Lecture 7 - Basics of Electron Microscopy PDF

Basics of Electron Microscopy Xiao Tianshu [email protected] Important notice Quiz 2: Content of week 8 – 10; 10-15 MCQs through NTULearn, available from 2:30 pm to 11:59 pm of Oct 30. Two essay questions will be released in NTULearn on Oct 30. Please submit the h...

Basics of Electron Microscopy Xiao Tianshu [email protected] Important notice Quiz 2: Content of week 8 – 10; 10-15 MCQs through NTULearn, available from 2:30 pm to 11:59 pm of Oct 30. Two essay questions will be released in NTULearn on Oct 30. Please submit the hard copy before noon of Nov 6. (Dropbox in lounge room) ~ Final exam: Content of week 8-12; ukf HPLC & FPLC : 38 MCQs and 2 essay questions. Revision in the lecture of week 13: quiz paper, key points, etc. Why are things visible? Relative size https://bio.libretexts.org/Bookshelves/Introductory_and_General_Biology Microscope  Microscope ~ to sample amplify Stage image Why are tiny objects invisible?  Airy disk and Rayleigh criterion Resolution is defined as the smallest distance between two points that can be differentiated. e distance betw 2 centres radius disk determine = of airy disk diry : Human eye has a resolution of about 0.1-0.2 mm. resolute Diameter of central disk disk pattern 2 laser points projected airy isa wh care abt onto image place , generated byI light size of 14 pdcA disk patterns source minimum as it 2 airy e on plane determines resolut I once beam is projected on to limit of i equipment & disk is e light Re approaching elo we image plane airy source disk , 2 airy. pattern you see one image plane distance is decreasing gradually First minimum ~ 2 - 9 e smallest distance that allow us to highest light. differentiate e 2 laser points here is airy => disks are concentric rings intensity in e known as resolute patterns middle called first minimum  ength radius of airy disk light beam - LX sin & goes through = (XO a slit size of "G x of light beam -- - first minimum in e - opening less through which light passes radius of dinmetaigh 4 A It min. disk aperture size/lens size of airy - size slit size & determined by this formula Rayleigh criterion Sandwich Theorem: sin θ = tan θ = θ, when O is very small when θ is very small Respecially when e image place is very far away fro aperture  Rayleigh criterion for the diffraction limit to resolution states that two images are just resolvable when the center of the diffraction pattern of one is directly over the first r minimum of the diffraction pattern of the other. r= L x sinθ L Angular resolution, θ =1.22𝜆/𝐷; min For geometrical optics, when the focal length (f) is much longer than the diameter of lens (D), the radius of airy disk 1.22𝜆𝑓 𝑟= resolute is determined by 2 𝐷 & factors , a of light source determined by If & i) which is e structure of e equipment  Rayleigh criterion In order to see a tiny object , we want to1 e resolute limit Resolution in microscope ↳ by ↓ e X 0.61𝜆 𝑑= 𝜇 𝑠𝑖𝑛𝛽 & denominator is determined by microscope 𝜆 – wavelength µ - refractive index β – semi angle of collection 𝜇 𝑠𝑖𝑛𝛽 is called numerical aperture (NA) You can never achieve the theoretical diffraction limit due to the imperfection of microscope. https://analyticalscience.wiley.com/do/10.1002/micro.140/  Comparison Diff light sources Advantages Disadvantages Visible light No damage to sample Long wavelength (~400 ↳ light microscope , Easy to focus nm) leading to lower resolut compound microscope Eye wonderful detector scattered X-ray easily ~ X-ray Small wavelength (0.01-10 nm) Difficult to focus & it can go through I Good penetration Damage sample as it microscope material ~Electron Small wavelength (pm) Poor penetration has go penetrat ? - ↳ electron microscope Can be focused is Damage sample cannot go through -se are cha, , bulky sample Neutron Good penetrating power Difficult to produce and Small wavelength (pm) focus & neutrons have no charge as light sources o small I have high energy & often damages I sample  Electron Microscope In 1932, Reinhold Rüdenberg from Siemens-Schuckert patent the electron microscope. In 1937, first scanning electron microscope was invented by Manfred von Ardenne. In 1938, Siemens produced the Development of first commercial electron electromagnet in microscope. How to focus 1883 electron beam 1899  Electron microscopy Nobel prize 1986 Nobel prize 1986 Nobel Prize 1986 Scanning tunneling Scanning tunneling Development of electron microscope microscope microscope lens How does e achieve short wavelength resolute ? & Electron Vs Photon high Resolution in microscopy is limited to about ½ of the wavelength of the illumination source. Electron has a shorter wavelength than photon. ~ in e accelerator stack Wavelength of electron is determined by the accelerating voltage. accelerator stack - has high Planck’s constant & determines : voltage term 𝜆 = ℏ xf ebeam generated 2𝑚ⅇ𝑉 ~ Taccelerating cHae Mass of electron Accelerating voltage Charge of electron & only thing we can change When there is a very high voltage, the velocity of electron is comparable to light, so the ‘V’ in the equation needs to be corrected.  