1.13 Light and Fluorescence Microscopy and 1.14 Electron Microscopy Flashcards
- Who created light microscopes?
Jansen, compound microscope (inverted telescopes essentially, multiple lenses), 1597
Malpighi, described red blood cell ‘corpuscles’/taste buds, 1660
Robert Hooke, named cells after observing little subunits in cork under a microscope, 1663
van Leeuenhoek, ‘father of microscopy’, improved lenses and improved microscopes, 1673
What is a cell?
A single compartment or unit, enclosed by a border, wall or membrane - the smallest metabolically functional unit of life. There are both plant and animal cells.
Define magnification.
The ratio between the apparent size of an object/is size in an image and its true size, so has no units/is dimensionless. Related to optics, used for scaling in microscopy.
Define resolution.
The minimum distance between distinguishable objects in an image. Loosely used in microscopes to describe their resolving power.
Are magnification and resolution the same?
No, resolving power depends on the wavelength of magnifying rays and magnifying beyond resolving power will make the object bigger but with no extra detail, essentially ‘magnifying the blur’.
What are metres used to measure?
Dimensions of individuals, using tape measures
What are millimetres used to measure?
Dimensions of organs and tumours, using rulers or hand lenses
What are micrometres used to measure?
Dimensions of organ layers, vessels, cell groups and organelles, using light microscopes or low magnification electron microscopy (EM)
What are nanometres used to measure?
Dimensions of membranes and molecular clusters, using higher resolution electron microscopy (EM)
What are the relative magnitudes of nano-, micro-, milli- and metres?
Metre: 1 Millimetre: 1x10^-3 Micrometre: 1x10^-6 Nanometre: 1x10^-9 Each related by x10^-3 to the next
What are used as standard indicators on microscopic slides and why?
Red blood cells - this is because they have a fixed size of 7 micrometres.
What do higher resolution microscopes reveal in cells?
Smaller internal structures; organelles, such as nuclei (and the constituents of), mitochondria, ribosomes, storage ‘granules’, and even smaller organisms such as bacteria.
What are the three sources from which a sample can be obtained?
- After death/post mortem
- At operation (biopsy = sample)
- Other, e.g. needle or scraping
- Give two examples of biopsy.
- Surgical - tissue is surgically removed, solid tissue samples.
- Needle aspiration - a thin, hollow needle is inserted into a region of abnormal cells or fluid, removing some cells for investigation like in other types of biopsy
Why can’t whole samples be studied?
Due to 3D structure, often will be too thick to study under a microscope (too much information). Instead, slices called sections should be made to reduce information, or the sample studied from the outside using scanning techniques.
What is the most common type of microscope sample?
A section
What are the two types of section?
Cross section (transverse section - TS) Parallel to the long axis of the structures within the sample (longitudinal section - LS)
What are the issues with preparing samples from human/animal tissue?
- Cell decay, this needs to be stopped immediately in order to preserve the sample.
- Loss of structural detail.
- Autolysis, aka breakdown of the cell by its own chemicals
How are samples prepared for light microscopy?
- Chemical cross-binding called FIXATION
- Placed at a low temperature (CRYO)
- Fixed samples need support before cutting so embedded in a WAX BLOCK
- Sample is currently too thick so is then cut into sections, now allowing light to easily pass through (cut by MICROTOME, very thin slices/samples produced as a result).
- Often poor contrast within the sample, section STAINING solves this issue, but sample first needs to be dewaxed by a suitable chemical before staining can occur.
Now ready for study!
What is histochemistry?
Staining of cells using specific chemicals
What is immunohisto(cyto)chemistry?
Localisation and identification of specific tissue antigens using labelled (and therefore visible) antibodies.
((similar to the idea of ELISA testing))
What is in situ hybridisation?
This is the localisation and identification of specific nucleic acid sequences in situ (in the sample) using hybridisation (i.e. formation of a hybrid DNA or RNA sample) through addition of a labelled molecule sufficiently similar to bind with the target sequence.
What are the colours seen in different structures as a result of H&E staining?
Nuclei/structures rich in nucleic acids stain purple and most proteins stain blue (for example, cytoplasm of muscle, red blood cells, epithelial cells)
- What are the two components of H&E stains and what are their properties?
Haematoxylin - is basic, so binds to basophilic structures, such as nucleic acids
Eosin - is acidic, so binds to acidophilic structures, such as mitochondria
- What can go wrong during the preparation of specimens/samples?
Artefacts can appear - if carried out poorly, other structures can be trapped within the specimen during the preparation process.
Lipids are also usually dissolved and lost during the preparation process, effecting the structure.
What is resolution dependant upon?
The wavelength of the medium used by the equipment - for example, in light microscopy, the resolution is the same as one wavelength of light
What are the main types of microscopy?
- Light microscopes (LM)
- Electron microscopes (EM), two types: scanning (SEM) and transmission (TEM)
- Fluorescence microscopy (FM)
- Confocal scanning laser microscopy (confocal/CF)
What is light microscopy?
The viewing of specimens through the use of visible light transmitted through the section.
Light from source:
- is focussed by condenser lenses
- passes through section
- section detail is then magnified by objective lens
- further magnified by eyepiece lens
- eyes can DIRECTLY see contrast in magnified image
- What are some useful features of light microscopy?
