Microscopy Flashcards
Key terms: Magnification
Magnification is the number of times larger an image appears to be compared to the actual specimen which enables further detail to be seen. Magnification is limited by the resolution of the microscope.
Total magnification of the specimen
total magnification of the specimen=eye piece magnification (x10) x objective magnification (x4,x10,x40)
Key terms: Resolution
Resolution is the ability to distinguish between 2 separate points (determines the clarity of the image)
Why does an electron microscope have higher resolution?
EM have higher resolution as they use a beam of electrons which has shorter wavelength.
Why do we preserve specimens?
-enables them to be cut into sections to observe under a microscope
-enables them to be treated with a variety of different stains to distinguish between different types of cells, living and dead cells and different chemicals or metabolic processes.
-enables different structures to be revealed
Function of eye piece on light microscope
-magnifies specimen
-can be dismantled to insert an eye piece graticule to enable specimens to be measured accurately
Function of barrel on light microscope
-passes light from objective lens to eyepiece
Function of turret
-holds objective lens
-rotates to enable selection of objective lens
Function of objective lens
-magnifies and resolves specimen
Function of stage
-supports slide in correct position at 90 degrees to illuminate source
-enables light to pass through specimen
Function of condenser
-focuses light from illuminator source onto specimen
Function of iris diaphragm
-controls the level of light reaching specimen
-best definition achieved with lower light intensity
function of substage illumination
-source of illumination
-blue light bulb can be used to use light of shorter wavelength to improve resolution
How to prepare a temporary slide for observing using a light microscope?
-fixation: use 70% alcohol
-staining: use few drops of appropriate differential stain
-mounting: cover with cover slip to exclude dust and air
Advantages of preparing a temporary slide
-rapid and simple procedure (no complex apparatus or skill required)
-can mount specimen in glycerine to prolong examination period
How to prepare a permanent slide for observing under a light microscope? 1
1) Fixation
-preserves specimen in lifelike condition
-minimises distortion
-chemicals such as alcohol or acetic acid are added to make proteins and nucleic acids insoluble, fixing them in position
Preparation of permanent slide 2
2) Dehydration
-removes traces of water from fixed material
-achieved by placing the specimen in increasing concentration of alcohol
Preparation of permanent slide 3
3) Clearing
-addition of xylol removes dehydrating alcohol
-ensures material is made transparent
Preparation of a permanent slide 4
4) Embedding
-supports material so it is firm enough for sectioning
-can be resin, plastic or wax
Preparation of a permanent slide 5
5) Sectioning
-use a microtome to cut fine slices embedded specimen
so that light can pass through the specimen
Preparation of a permanent slide 6 7
6) Differential staining
-improves contrast between different tissues and structures
-can be permanent or temporary
7) Mounting
-embeds and protects material
ensures material can be observed over a long period of time
Iodine-KI solution
-for plant specimen
-temporary stain
-colour is blue-black
-used in starch
Aniline sulfate
-for plant specimen
-temporary stain
-yellow colour
-used in lignin
Toluidine
-plant specimen
permanent stain
red/purple
lignin and cellulose
Eosin
-for plant specimen
permanent stain
red colour
used in cellulose
Methylene blue
-animal specimen
-permanent stain
-blue colour
-used in Nuclei reticulocytes
Leishman’s stain
-animal specimen
permanent stain
red pink blue
red blood cells, nucleus of white blood cells
Haematoxylin
-animal specimen
permanent
blue
nuclei of animal cells
Advantages of using differential stains
-most biological specimens are colourless and almost transparent so differential stains make it easier to observe tissues/cells/chemicals
-when observing plant tissue= allows observer to distinguish chemicals enabling the identification of different tissue types such as xylem vessels from phloem
-when observing animal tissue, it allows observer to distinguish between different types of white blood
cell
-improves contrast between structures
Advantages of LM
-low skill set needed by user
-can be transported to use in field work
-can observe living organisms
-relatively inexpensive so available for schools and colleges
Disadvantages of LM
-low resolution
-limited magnification
many internal cellular structures can’t be seen such as ribosomes, cristae
Romanowsky Stains-Leishman’s stain
Allow blood smear to AIR dry
Fix with methanol
Flood the slide with Leishman’s stain
Leave for 2 mins
Dilute with distilled water
Leave for 7-10 minutes to AIR dry
Wash with DISTILLED water until the smear appears pale pink to the naked eye
Key points of cell theory
A cell is the basic unit of all life forms.
All living organisms are made up of one cell (unicelluar organisms) or more than one cell (multicelluar)
Metabolic processes occur inside living cells.
All new cells are derived from pre-existing cells.
Cells possess genetic material which can be passed on to their daughter cells.
A cell is the smallest unit of an organism capable of surviving independently.
What is the impact of microscopes on biology?
