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Imaging in Biomedical Research > Electron Miscroscopy > Flashcards

Electron Miscroscopy Flashcards

1
Q

What is the definition of resolution?

A

Ability of the microscope to distinguish separate and distinct objects

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2
Q

What occurs to the resolution when the wavelength of light is small enough?

A

Two objects distinguishable as separate and distinct

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3
Q

What occurs to the resolution when the wavelength of light is too long?

A

Two objects not distinguishable as separate and distinct

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4
Q

What is the resolution range of electron microscopy?

A

0.05-0.1nm

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5
Q

What is the resolution range of X-rays?

A

0.1-1nm

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6
Q

What is the resolution of light microscopes?

A

200nm

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7
Q

What is the resolution that visable light begins?

A

400nm

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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

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9
Q

What x of magnification can electron microscopy go to within the microscope?

A

100-1000x magnification

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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
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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
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12
Q

Who built the first scanning microscope?

A
  • Max Knoll 1935
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13
Q

What are 7 components to the electron microscope?

A
  • Electron gun
  • Condenser lens
  • Aperture
  • Stage
  • Objective lens
  • Projector lens
  • Viewing screen
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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
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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

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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
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17
Q

What are two types of transmission EM?

A
  • Single particle cryoEM
  • Electron tomography
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18
Q

What are two types of scanning EM?

A

Serial blockface SEM

  • FIB (focused ion beam) SEM
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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

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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
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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

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22
Q

What are 3 advantages of SEM?

A
  • 3D-effect surface visualisation
  • Relatively quick
  • Cheap
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23
Q

What is a downside of SEM?

A

The resolution is fairly low

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24
Q

What does a transmission EM do?

A

Transmits electron beam through the sample

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25
Q

What is the grey scale in a TEM the result of?

A
  • 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
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26
Q

What is an essential requirement in TEM?

A

The sample has to be really thin in order to allow the electrons to pass through the sample

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27
Q

What is the spacing resolution within TEM imaging?

A

10-20nm

Very good resolution

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28
Q

Why is there a vacuum in in conventional EM specimens?

A

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

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29
Q

Why must conventional, biological EM specimens be dehydrated?

A
  • Biological specimens consist of around 80% water
  • This means they will not react well within the vacuum and must be dehydrated
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30
Q

Why must samples be fixed in conventional EM specimens?

A
  • They can lose a third of their dry mass from electron bombardment
  • They need to safely dehydrate them without losing their structural properties
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31
Q

How are conventional EM samples fixated?

A
  • Using chemicals to stabalise molecular structure by cross-linking proteins
  • Such as gluta-aldehyde
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32
Q

How are conventional EM samples dehydrated?

A
  • The water content in the specimen is replaced with a solvent
  • Such as alcohol
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33
Q

How are conventional EM samples embedded?

A
  • 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
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34
Q

How does sectioning occur for a conventional EM sample?

A
  • Produces 60-90nm thin slices using an ultra microtome
  • Or thick sections around 200nm for 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
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35
Q

How does staining occur in conventional EM?

A
  • Heavy metals used to increase the contrast and scatter electrons
  • Usually lead or uranium
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36
Q

What are the advantages of freezing samples?

A
  • Preserve native ultrastructure eg protein structures, membrane organisation- reduces artefacts seen in dehydration
  • Can image to high/atomic resolution
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37
Q

How are small samples such as proteins, virsuses and some bacteria frozen for TEM?

A
  • Isolated in a solution the plunge freeze straight into liquid ethane
  • 5-10micrometers
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38
Q

How are larger samples such as becteria, cells, tissue cells and small organisms frozen for TEM?

A
  • 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)
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39
Q

Why can plunge freezing not work for larger samples?

A

The freezing does not occur rapidly enough

Causes crystals to form and there is not good enough preservation of internal features

40
Q

What is an issue with TEM?

A

We get a 2D image of a 3D sample

41
Q

What is single particle cryo-electron microscopy for?

A

The analysis of proteins, complexes, fibrils and viruses

(anything small enough to be frozen in the plunge method- cannot use slicing)

42
Q

Outline how cryoEM and single particle analysis works to overcome the issue of dimension?

A
  • 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
Q

What did Richard Henderson develop?

A

Direct Electron counting detectors

44
Q

What are Direct Electron counting detectors

A

They can read-out images fast enough to see the individual electrons hitting the sensor

45
Q

What is an advance that direct electron counting detectors allow for?

A
  • 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
Q

What is Volume EM and what is it used for?

A

Collective term for 3D imaging of cell or tissue samples which includes (cry)-electron tomography, serial blockface imaging

47
Q

What is electron tomography?

A
  • Collect a series of images at different tilted angles in the TEM (tilt series)
  • Computationally reconstruct a 3D volume
  • Can contour around features
48
Q

How thick is an electron tomography sample?

A

Around 200nm

49
Q

How is an electron tomography sample prepared?

A
  • Either conventional dehydrated way
  • Or high-pressured slice
  • Plastic embedded dehydrated image is more likely to have artefacts and limited resolution
50
Q

How are frozen samples sliced?