Electron Microscopy Advantages: Much higher magnification, e.g. x50000; Optical microscope < x1000 Resolving power, angstrom (0.1 nm) level; Optical microscope ~200nm Disadvantages: larger than light microscope Costly ~ Size of the instrument Complicated operation Image acquisition needs to be done in vaccum.  Basic components pac e-beam ~ providehigh vola,a▪ e pace enter a on electric field ~ Lenses in electron microscopes can be strong classified into three systems ; & equip e-beam i high speed ▪ The condenser lens – direct and focus the & xf e beam becomes electron beam into the sample short ▪ Controls the beam intensity, spot size and beam coherence ▪ The objective lens - produces a magnified real image of the sample and is used for focusing. a L need furthe ▪ The projector lens -allow easier control of the magnification and projects the magnified & image onto a detector. detector : receive e signal of EM , process ita convert it to an image  sass ~ beam Control the movement of beam & Electron can movement control i of e microscopy beam e-beams do not similar basic go i structure through sample, instead they are scattered 2D image by surface i poed of e sample & generate an image  Information carried by electron What is I interacti betwee e-beam & sample ? Elastic scattering occurs when there is no loss of energy of the incident primary - x fe electron. Elastically scattered electrons can e-beam 4 scattered e- ↑ change direction but do not change their is not wavelength. Inelastic scattering occurs changed when there is loss of energy. - X of e-beam is changed Scattering of electron carries information of inner struct of sample As I direct of e- the sample. The phase change created by beam crefract) scattering results in contrast in image. - create contrast to see inner struct - < Blue & Pink - TEM  ~ ebeam goes through ~ e-beam does not : sample go through sample Biological transmission electron microscope on the left and a scanning electron microscope on the right. According imaging modes in transmission and scanning electron microscopy. A thin sample is transmitted by the electrons to form a projected image of the sample in TEM. A bulk sample is scanned with a focused beam of electrons in an SEM providing a surface view. ~ e surface is detected morphology of Images show Hela cells, thin section of a fixed, dehydrated, plastic embedded cell culture (70 nm thickness) on the left, and freeze-fractured Hela cell imaged in the 2D image inner frozen state in a cryo SEM on the right. show struct 3D image. of surface I e sample  Electron microscope Transmission electron microscopy (TEM) -electrons that are transmitted through the ultra-thin sample ▪ 2-D images ▪ Provide details about their internal composition. Scanning Electron Microscopy (SEM) – secondary electrons, backscattered electrons and characteristic X-rays that are scattered from sample ▪ 3D image of sample surface ▪ Samples that are used for SEM need to be electrically conductive -biological samples need special preparation. ▪ They must be fixed, dried, and coated with a thin layer of a conducting material.  SEM Images of EM TEM A C Hela cell SEM TEM Electron microscopy image of SARS-CoV. Photo B D credit to Dr. Fred Murphy. This media comes from the Centers for Disease Control and Prevention's (CDC) Public Health Image Library (PHIL), identification number 4814 Transmission electron micrograph of West Nile virus. Courtesy of CDC (Dr Edwin P Ewing, Jr). Insect  EM sample sample preparate Samples of EM must be immobilized through: Chemical fixation Cryofixation is to immobilise I sample purpose Dehydration to get a clear image Sectioning Negative staining Heavy metal staining problem of e Freeze-fracture one chemical treatments is that most samples require i native structure which