-enables scientists to see and examine cells in detail
-opened up new fields of science
Electron microscopes
Electron microscopes:
-use a beam of electrons which have shorter wavelength
-electron beam is focussed by electromagnetic condenser lens
-achieves greater resolution than LM which enables higher magnification which enables finer detail to be seen
-electrons focussed on fluorescent screen which emits visible light where electrons hit
-image converted to photomicrograph
-final images are always black, grey and white
Type of electron microscope: Transmission EM
TEM:
-produces images of cell ultrastructure and smaller organelles
-specimens must be very thin—>use ultra-microtome as electrons have to pass through the specimen
-specimens require complex staining process
-heavy metals used to stain specimen as atoms of heavy metals have large, positive nuclei that scatter electrons
-artefacts can occur due to the preparation techniques
-scattered electrons don’t hit the fluorescent screen which means there is a dark area on image
cell structures appear as dark images
2D and black and white images
higher magnification=x500,00higher resolution=0.1nm
enables identification of organelles and observation of internal structures
provides detail of surface composition
Scanning Electron Microscope
-produces image of cell surface and topography
-beam of electrons is passed backwards and forwards over specimen in regular pattern
-electrons reflected off the surface of the specimen
pattern of scattered electrons reflects the contours of the specimen
no internal structures observed-only external detail
information processed by computer to generate 3D image
max magnification-100,000
lower resolution than TEM but higher than LM at 10nm
Confocal Laser Scanning Microscope
-can produce focused images of thick specimens at various depths due to optical sectioning
-point source of light is detected on to the object plane
point of emitted fluorescence light or reflected light from the specimen is directed through the director pinhole
fluorescent light is enhanced using photomultiplier
fluorescent light displayed on computer screen as a pixel
specimen is scanned point by point and line by line-very thin, blur-free optical sections are recorded one pixel at a time
-series of images is combined to form an image stack
How are fluorescent markers used to detect biological chemicals/structures?
-fluorochromes are attached to antibody that is specific for one antigen on or in the cell
-fluorochromes can also be tagged to a chemical that binds specifically to a component of the csm, DNA or other structure.
each fluorochrome has its own peak excitation and emission.
lasers with different wavelengths are used depending on which fluorochromes are used.
laser excites the fluorochrome causing the tagged cells to fluoresce.
this fluorescence is counted by the detector.
this specific light scattering and fluorescent characteristics of each cell as they pass the laser beam is used for counting and sorting.
Images formed by CLSM
Images are high resolution as very thin sections are examined and the light from elsewhere is removed
2D images as the illuminating spot is moved across the surface of the specimen
can be 3D when images are created at different focal planes.
Advantages of Electron Microscope
-greater resolution
-higher magnification
-finer ultrastructure and detail seen e.g ribosomes
Disadvantages of EM
-specimen has to be placed in a vacuum therefore must be dead
highly expesnive so prohibits use in schools
high skill set required as training is needed before use
must be used in specialist room due to electromagnets within the EM
more complex process to prepare specimen e.g must be mounted on copper grid rather than glass slide
more prone to artefacts due to complex preparation procedure
Advantages of CLSM
-Ability to reduce or eliminate background information away from the focal plane to reduce to image degradation
capability to collect a series of optical sections from thick specimens as various depths by optical sectioning
high resolution images
3D reconstructions
living cells can be used
any out-of-focus light is blocked for a clearer image than standard LM
How to use a LM?
-Insert x10 eyepiece lens into eyepiece into top of barrel
-Place slide on stage and secure in position using stage clips
-Select lowest power objective lens on the turret.
-Whilst looking at the stage and slide from the slide of the microscope: use coarse adjustment knob to bring stage up to the highest setting beneath the slide
-Look down the eyepiece: using the fine adjustment knob move the stage slowly downwards until specimen is roughly in focus.
-Adjust very slowly with the fine adjustment knob until the specimen is in clear focus.
If a greater magnification is required, ensure that the specimen is in the centre of the field of view.
Select higher objective lens ( medium power x10)
Refocus the specimen by adjusting the fine adjustment knob very slowly until the specimen is once again in clear focus.
Repeat at higher power (x4) if required.
Erythrocytes: typical abundance, size and cytoplasm contents
-approx 5 million per mm cubed
6.2-8.2 micrometre diameter
2-2.5 micrometre thick
-contains approx 250 million molecules of haemoglobin
-lack nucleus
-lack most other organelles
-contain carbonic anhydrase
-biconcave shape-flexibility to squeeze through capillaries
-large SA:vol
Erythrocytes
-Function
-Details of production
-transport oxygen as oxyhaemoglobin from lungs to tissues
-transport carbon dioxide from tissues to lungs
-produced in bone marrow by erythropoiesis which is stimulated by the hormone erythropoietin (EPQ)
-Immature rbc=reticulocytes
-Last approx 100-200 days
