1
Q
What is the definition of resolution?
A
Ability of the microscope to distinguish separate and distinct objects
2
Q
What occurs to the resolution when the wavelength of light is small enough?
A
Two objects distinguishable as separate and distinct
3
Q
What occurs to the resolution when the wavelength of light is too long?
A
Two objects not distinguishable as separate and distinct
4
Q
What is the resolution of electron microscopy?
A
0.05-0.1nm
5
Q
What is the resolution range of X-rays?
A
0.1-1nm
6
Q
What is the resolution of light microscopes?
A
200nm
7
Q
What is the resolution that visable light begins?
A
400nm
8
Q
What is the definition of an electron microscope?
A
A microscope that uses electron beams, electrostatic and electromagnetic lenses to produce an enlarged image of very high magnification
9
Q
What x of magnification can electron microscopy go to within the microscope?
A
100-1000x magnification
10
Q
Who came up with the concept for electron microscopy?
A
- Louis de Broglie 1924
- Wanted to look at whether electrons could be diffracted in the same way as light properties
- He showed that electron beams behave similar to light waves
11
Q
Who built the first electron microscope?
A
- Max Knoll and Ernst Ruska 1931
- Transmission microscope
- Built the first prototype
- At the time people knew there were small pathogenic diseases such as those causing polio
12
Q
Who built the first scanning microscope?
A
- Max Knoll 1935
13
Q
What are 7 components to the electron microscope?
A
- Electron gun
- Condenser lens
- Aperture
- Stage
- Objective lens
- Projector lens
- Viewing screen
14
Q
What does the electron gun do and how?
A
- Traditionally a tungsten filament which is super-heated to 2700 degrees
- This generates electron admittance from the filament
15
Q
What do the three lenses and the aperture do in the electron microscope?
A
Direct the bram through the column and focus onto the sample at the specific height where it is inserted on the stage
16
Q
What happens at the viewing screen on a traditional EM and how are newer ones different?
A
- Image formation happens here
- Now have detectors in various places and imags are recorded onto these
17
Q
What are two types of transmission EM?
A
- Single particle cryoEM
- Electron tomography
18
Q
What are two types of scanning EM?
A
Serial blockface SEM
- FIB (focused ion beam) SEM
19
Q
What does a scanning EM do?
A
Scans the surface of the specimen with an electron beam to produce a 3D-effect image
20
Q
How does the SEM work?
A
- Electron beam rastered across the surface of the specimen using scanning coils in and X and Y motion
- Secondary electrons are bounced off the surface of the sample and are scattered and then picked up by the camera
21
Q
What is a main preparation technique in SEM?
A
Put a conductive coating on the sample to prevent accumulation f charge and static electric fields
Usually a layer of gold
22
Q
What are 3 advantages of SEM?
A
- 3D-effect surface visualisation
- Relatively quick
- Cheap
23
Q
What is a downside of SEM?
A
The resolution is fairly low
24
Q
What does a transmission EM do?
A
Transmits electron beam through the sample
25
What is the grey scale in a TEM the result of?
- The contrast between electron dense parts of the sample and non-dense parts
- Electrons pass through the less electron dense areas more readily
- Less electrons pass through electron dense areas
- Properties of the sample in terms of electron density of the material determines the grey scale
26
What is an essential requirement in TEM?
The sample has to be really thin in order to allow the electrons to pass through the sample
27
What is the spacing resolution within TEM imaging?
10-20nm
Very good resolution
28
Why is there a vacuum in conventional EM specimens?
In order for electrons to get from one end of the microscope to the other viewing end. If no vacum, the air would scatter the electrons
29
Why must conventional, biological EM specimens be dehydrated?
- Biological specimens consist of around 80% water
- This means they will not react well within the vacuum and must be dehydrated
30
Why must samples be fixed in conventional EM specimens?
- They can lose a third of their dry mass from electron bombardment
- They need to safely dehydrate them without losing their structural properties
31
How are conventional EM samples fixated?
- Using chemicals to stabalise molecular structure by cross-linking proteins
- Such as gluta-aldehyde
32
How are conventional EM samples dehydrated?