requires native conditis e. g agreors. buffer wate , , sable pH this hinders I applicate of EM  addresses e ~ I problem as Cryo-EM sample is fixed using a cryogenic temp , sample is fixed by freezing this reservesI native conformate ofI bio sample ↓  this damages I sample Vitrified water - as to be it forces re-oriented I sample molecules I even moleculesreamaged a pattern distort native e sample of conformate normal freezing causes crystallizati of water ice form - in Vitrified water: water molecule is distributed randomly in vitrified ice. I ethare is Plunge freezing: too fast for crystal to form. I in native fo are,is samples called stay A conformate this freezing process & ethare extremely provides low special an is very temp also called cryogenic temp i => process is too fast to e allow format of ice crystal , sample & water molecules are frozen native immediately in conformati I no crystallisat process Bojic et al. BMC Biology. 2021  to - doesI freezing get vitrified sample Automated vitrobot Accurate temperature and humidity control Precise blotting time and force Sample is vitrified in liquid ethane.  ~ Cryo-TEM World under cryo-EM Cryo-TEM image of GroEL Image of frozen, hydrated epsilon15 suspended in amorphous ice at bacteriophage. Jiang et al., Nature 439: 50000× magnification 612-616, 2006 https://www.gatan.com/  Key points ~ resolute limit Diffraction limit Why electron Advantage of electron microscopy Imaging technology to visualize protein, large complex and sub- cellular structure Difference between TEM and SEM Technologies of protein structural biology Nuclear magnetic resonance (NMR) (1940s); X-ray crystallography (1910s); Cryo-EM (1980s). X-ray crystallography hard it might be to get ~ a crystallised P (depends on P itself) Require crystallization Sample in high concentration 10-20 mg/ ml. Detect diffraction of electron. Difficult for transmembrane protein or protein with conformation heterogeneity.  density map parystallised illuminated by X-ray beam a generates diffract : pattern raw data structure T complicated process  NMR Sample in solution Sample concentration ~0.1-1 mM Detect signals of radioactive nuclei, 1H, 15N and 13C, etc. Suitable for small protein (< 50 KDa), chemical compound, DNA, lipid, etc. Advantage in protein dynamics and protein-ligand interaction. 1H–15N HSQC spectrum of a fragment of an isotopically labelled protein bigger molecules ( > 50kDa) will have a lot atoms NleG3-2 & espectrum will be very crowded & peaks of each nuclei cannot be resolved Wu et al. 2010. PLoS Pathog. sample exposed to magnetic excites field a radiofrey" nuclei of e atom Cryo-EM sample is vitrified amt of ~ Cryogenic sample ~tiny Sample concentration ~1mg/ml, 3µl Psample Directly capture images - structure reconstruct no needed Suitable for large protein (> 200 KDa) Advantage in large complex, transmembrane protein, protein with conformational heterogeneity. give greater scattering ofa molecules beam large , visible making easily  Single particle analysis Low resolution High resolution Before 2013 At present 3 (Angstrom) , 0 3 am. can see more details ! Credit: Veronica Falconieri and Sriram Subramaniam, National Cancer Institute (NIH) via Flickr, CC BY-NC 2.0 What factors allows cryo-EM to achieve such a high resolute ? 1st barrier is Radiation damage lead to radiate of secondary a break down e chemical bonds - density of side-chain is degraded die to chemical Energy of electron can transmit to bond breakdown sample and cause radiation damage. no damage damage Detailed information is lost. further breakdown of chemical ~ air bubble Radiation damage is a limiting bonds pdcq caused by free , radicals, radiath factor of high resolution. H (air damage gas bubbling in e sample) Mishyna et al. 2017 Micron  to allow P ~ Protein in solution to remain native in conformat Protein molecule are freely mobile in solution. Diff movements that occur in P solute : Xu et al. 2015. The Journal of Chemical Physics  Take photos of protein I p from ~ including water molecule this protects Protein molecule is fixed by flash freezing. radiate damage & Pictures of millions of particles are taken by cryo-EM. keep itinitsnatire take cryo-EM scans shorter time as all P molecules are identical as they are purified P. : one scan is enough as p it captures many relieved in cryo-EM moleues i samples problem is , from are exposed to sample is frozen in cryogenic temp this protects sample , radiati damage, less