Advanced Molecular Cell Biology (AMCB) PDF

Advanced Molecular Cell Biology u Advanced imaging techniques Resour for life science research Topics: T one Advanced Light Microscopy Advanced Electron Microscopy Paul Verkade School of Biochemistry ...

Advanced Molecular Cell Biology u Advanced imaging techniques Resour for life science research Topics: T one Advanced Light Microscopy Advanced Electron Microscopy Paul Verkade School of Biochemistry  Learning objectives Intended learning outcomes cell imaging lectures To understand resolution in terms of microscopy and the difference between resolution, magnification, and detection. To be able to explain the principles of fluorescence microscopy and sample labelling. To be able to explain, using examples, how we can image samples in 2D and 3D and gain quantitative information relating to dynamics. To understand and explain the principles of super-resolution microscopy and light sheet microscopy. To understand and explain the principles of Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) To understand and explain the advantages of Correlative Light Electron Microscopy (CLEM) To understand and explain the principles of 3-dimensional Electron Microscopy techniques To appreciate how the techniques learned in these lectures could be applied to other topics across the course.  Learning objectives Required background knowledge (all covered in earlier years and A level) Basic principles of light microscopy Fluorescence light microscopy Green fluorescent protein (GFP) Basic principles of live cell imaging Basic principles of Electron Microscopy Difference between Scanning and Transmission Electron Microscopy For reference see the video sections on Blackboard Life science research is based on Imaging  Advanced Molecular Cell Biology Advanced Light Microscopy Topics: - Super resolution light microscopy - Light sheet microscopy - Lattice Light sheet microscopy Paul Verkade School of Biochemistry Introduction  What do you notice? Problem : killing gluorophores · & cell over time illuminate from other side-phoodamage Soluhan : light sheet microscopy VSVG tso45-GFP + tubulin-rhodamin  Light Sheet Microscopy Imaging in 3D with less photodamage Sheet plane imaging scanning using a sheet of light instead of a point Long term imaging with reduced photodamage - Great example of data analysis / image rendering Keller et al., (2008) Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322, no. 5904, pp. 1065-1069  Light Sheet Microscopy Less light = less damage Confocal Light sheet Less photodamage is much better for long term imaging – now experimental model organisms can be imaged over days of development. Light Sheet Microscopy Imaging Zebrafish Development – raw data. "gentle imaging" Key issue is how to process these image data sets to produce quantitative & meaningful data  What do you notice? 20nm microtubule - Image is lacing microtubules VSVG tso45-GFP + tubulin-rhodamin  Resolution Resolution (not magnification!) is the ability to separate two objects optically Unresolved di as Partially resolved X : wavelengh In sinx-now goodis miroscope ~ 1 4 : Resolved Resolution Unresolved Partially resolved Resolved  Resolution Unresolved Partially resolved Whether you can see an object does not depend on its size but its brightness Resolved Light Microscopy Higher resolution? Selective illumination – TIRF Beyond the diffraction limit Electron microscopy  Light Microscopy Total Internal Reflection Fluorescence (TIRF) Microscopy very slight angle in light comes a & > illuminates 100m only Stephens and Allan (2003) Science, 300, 82-86  Light Microscopy Total Internal Reflection Fluorescence (TIRF) Microscopy Fusion of VSV-G with the plasma membrane. D. Toomre Super Resolution Light Micoscopy  Super Resolution Light Micoscopy Structured illumination STED PALM/SFORM © 2010 Schermelleh et al. Schermelleh L et al. J Cell Biol 2010;190:165-175  Super Resolution Light Micoscopy STimulated Emission Depletion (STED) Hell  STED + - - https://www.technologynetworks.com/neuroscience/articles/what-is-super-resolution- microscopy-sted-sim-and-storm-explained-328572 Yamanaka M et al. Microscopy (Tokyo) 2014;63:177-192  STED Drawback ↳ not continuous · so not complete Neurofilaments in human neuroblastoma observed by (a) confocal and (b) STED microscopy. Yamanaka M et al. Microscopy (Tokyo) 2014;63:177-192 © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: [email protected]  Super Resolution Light Micoscopy PhotoActivation Localization Microscopy (PALM) 3 S Stochastic Optical Reconstruction Microscopy (STORM) Moerner / Betzig - activation Single moleucle localisation +  PALM Statistical solutions – predicting the origin of a point of light. & localisation Fit the data to a model of a 2D Gaussian very precise Predicts centre of the fluorophore Requires exceptional signal-to-noise ratio a) reduce noise b) reduce number of fluorophores visible at any one time  PALM Photo- Activation of single fluorophores  PALM Point Spread Function Activation of single fluorophores  Super Resolution Light Micoscopy https://www.youtube.com/watch?v=uGoae-weHLw  Super Resolution Light Micoscopy From: Fluorescence Microscopy: Super-Resolution and Other Novel Techniques, 2014, Pages 199-212  Light Sheet Microscopy Newest developments – lattice light sheet Live imaging of actin comparing “SIM” and “Lattice Light Sheet”. Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution. Chen et al (2014) Science, DOI: 10.1126/science.1257998  Protein Dynamics Tools to monitor protein and cell dynamics dynamic activation selective photobleaching “Normal” GFP can be easily photobleached by high power lasers Engineered forms can be switched on and off or from green to red.  