Microscopes Flashcards
Lecture 1 (Complete)
Why is it useful to determine the structure of cells, organelles and molecules?
Structure tends to relate to function, so knowing structure makes it easier to work out function
List the light microscope types
Brightfield
Fluorescent
Advanced
Confocal
List the electron microscope types
Transmission
Scanning
When is molten wax used in the preparation of specimens for light microscope viewing?
Before it is cut using the microtome, in order to support tissue and prevent distortion when it is cut
Why do tissues need to be fixed for viewing under light microscopes?
To stabilise tissue to prevent autolysis and degradation
What problems are associated with Brightfield microscopy?
- Most cells, tissues and cellular structures are colourless & transparent without using dye to stain them
- Most tissues are too thick so must be preserved/fixed, embedded and sectioned to view
- These actions can alter cell structure/molecules, and means only dead cells can be viewed
What width does the microtome cut sections into for viewing under a light microscope?
5 micrometers
What width does the ultramicrotome cut sections into for viewing under a electron microscope?
10-50 nanometers
What is a microtome?
A machine that cuts the specimen into very thin sections (5 micrometers) for microscope viewing
What are sections placed on for viewing them under most microscopes?
A microscope slide
What are sections placed on for viewing them under TEMs (transmission electron microscopes)?
Copper mesh grid
What do chemical stains (used to absorb light and allow visible images from light microscopes) usually bind to?
A certain class of molecules, rather than a specific molecule within the class
(eg; proteins in general, rather than a specific protein)
Give an example of a chemical stain for visualising cells / tissues under a light microscope
Haematoxylin (nucleaic acids)
Eosin (Protein)
What is Hematoxylin used for?
Hematoxylin stains nucleic acids a deep blue-purple colour
What is Eosin used for?
Eosin stains proteins a pink colour, nonspecifically
What are some positives about fluorescent microscopy?
- Fluorescent dyes can associate directly or indirectly with specific cellular molecules
- Multiple fluorescent dyes can be used simultaneously
- The fluorescent dyes “glow” against dark backgrounds
- Dye can attach to antibodies - immunolabelling
- Cells may be fixed or living
- More sensitive than brightfield microscopes since targets emit their own light through fluorescing
True or False:
Brightfield microscopes allow living cells to be viewed
False:
Brightfield microscopes allow only dead cells to be viewed
True or False:
Brightfield microscopes allow only dead cells to be viewed
True
True or False:
Fluorescent microscopes allow living cells to be viewed
True
True or False:
Fluorescent microscopes allow only dead cells to be viewed
False:
Fluorescent microscopes allow both living and dead cells to be viewed
What is a positive for a brightfield microscope?
it is relatively inexpensive
What type of light is used for fluorescent microscopes?
UV, to irradiate the stain to make it fluoresce
What type of light is used for brightfield microscopes?
Visible light
What is the purpose of immunolabelling?
To identify the location and quantity of a specific antigen in a tissue
Describe the general idea / principle of immunolabelling
- Want to identify location and quantity of antigen A in a tissue
- add primary antibody and allow it to bind to antigen A
- add marker-coupled secondary antibody and allow it to bind to the primary antibody
- observe markers
Why are the secondary antibodies labelled instead of the primary antibodies?
- Primary antibodies more limited in quantity, labelling & purifying them would be expensive
- Easier to label large amounts of secondary antibodies to get good final yield
- Secondary antibodies can detect multiple / different specificities
- More than one secondary labelled antibody can bind to primary antibody so signal is enhanced
What are the steps for immunolabelling?
- Prepare sample & place on microscope slide
- Incubate with primary antibody
- Wash away any unbound antibody
- Incubate with fluorochrome-conjugated secondary antibody
- Wash away any unbound antibody
- Mount specimen and observe in fluorescent microscope
What fluorochrome-conjugated drug binds actin filaments?
Rhodamine-labelled phalloidin
What is Rhodamine-labelled phalloidon?
A fluorochrome-conjugated drug that binds to actin filaments
What is confocal microscopy?
Optical sectioning instead of physical sectioning using lasers.
This means instead of having everything the light travels through in a tissue super-imposed together, a thin section on one focal plane can be shown in more detail
What is a negative of confocal microscopy?
It is very expensive, confocal fluorescence in the £100,000 range instead of the £1000s range for conventional fluorescence.
What are the positives of Advanced light microscopy?
- Permits observation of transparent living cells (useful for observing processes like cell migration, mitosis)
- Light phase shifts induced by the specimen are used to exaggerate very small differences between refactive indexes of components of cells and generate contrast
- Can have phase contrast (refracted & unrefracted light)
- Can have differential interference contrast (two light beams)
What super-resolution light microscopes break the resolution limit (200nm) ?