radiat? allowing cryo-EM to achieve high resolute  utilise purified P sample ~ large Single particle analysis containing quantity of molecules 2D projection 3D structure require many diff views largequantities diff orientat of e particle allows us to derive =P 30 Struct molecule frozen. each is in a specific orientati each giving an , image  another method of cryo-EM ~ to characterise O Struct Tomography. & don't have to be purified I cross-sect just use sample , can Sample stage is tilting to record 2D images from different orientations. to generate 3D structure Cryo-ET allows us to characterize structure in the context of native environment. -most this method causes a higher asI same radiate damage Koning et al. Annals of Anatomy 217 subjected to multiple scans sample is (2018) 82–96.  Key points Advantages of cryo-EM: -Large molecule > 200 Kda, transmembrane protein -Frozen sample -Tiny amount of sample Vitrification can retain the native form of protein molecule. 2D projections are reconstructed to 3D structure.  also contributes to - high resolute image of EM Instrumentation e of microscope e g. operature.  ~ polies e-beam Electron gun ~ quality of t beam paced not go enough, cannot fulfill requirements of high resolute M 1. Heated Tungsten ~ still not ideal 2. Heated Lanthanum hexaboride (LaB6) 3. Tungsten field emission gun (FEG) ebeaus paced & intest & expensive , provide most oncethere is e isplad ↑ a high qualitye beam e-current , from e tip f filament ↑ additional electric ↑ & bottom field e-que to of Wash = ! ↑ attracti e- I allowing -very sharp e- to gradually escape e-escape frm e frm e tip tip , this ensures quality of beam https://www.thermofisher.com/blog/materials/electron-source-fundamentals/  Magnetic lens Electric current in the wire creates magnetic field. The magnetic field guides the electron and focus the beam. The magnetic lens is different from optical lens because the electron beam rotates in the lens. e-rotates & approaches diff betwe magnetic optical lens [LM) lens & focal pt of e lens lens e · , is that I image paed by magnetic to e focusing et-beam is rotated in a certain & inverted , but in CM , focal pt. image is simply  Vacuum Molecules in air can cause electron scattering, interfering with coherence of electron beam. light mas na b what is high Ehasveny Spatial and temporal coherence: all the electron arrives on the quality e sample at the same time and from the same direction. beam? colerance ↳ high e- e- e. g. air interfere e- is movement of , beam is distorted , direct? AsI arrive & they sample& diff time , colerance compromised by air molecules  Detector used to defect & receive convert e cannot be & 1. Fluorescence Screen ~ signal digitalisede image it to an image 2. Photographic film involves chemical rxtes a cannot - , digitilised image 3. CCDs (convert electron to photon) - can 4. Direct electron detector (directly count digitilised image the electron as charge) digitise image directly receive e- signal ~ convert e - - > photon -photon Through travel FOL & receive photon signal 2) multi-step process, each step causes some to digitilised image It direly detest seis to digitilised image , ensuring I quality of image loss in informat ~ high voltage , short x & high resolut  dependent on software ~ Data collection and processing mesh each hole can trap a small piece of vitrified sample  Data as a movie Beam induced motion causes the vitrified ice to move, leading to a blurry image. & subtle movement & low resoluti Sample stage may drift. frames To solve this : mulconsisting of t i p l e Cryo-EM data is recorded as a movie and needs to be aligned to an image. & i movie back to each frame of The motion of sample can be corrected restore high resolute image during data processing. Brilot et al. 2012. J. Struct. Biol.  