A

Cryo-FIB (focused ion beam) imaging

51
Q

How does cryo-FIB milling work?

A
  • Inside the microscope slices off little lamellae
  • Transfers to another microscope such as electron tomography
52
Q

How does serial blockface SEM imaging work?

A
  • SEM has a microtome inside
  • Image the surface of tissue block collecting back-scattered electrons
  • Remove another section then image again
  • Repeat thousands of times
53
Q

How are serial blockface SEM samples prepared?

A

Same as TEM

54
Q

What are 3 pros of EM?

A
  • High magnification
  • Higher resolution
  • Large complexes/tissues
55
Q

What are 4 cons of EM?

A
  • Expensive
  • Requires vacuum
  • Need stable environment
  • Skill needed to operate it
56
Q

See pg 29 of electron microscope lecture for comparison of light and electron microscopes

A
57
Q

Was what a main driving factor behind the development of EM?

A

Could not image viruses as were too small

58
Q

What is the magnification range of EM?

A

5-300nm

59
Q

Why is EM ideal for imaging viruses?

A
  • 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
Q

What is Polio’s full name?

A

Poliomyelitis

61
Q

Who discovered polio and when?

A
  • 1908
  • Landsteiner and Popper
62
Q

What does polio cause?

A
  • Progressive muscle weakness
  • Paralysis (1:200 irreversible)
  • If diapraghm is effected, individuals cannot breathe
  • 15-30% fatality rate
63
Q

When was polio visualised by the EM?

A

1952

64
Q

What did EM show us about polio?

A
  • Cryo-EM reconstruction shows poliovirus 135S particles poised for membrane interaction and RNA release
  • Butan at al 2014
65
Q

How was it understood that polio was not a bacteria but something else?

A
  • 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
Q

What was discovered (using EM) about chicken pox versus small pox?

A
  • 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
Q

What does the small pox virus do?

A
  • Localises to blood vessels in mouth and throat
  • Cases disfiguring rash
  • Blindness
  • Can be lethal
68
Q

Why is single particle cryoEM such a good technique for viruses?

A
  • 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
Q

When were some of the first images of Covid-19 taken?

A

Jan 2020

70
Q

What is the strucutre of covid-19?

A

A coronavirus- coronal spikes sticking out from the surface of the virus

71
Q

What was the region of interest within covid-19?

A

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
Q

When was the S proteins of covid-19 first imaged and how?

A
  • 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
Q

What did the imaging of the S protein of covid-19 allow?

A
  • Aided the generation of vaccines by providing the structural blue-print
  • Can see the interactions occuring between the host cell and the virus
74
Q

What does SARS stand for?

A

Severe Acute Respiratory Syndrome

75
Q

Why did the knowledge of SARS aid the treatment of covid-19?

A
  • Scientific community dealt with SARS pandemic in 2003
  • Spread to 37 countries (starting in China)
  • Identified SARS using EM
76
Q

Why did SARS not have the same inhibitors or vaccines as for covid-19?

A
  • 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
Q

What is the structure of SARS?

A
  • Architecture is of the coronavirus perfusion spike protein
  • Beniac et al 2006
78
Q

Why is EM instrumental in virology?

A
  • 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
Q

What are connective tissue diseases?

A
  • 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
Q

What are 5 symptoms of Urban-Rifkin-Davis syndrome?

A
  • 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
Q

What was the initial propsal for the cause of Urban-Rifkin-Davis syndrome?

A

Disease with excess folds (Cutis Laxa)

82
Q

How was Urban-Rifkin-Davis syndrome identified as not being cutis laxa?

A

Weren’t mutations in the genes causing this disease

83
Q

How did EM identify the mutant gene in Urban-Rifkin-Davis syndrome?

A
  • 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
Q

What was the conclusion of Urban-Rifkin-Davis syndrome genetics?

A
  • 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
Q

What id LTBP4 and what is its normal function?

A
  • 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
Q

Why is EM instrumental in connective tissue diseases?

A
  • 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
Q

What is common to all neurodegenerative diseases?

A

The assembly of proteins into amyloid that form disease-specific structures

88
Q

What is Alzheimer’s disease (AD) characterised by?

A

Deposition of beta-amyloid fibrils and tau tangles with disease-specific conformations

89
Q

Outline AD

A
  • 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
Q

What is normal tau function?

A
  • Microtubule associated protein
  • Regulates the assembly and maintenance of microtubules
91
Q

What does malfunctioning tau cause?

A

Forms insoluble filaments that accumulate as neurfibrillary tangles in AD

92
Q

Outline how in situ beta-amyloid and tau was visualised

A
  • 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
Q

What is the structure of beta amyloid as seen by Gilbert et al?

A
  • AB filaments in the plaques were mostly linear in one direction
  • They could also be branched
94
Q

What is the structure of tau filaments as seen by Gilbert et al?

A
  • Bigger than AB 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
Q

Why was EM instrumental in neurodegenerative diseases?

A
  • 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
Q

Overall, why is EM instrumental for diseases?

A
  • 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 (9 decks)

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