- The water content in the specimen is replaced with a solvent
- Such as alcohol
33
Why are conventional EM samples embedded?
- In order to make a sample thick enough to slice through put samples in resin
- The sample polymerises and makes the tissue a solid block, protecting the shape of it to enable sectioning
34
What determines the thickness of slices in electron microscopy and how does it vary for standard imaging versus tomography?
- Dependent on the powerfulness of the microscope- there is a balance between the sample being thin enough and a more powerful microscope, in which a thicker sample could be used and the electrons would still pass through.
- Produces 60-90nm thin slices using an ultra microtome
- Or thick sections around 200nm for tomography
35
What are used as stains in conventional EM?
- Heavy metals used to increase the contrast and scatter electrons
- Usually lead or uranium
36
What are the advantages of freezing samples?
- Preserve native ultrastructure eg protein structures, membrane organisation- reduces artefacts seen in dehydration
- Can image to high/atomic resolution
37
How are small samples such as proteins, virsuses and some bacteria frozen for TEM?
- Isolated in a solution the plunge freeze straight into liquid ethane
- 5-10micrometers
38
How are larger samples such as becteria, cells, tissue cells and small organisms frozen for TEM?
- High pressure freezing, pressurise the sample while freezing to force the cold cryogens into the sample so it is frozen all the way through
- Cryo- FIB (focused ion beam) then mills out a region of the sample (as too thick for electrons to get through)
39
Why can plunge freezing not work for larger samples?
The freezing does not occur rapidly enough
Causes crystals to form and there is not good enough preservation of internal features
40
What is an issue with the images TEM produces?
We get a 2D image of a 3D sample
41
What is single particle cryo-electron microscopy for?
The analysis of proteins, complexes, fibrils and viruses
(anything small enough to be frozen in the plunge method- cannot use slicing)
42
Outline how cryoEM and single particle analysis works to overcome the issue of dimension?
- 2D images are scanned and arranged according to orientation
- Align and average images within an orientation class
- Reconstruct the 3D density map of the protein
(can see every atom within the structure)
43
What did Richard Henderson develop?
Direct Electron counting detectors
44
What are Direct Electron counting detectors
They can read-out images fast enough to see the individual electrons hitting the sensor
45
What is an advance that direct electron counting detectors allow for?
- Can account for the movement of the sample.
- As soon as the beam hits the surface, the sample starts to degrade
- Can record movies- images in fractions of seconds and see the deterioration process
- Can work out the time of deterioration and see the detailed image beforehand
46
What is Volume EM and what is it used for?
Collective term for 3D imaging of cell or tissue samples which includes (cry)-electron tomography, serial blockface imaging
47
What is electron tomography?
- Collect a series of images at different tilted angles in the TEM (tilt series)
- Computationally reconstruct a 3D volume
- Can contour around features
48
How thick is an electron tomography sample?
Around 200nm
49
How is an electron tomography sample prepared?
- Either conventional dehydrated way
- Or high-pressured slice
- Plastic embedded dehydrated image is more likely to have artefacts and limited resolution
50
How are frozen samples sliced?
Cryo-FIB (focused ion beam) imaging
51
How does cryo-FIB milling work?
Uses a focused ion beam to slice thin lamellae from a frozen specimen, which are then transferred to another microscope (e.g., for cryo-EM or tomography) for imaging.
52
How does serial blockface SEM imaging work?
* Uses an SEM with a built-in microtome to image the surface of a tissue block, slice by slice.
* The process is repeated thousands of times, and 2D images are reconstructed into a detailed 3D model.
53
How are serial blockface SEM samples prepared?
Same as TEM
54
What are 3 pros of EM?
- High magnification
- Higher resolution
- Large complexes/tissues
55
What are 4 cons of EM?
- Expensive
- Requires vacuum
- Need stable environment
- Skill needed to operate it
56
Compare Light and Electron Microscopes on 5 axes.
**Light Microscope:** Uses light, glass lenses, no vacuum, lower resolution, and is cheaper.
**Electron Microscope:** Uses electrons, electromagnetic lenses, requires a vacuum, higher resolution, and is more expensive.