Motion correction a Individual frame 1 ar datarecord consisting of eq 38 frame , each frame records a resolut subtle movement resolved of I sample data during can processing we , align particles : of each frame& compilea frames Precise patch motion into stack image a correction Each movie comprises of 38 frames - Total dose per frame ~1.4 e-/Å2 informate cannot be 100 % delivered to e sensor CTF corruption Contrast transfer function is a sinusoidal function of spatial frequency, which mathematically describes how aberrations in a transmission electron microscope (TEM) modify the image of a sample. It is dependent on defocus, spherical aberration coefficient. CTF can be estimated and corrected during data processing. sinusoidal funct ~ signals are delivered form of forview transformat: in a & diff spatial free" Giz-axis) signal corrupted by CTP signal 1101 a 7 S n of image CTF modulates lost of informat/signal https://www.globalsino.com/EM/page4236.html  ~ wha is e factorsthatench CTF function set to correct can defocus -3.0 µm CTF defocus -0.75 µm to get a higher & lower defocus value , i contrast resolut of particle is compromised more difficult to identify particle https://cryoem101.org/  CTF corruption Contrast transfer function is a sinusoidal function of spatial frequency, which mathematically describes how aberrations in a transmission electron microscope (TEM) modify the image of a sample. It is dependent on defocus, spherical aberration coefficient. CTF can be estimated and corrected during data processing. funct to shift left e defocus value causes : CTF M - informat ↑ defocus value e =>adjustdeforthat causes defocus e : smaller reserves informati in this region will => however, a a CPF funct to shift left & & details e particle of & it can give you info & resolute maximised , be distorted , this also higher resolute of I image DESPITE If giving is to combat CPF compromises contrast compromised contract & some info loss in front reg corrupt https://www.globalsino.com/EM/page4236.html  CTF corruption Contrast transfer function is a sinusoidal function of spatial frequency, which mathematically describes how aberrations in a transmission electron microscope (TEM) modify the image of a sample. It is dependent on defocus, spherical 2 aberration coefficient. CTF CTF can be estimated and corrected during data processing. signal of image is compromised byITF corrupth decided = FFX CTF CTF corrEHE however not all informate can be will be restored , info & 0 point permanently loss https://www.globalsino.com/EM/page4236.html  Useful software of data processing Large dataset: thousands of micrographs; Workstation with GPU or GPU server is required; Software: EMAN2, RELION, CRYOSPARC  Summary Rayleigh criterion Diffraction limit, wavelength of electron beam Electron compared with other illumination source SEM and TEM Important application of cryo-EM in structure biology Difference between NMR, X-ray crystallography and Cryo-EM. Important components of EM Why do we want to see biomolecules? Reference list Transmission Electron Microscopy, David B. Williams , C. Barry Carter https://nccat.nysbc.org/activities/nccat-remote- learning/em-reading-list/ Single-particle Cryo-EM of Biological Macromolecules, Robert M Glaeser, Eva Nogales and Wah Chiu, Published May 2021 Physical Principles of Electron Microscopy - An Introduction to TEM, SEM, and AEM, R.F. Egerton https://guide.cryosparc.com/  Q&A The wavelength of the electron beam under 300 KeV is 2 pm. What is the wavelength when there is a 10 KeV field? Let’s assume that the accelerating voltage won’t be affected by the velocity of the electron. Why is neutron difficult to be focused? What image you can get under TEM for the gold nanoparticle as below? Which type of molecule is easier to get a high-resolution structure under EM? Asymmetrical, trimeric (C3 symmetrical) or heptameric (C7 symmetrical) What is the advantage of cryoEM over X-ray crystallography and NMR? Why do you think cryo-EM can have better resolving power?  Q&A The wavelength of the electron beam under 300 KeV is 2 pm. What is the wavelength when there is a 10 KeV field? Let’s assume that the accelerating voltage won’t be affected by the velocity of the electron. Formula in page 15, 10.95 pm X- mevJ2 Why is neutron difficult to be focused? They are not charged, difficult to be manipulated by lens. What image you can get under TEM for the gold nanoparticle as below? Think about the feature of TEM image in page 23, TEM image is 2D projection. inner firuct of sample RHS look symmetrical LHS : = same , I no. Of views we need to Which type of molecule is easier to get a high-resolution structure under EM? Asymmetrical, devive e trimeric (C3 symmetrical) or heptameric (C7 symmetrical). C7 is easier than C3, C3 is easier 3D Struct than asymmetrical. & ↓ no of particles reg to reconstruct · What is the advantage of cryoEM over X-ray crystallography and NMR? Page 32-37 3D Struc Why do you think cryo-EM can have better resolving power? Cryogenic temperature, advanced equipment, data processing software, computation power by GPU. Thanks