and clear resolution
Properties:
- dead specimens (vacuum)
- can see more detail of cell ultrastructure
Describe the use and properties of LSCM. (F)
Use:
- diagnosis of diseases of the eye and endoscopic procedures
- used for drug development
- virtual biopsies
Properties:
- living/dead specimen
- can see distribution of molecules within cells
- non-invasive
State the features of the images produced from light microscopes.
- true colour
- living/dead specimen
- halide stains (?)
- 2D image
State the features of the images produced from SEM.
- false colour
- dead specimen (vacuum)
- heavy metal stains
- high magnification and resolution
- 3D image
State the features of the images produced from TEM.
- false colour
- dead specimen (vacuum)
- heavy metal stains
- high magnification and resolution
- 2D image
State the features of the images produced from LSCM.
- true colour
- living/dead specimen
- fluorescent dye
- 2D/3D (can scan in layers)
Explain how to use a light microscope to view a specimen at low and high powers.
- change the objective lens to a different power
- adjust resolution with coarse and fine focusing knob
Describe how to produce a temporary wet mount of living tissue.
- suspend specimen in liquid (e.g. water or an immersion oil)
- place a cover slip on from an angle
Describe and explain the characteristics of a good slide preparation.
- suitable stain so can differentiate between different parts of the sample
- no air bubbles
- evenly and well stained
- thin specimen
- no artefacts
Explain why slide preparations need to be thin. (F)
- so light can shine through
- details can be seen
Explain how to use a stage micrometer to work out the distance represented by the small divisions in an eyepiece graticule under 3 different objective lenses. (F)
1 graticule division = number of micrometres/number of graticule divisions
Explain how to use a stage micrometer and eye-piece graticule to add a scale bar to a drawing.
- measure feature of speciment with eyepiece graticule
- convert to micrometres using calibration done with stage micrometer
- draw scale bar on drawing, matching the length of the feature you measured and label with micrometres
Explain how to use a stage micrometer and eye-piece graticule to calculate the size of a specimen.
graticule divisions x magnification factor = measurement
Describe how to choose an appropriate number of significant figures, or decimal places to present data.
Use the same decimal places/significant figures as all of the other data is given in
Explain how an adjustment to the “plane of focus” can alter what is viewed within a cell. (F)
Because a cell is 3D, the shapes can look different from different angles i.e. the mitochondria is sausage shaped, but can appear spherical if viewed from the end.
Explain how a tissue slice might be misleading due to the very thin nature of the slice.
Can be distorted.
Explain why staining is useful for light microscopy. (F)
Provides contrast between different parts of the sample, so can be more easily identified.
Describe the properties a stain needs to have to be useful for light microscopy. (F)
- binds to the sample
- coloured
Describe how to prepare a stained specimen for viewing under a light microscope.
- place on a slide to air dry
- heat-fix by passing through a flame
- will adhere to slide and take up stain
State the rules for biological drawings. (F)
- title
- magnification stated
- sharp pencil
- white, unlined paper
- use as much of the paper as possible
- smooth, continuous lines
- do not shade
- clearly defined structures
- correct proportions
- no arrow heads on label lines
- label lines parallel to top of page and drawn with a ruler
State the magnification formula and represent the magnification formula in a triangle diagram. (F)
magnification = size of image/actual size of object
Explain how to use the magnification formula. (F)
- rearrange so what you want to find is the subject of the equation
- substitute values in and solve
State the symbols used for millimetres, micrometres and nanometres. (F)
millimetres: mm
micrometres: μm
nanometres: nm
Explain how to convert measurements from one unit into another.
- find the difference between the units
- if converting to a bigger unit, divide the measurement by the difference
- if converting to a smaller unit, multiply the measurement by the difference
Explain how to represent numbers in standard form.
- number before the decimal place can be from 1 to 9
- count how many places decimal place moved
- if moved left, power of 10 is +ve
- if moved right, power of 10 is -ve
Define the terms “resolution”. (F)
The ability to see individual objects as separate entities.
State the difference between magnification and resolution.
Magnification is how much the image has been enlarged, whereas resolution is the ability to see detail.
State the resolution and useful maximum magnification of light microscopes.
- 200nm
- x 2000
State the resolution and useful maximum magnification of SEMs.
- 3-10nm
- x 100 000
State the resolution and useful maximum magnification of TEMs.
- 0.5nm
- x 500 000
Define the term “magnification” (F)
How many times larger the image is than the actual size of the object being viewed.