57
Was what a main driving factor behind the development of EM?
Could not image viruses as were too small
58
What is the range of resolution in EM?
5-300nm
59
Why is EM ideal for imaging viruses?
- Normally already in a liquid form such as saliva
- Sample preparation can therefore be rapid by just staining or vitrification and imaging
- No need for fixation, sectioning etc
60
What is Polio's full name?
Poliomyelitis
61
Who discovered polio and when?
- 1908
- Landsteiner and Popper
62
What does polio cause?
- Progressive muscle weakness
- Paralysis (1:200 irreversible)
- If diapraghm is effected, individuals cannot breathe
- 15-30% fatality rate
63
When was polio visualised by the EM?
1952
64
What did EM show us about polio?
- Cryo-EM reconstruction shows poliovirus 135S particles poised for membrane interaction and RNA release
- Butan at al 2014
65
How was it understood that polio was not a bacteria but something else?
- Took spinal fluid from someone who dies of polio
- Used a filter that bacteria could not pass through (trapping the bacteria)
- Injected the filtered spinall fluid into monkeys
- They developed the disease despite there being no bacteria in the sample
66
What was discovered (using EM) about chicken pox versus small pox?
- Was originally thought that they were of the same family due to similar nature of pustule formation
- Imaged the virsuses to find structural differences between the two
- Showed chicken pox had envelopes of protein coats and a nucleocapsid of genetic material
- 1948
67
What does the small pox virus do?
- Localises to blood vessels in mouth and throat
- Cases disfiguring rash
- Blindness
- Can be lethal
68
Why is single particle cryoEM such a good technique for viruses?
- Trap the virus in a thin layer of ice on the EM grid
- Image each molecule trapped in different orientations within the 2D grid
- Computational method assigns different orientations to different classes
- Can then back-project to a 3D object (reinstating a 3D image of the virus)
69
When were some of the first images of Covid-19 taken?
Jan 2020
70
What is the strucutre of covid-19?
A coronavirus- coronal spikes sticking out from the surface of the virus
71
What was the region of interest within covid-19?
Working out the structure of the spike proteins on the surface of the virus as this allows the interaction and invading of the human cell
72
When was the S proteins of covid-19 first imaged and how?
- Feb 2020- released in March
- Used cryoEM
- Took 3000 images in 24 hours
- Worked out the orientation of the particles and then presented the 3D structure
- Wrapp et al 2020
73
What did the imaging of the S protein of covid-19 allow?
- Aided the generation of vaccines by providing the structural blue-print
- Can see the interactions occuring between the host cell and the virus
74
What does SARS stand for?
Severe Acute Respiratory Syndrome
75
Where did the previous knowledge of SARS come from (Pre-Covid 19)?
How was it identified?
- Scientific community dealt with SARS pandemic in 2003
- Spread to 37 countries (starting in China)
- Identified SARS using EM
76
Why did SARS not have the same inhibitors or vaccines as for covid-19?
- Before the resolution revolution so didn't have the tools or techniques to apply the cryoEM method effectively
- Took a few hundred images in 24 hours as did not have the fast electron counting detectors
- Limited the resolution
77
What is the structure of SARS?
- Architecture is of the coronavirus perfusion spike protein
- Beniac et al 2006
78
Why is EM instrumental in virology?
- Diagnostic (fairly rapid results)
- Discovery and design of antiviral agents
- Magnification and resolution excellent- every single amino acid side chain could be seen within the protein
- No need for organism specific reagents such as antibodies so can image directly
79
What are connective tissue diseases?
- Connective tissues such as skin, tendons, lung are mainly composed of large extracellular matrix polymers such as collagen
- Becasue they are found everywhere, diseases cause multi-organ pathologies
- Rare and difficult to diagnose and may only present in adulthood
80
What are 5 symptoms of Urban-Rifkin-Davis syndrome?