- Can see colour due to image being detected by rod and cone cells in eye and as visible spectrum used
- Samples can be directly viewed (and can be live specimens)
- Several special formats like fluorescence
- Used to be recorded by sketching, images can now be taken instead - this also allows phase contrast to be enhanced
What is the resolution of a light microscope?
0.2 micrometres (200 nm)
- Clinical relevance for H&E staining?
Irregularly shaped/enlarged nuclei are often seen in cancerous cells (example given in cross section of Barrett oesophagus, caused by reflux of acidic stomach contents and replaces squamous stratified epithelium with columnar, massively increases likelihood of adenocarcinoma of the oesophagus, common malignant epithelial tumour).
What are the colours seen in trichrome staining/Masson’s trichrome?
Three-colour staining protocol:
Collagen is blue or green
Cytoplasm is pink
Nuclei are brown/black/dark purple/dark red
What is an important use for trichrome staining?
Allows cells to be distinguished from surrounding connective tissue
What is fluorescence microscopy?
Uses light
- Lamp produces light for excitation
- Microscope focusses light on to section and collects fluorescence emission light
- Sample is preserved (chemically or by freezing)
- Sample is labelled to tag/label specific molecules, live fluorescent proteins (bioluminescence) or antibodies targeting specific proteins (immunofluorescence) used
- Different dyes emit visible light of different colours/wavelengths
What are the similarities between fluorescence and light microscopy?
- Both use visible spectrum as medium
- Both use the same eye pieces
- Both use the same method of visualisation and recording of images
What is GFP?
Green Fluorescent Protein - isolated from jellyfish (aequorea victoria), allows live imaging. Excitation and 395 nm, emission at 509 nm.
There are many colours available (red, yellow, blue etc)
- Who discovered GFP and when did they win a Nobel prize?
Chalfie, Shimomura and Tsien, 2008 Nobel prize for chemistry
What is immunofluorescence microscopy?
- Antibodies bind to specific target protein region
- Direct (attached to antibody) or indirect (another antibody carrying molecule necessary) labelling of target region by fluorescent marker
- Image by fluorescence microscopy, obtained through excitation and emission wavelengths
What is TEM?
Transmission electron microscopy ( Ruska in 1931, first prototype)
- Electron beam used to penetrate/illuminate sample, lens arrangement like LM to magnify (condenser/objective combo)
What is the resolution of TEM?
0.5 to 10nm
What are the differences between LM and TEM?
- TEM occurs in a vacuum, as electrons are absorbed by any material they meet, not needed in LM
- TEM has electromagnetic lenses, LM does not
- Final image of TEM cannot be directly viewed by human eyes like it can in LM, electrons instead create an image by interacting with a phosphorescent screen which can then be viewed, now recorded as a digital image
- Although both use an illumination/ray diagram technique, LM is ‘lit’ from the bottom whereas TEM is ‘lit’ from the top
- What are contrast stains used in TEM?
Heavy metal salts, such as uranium and lead - the salts scatter the electrons, producing contrast
What is the thickness of plastic resin sections used in TEM?
50-100nm thick
What is the thickness of sections used in LM?
5-50 micrometres thick
What sort of sections are needed for TEM?
- Ones with more carefully controlled fixation, need to preserve fine detail resolvable by electron beam
- Better support needed as thinner sections (so embedded in resin, not wax)
Can TEM be used to create 3D interpretations of samples?
Yes, through serial sections, provides a lot of information but takes forever so is NOT frequently used.
What is confocal scanning laser microscopy (CF)?
- Laser light, point-like source
- Often uses fluorescence microscopy
- Can use a thicker sample than conventional LM
- Scanned beam focusses at different levels of the section
- Image produced by a detector and a photomultiplier system
- Cathode ray tube (CRT) screen used for imaging
- Can be used to view 3D structures, optical slicing so no serial sections needed (takes less time), not all optical sections need to be shown
What is FISH?
Fluorescent in situ hybridisation, DNA probes used onto chromosomes (in metaphases). Locates genes.
- What are dissecting (light) microscopes?
- Allows external view of a sample
- Use visible light
- Light reflected from a thick sample
- Magnifying lenses and eye-pieces above sample
- Limited resolution
- Useful for initial study of some specimens
- Recorded as LM
- What is capsule endoscopy?
Patient swallows pill-sized capsule containing:
- Camera
- Light-emitting diode (LED)
- Batteries
- Radio-transmitter
Data goes to recorder belt at waist, capsule secreted and recovered (endoscopy can also be done, camera on end of optical fibre inserted into patient)
What is SEM?
Scanning electron microscopy
- Electrons scanned/used as a probe across a sample surface
- Thick sample, electrons interact with it and emitted signal goes to detectors and then a screen
- Single cells can be viewed externally
What is the resolution of SEM?
0.2-2nm (depends on diameter of the probe and volume of interaction with sample, high and low magnifications available)
How are samples produced for SEM?
- Fixation as for LM and TEM
- Sample may be very large, glued onto rivet or unsupported
- Scanned with electrons (charge could build up)
- Conducting coating applied to minimise build-up of electrons which would produce areas lacking in detail
What structures are able to be viewed under LM?
Nuclei, cell membranes and cytoplasm
What structures are able to be viewed under EM?
Most organelles (mitochondria, Golgi apparatus, endoplasmic reticulum, nucleolus/nuclear pores, bilayer of lipid membranes, viruses, macromolecules, structures within organelles)