- Structured illumination microscopy (SIM)
- Simulated emission depletion microscopy (STED)
- Photo-activated localisation microscopy (PALM)
What is the resolution limit for conventional light microscopes
200 nm
What microscopes have significantly higher resolution than super-resolution light microscopes?
Electron microscopes
Why do electron microscopes only work for dead material?
Because a hard vacuum is required for the electrons to go through specimen and scatter
What resolution can transmission electron microscopes reach?
TEMs can reach 0.2-0.1 nm resolution
What are three special techniques associated with transmission electron microscopes (TEMs)?
- Metal shadowing / negative staining (visualise molecules, viruses, cell components)
- Cryoelectron (visualise unfixed, unstained samples)
- Freeze fracture (visualise membrane interior)
What is the viewing screen for TEMs made of?
Usually a photographic film or phosphorus-based material which will fluoresce when electrons interact with it
When is a TEM wholemount used?
If a specimen is ultra-thin it can be mounted directly onto an electron microscope grid (no prep required). Only molecules, bacteria and viruses are small enough.
What is a TEM wholemount?
Mesh, (often copper) coated with a 10nm thin film of formvar (a plastic). Used for ultra-thin specimen mounting.
How are specimens that are too thick to be mounted directly usually prepared for viewing under a TEM microscope?
Specimen undergoes primary fixation, washing, secondary fixation, dehydration, infiltration with resin, polymerisation of resin, embedding, sectioning and staining with an electron-dense material
Why do specimens need special preparation for being viewed under a TEM?
To ensure specimens can withstand the rigors of the vacuum and irradiation, preserving the in vivo structure of the samples
Why do specimens for TEMs have to be extremely thin for viewing?
The electrons pass through the specimen and diffract, but they have poor penetrating power so specimens must be thin enough for them to pass through
What is an ultramicrotome?
Usually used for electron microscope sections, cuts them very thinly to prepare them for viewing
What knives are used for ultra-thin sectioning, for electron microscopes?
Glass (though wears out quickly so need regular replacements)
Diamond (Atomically perfect edge, last a long time, expensive £5,000-£10,000)
How is contrast improved for TEM images?
Heavy metals such as lead, depleted uranium or tungsten are used to scatter electrons (colour dyes are useless here)
How close to the theoretical limits of electron microscope resolution are we?
Nowhere near the limits
Describe X-Ray crystallography
X-rays pass through a crystalised protein and scatter. The diffraction pattern produced is used to deduce the atoms’ positions.
Describe TEM Cryo-electron microscopy
Electron beam fired at frozen protein solution. The emerging scattered electrons pass through a lens, magnifying their image on the detector. Structure worked out from this.
What positives does cryo-electron microscopy have over X-ray diffraction?
It is faster (weeks not years) and easier to determine protein structures
Discuss similarities between the Scanning electron microscope (SEM) and Transmission electron microscope (TEM)
Both operate in a vacuum
Specimen is usually fixed using the same fixatives
Discuss how the Scanning electron microscope (SEM) is different to the Transmission electron microscope (TEM)
After the specimen is fixed for SEMs, it is dehydrated and gold coated to protect from the electron beam
The SEM achieves a resolution of 1nm
The SEM gives a 3D image of the surface only
The SEM is half the size of a TEM
What are scanning electron microscopes used for?
To examine the surface features of samples such as tissues, cells or isolated cell parts
Give general advantages to electron microsopy
Over 1000x greater resolution than light microscopy
Only way to visualise nanoparticles (nanotechnology and nanomedicine)
Cryo-TEM now has equal resolution to X-ray diffraction (and is improving)
How do scanning electron microscopes work?
Electron beam directed via scanning coils through an electromagnetic objective lens to reflect off of the specimen to the detector
How do transmission electron microscopes work?
Electron beam directed through condenser lens through specimen. After this they diffract, are focussed by an electromagnetic lens, and projected via a projecting lens onto a viewing screen
How does a conventional brightfield light microscope work?
Light from lamp shines up through condenser lenses, through the specimen on the stage, through objective lenses and is reflected via a prism through the ocular lens into the eye. The lenses magnify it appropriately.
How does a fluorescent microscope work?
UV light shines through an excitation filter, reflects via a dichroic mirror through the objective lens to the specimen, which irradiates the stain causing it to fluoresce. The light emitted from the stain shines up, passes through the dichroic mirror, through barrier filters and into the eyepiece and eye.
In a fluorescent microscope what is the purpose of a barrier filter?
To prevent UV light from entering the observer’s eyes?
What is a fluorochrome?
A fluorescent dye
Give some examples of fluorochromes
Fluorescein (green)
Rhodamine (red)
What is Nomarski microscopy referring to?
Advanced differential-interference-contrast microscopy (he invented it)