- Patients have loose, redundant skin
- Esophageal tortuosity (Meandering oesophagus)
- Hyperinflation of lung and bell shaped chest
- Diaphragmatic hernia (diaphragm has a gap so tissue above and below was coming through in the wrong place)
- Stomach diverticula (extra bulge)
81
What was the initial propsal for the cause of Urban-Rifkin-Davis syndrome?
Disease with excess folds (Cutis Laxa)
82
How was Urban-Rifkin-Davis syndrome identified as not being cutis laxa?
Weren't mutations in the genes causing this disease
83
How did EM identify the mutant gene in Urban-Rifkin-Davis syndrome?
- Normal skin microscopy shows dense elastin fibre dents surrounded by a fine layer of microfibrils
- Patient (1mo) skin shows diminished elastin core, abundant microfibrils and globular elastin aggregates in the periphery
- Patient skin had similar skin phenotype to LTBP4 lung KO mouse
84
What was the conclusion of Urban-Rifkin-Davis syndrome genetics?
- Mutations in LTBP4 casue syndrome of impaired pulmonary, gastrointestinal, genitourinary, musculoskeletal, and dermal development
- Confirmed to be Urban-Rifkin-Davis syndrome through genetic testing
- Am J Genet 2009
85
What is LTBP4 and what is its normal function?
- Latent TGFbeta Binding Protein 4
- Required for formation of elastic fibres and regulation of TGF
- Binary interactions between elastic fibre protein (tropoelastin) and chaperoning proteins (fibulin5 and fubulin4) facilitate formation of globules and deposition onto microfibrils
- Linear deposition of elastin onto microfibrils to form elastric fibres
- Present in all elastic tissues
86
Why is EM instrumental in connective tissue diseases?
- Important tool to look at the pathology of tissue samples from patients
- Experienced microscopists and pathologists can recognise abnormal features
- Can provide clues to a diagnosis or suggest genes or pathways involved
87
What is common to all neurodegenerative diseases?
The assembly of proteins into amyloid that form disease-specific structures
88
What is Alzheimer's disease (AD) characterised by?
Deposition of beta-amyloid fibrils and tau tangles with disease-specific conformations
89
Outline AD
- Progressive neurological disease
- Characterised by plaques and tangles
- Reduction in ACh in the brain
- 944,000 affected in UK
- Most common cause of dementia
90
What is normal tau function?
- Microtubule associated protein
- Regulates the assembly and maintenance of microtubules
91
What does malfunctioning tau cause?
Forms insoluble filaments that accumulate as neurfibrillary tangles in AD
92
Outline how in situ beta-amyloid and tau was visualised
- Tissue autopsy taken from dead patient (6 hours postmortem)
- Thaw and acute brain slice preparation
- MX04 (fluorescent label that binds to amyloid) is added
- High pressure freezing biopsy
- Transfer the sample into cryo-FM-targeted cryo-sectioning device with FIB
- Focused ion beam mills of a layer of tissue to 100-200nm that TM can image through
- FIB uses fluorescence light microscope to visualise labelled amyloid and 'mill off' the correct area
- Transfer the whole grid to gather electron tomography data (rotate the image)
- Contour around the features
- Gilbert et al 2024
93
What is the structure of beta amyloid as seen by Gilbert et al?
- AB filaments in the plaques were mostly linear in one direction
- They could also be branched
94
What is the structure of tau filaments as seen by Gilbert et al?
- Bigger than Amyloid Beta so had more detail.
- Are unbranched and align with each other
- Took many averages of the elements to generate 3D structure and see the molecular data of the filament- seeing individual protein chains at high resolution
95
Why was EM instrumental in neurodegenerative diseases?
- To visualise the 3D architectures of beta-amyloid and tau fibrils in-tissue within human postmortem AD brain
- Very high resolution so protein structure could be observed
- Samples were frozen, hydrated and unstained so structural preservation is excellent
96
Overall, why is EM instrumental for diseases?
- Diagnostic- can point to which genes to screen; rapid results when imaging viruses
- Discovery and design of antiviral agents
- Magnification and resolution excellent- can identify subtle structural abnormalities; 3D structure of viruses
- Don't need speicifc antibodies to label proteins
Imaging in Biomedical Research
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