Protein Dynamics Photoactivation (also photoconversion) Express photoactivatable probe in cells Uses ENGINEERED Photoactivatable form of GFP Selectively photoactivate (e.g. here: the nucleus) Start imaging dynamics of photoactivated protein Monitor dynamics – e.g. here is nuclear export Fluorescence Fluorescence Inside nucleus Outside nucleus time time See Figure 9-30 Molecular Biology of the Cell (© Garland Science 2008)  Protein Dynamics Fluorescence Recovery After Photobleaching (FRAP) Cell labelled with ER marker fused to GFP. Photobleaching with high power laser. Monitor diffusion of the remaining fluorescent molecules back into that bleached area. Quantitative method. See Figure 9-31 Molecular Biology of the Cell (© Garland Science 2008)  Protein Dynamics Tools to monitor protein interactions and signalling: FRET FRET requires: spectral overlap dipole coupling FRET is not about transfer of photons it is about transfer of energy = dipole coupling FRET: Fluorescence Resonance Energy Transfer Dynamic Proteome, Mike Jones  Super Res and Dynamics summary: The resolution barrier in light microscopy has been broken in multiple ways..You can win a Nobel prize from your living room! Latest methods such as Lattice Light Sheet microscopy combine enhanced resolution with less damage By switching fluorophores on or off the dynamics of proteins can be studied  Advanced Molecular Cell Biology Advanced Electron Microscopy Topics: - Volume electron microscopy - Correlative microscopy Paul Verkade School of Biochemistry Electron Microscopy  Electron Microscopy Ernst Ruska (1906-1988) – Developed the electron microscope in 1930s – Received Nobel prize Physics in 1986  Electron Microscopy Keith Porter (1912-1997) – First person to visualise a cell in the electron microscope in 1945 – Awarded Nobel Prize in 1974  Cell Organelles Molecular Biology of the Cell, 4th edition 3D EM Standard TEM provides 2D images  3D EM Volume EM: The Quiet Revolution Comment What is volume EM? ! https://doi.org/10.1038/s41592-023-01861-8 Volume EM: a quiet revolution takes shape collection of techniques Lucy M. Collinson, Carles Bosch, Anwen Bullen, Jemima J. Burden, Raffaella Carzaniga, Cheng Cheng, Michele C. Darrow, Georgina Fletcher, Errin Johnson, Kedar Narayan, Christopher J. Peddie, Martyn Winn, Charles Wood, Ardan Patwardhan, Gerard J. Kleywegt & Paul Verkade Check for updates Volume electron microscopy (vEM) is a group of organelles within cells, to the communities of cells that make up tissues techniques that reveal the 3D ultrastructure of and the architecture of tissues that make up organisms. This makes vEM a critical tool for understanding biological complexity across scales. cells and tissues through continuous depths of Indeed, the development of vEM was originally driven by the quest to at least 1 micrometer. A burgeoning grassroots understand the connections in the brain, from individual vesicles that release neurotransmitters at synapses to entire neurons that make community effort is fast building the profile connections across different brain regions. Since the 1980s, vEM has and revealing the impact of vEM technology in delivered connectomes from model organisms including Caenorhab- the life sciences and clinical research. ditis elegans2, Drosophila melanogaster3–5 and Danio rerio6. However, complexity across scales is present in every organism and vEM is now being used throughout the life sciences, revealing the structural com- The awarding of three Nobel Prizes for imaging technologies between plexity of fertilization7 (Fig. 2), blood vessels8,9, muscles10, sensory 2008 and 2017 has highlighted the key importance of imaging in organs11,12, tumors13,14, pathogen-infected cells and tissues15,16, plants17,18 present-day life science research. The expression of proteins tagged and marine organisms19,20, to name but a few. with green fluorescent protein in living cells and organisms trans- formed the way in which life science research was conducted. The vEM workflows ability to visualize the dynamic nature of proteins in cells and tissues There are three main components that are common to all vEM work- was recognized with the Nobel Prize in Chemistry in 2008. This was fol- flows: (1) sample preparation, (2) imaging and (3) data. lowed by another Nobel Prize in Chemistry in 2014 for the development In the first step, the sample is prepared by chemical or cryogenic of super-resolution light microscopy technologies, in which Abbe’s fixation, followed by staining using heavy metal salts of osmium, lead resolution limit was finally broken to allow localization of fluorescently and uranium to add electron contrast to the membranes and make tagged molecules with a precision of tens of nanometers. Most recently, the sample more conductive. The sample is then dehydrated using the ‘resolution revolution’ in cryogenic electron microscopy (cryo-EM) a solvent, infiltrated with a liquid resin and the resin is polymerized has enabled the determination of the molecular structure of isolated using heat or UV light. This encases the cells and tissues like a mosquito Collinson, L.M., Bosch, C., Bullen, A. et proteins and protein complexes, and was recognized with the 2017 in amber, resulting in a hard block that can be sliced using a diamond Nobel Prize in Chemistry. There is now another imaging revolution knife or an ion beam. Slicing is an essential part of the vEM workflow al. Volume EM: a quiet revolution takes underway that reveals the exquisite complexity of cells and tissues because of the poor penetration of the electron beam into the samples. shape.Nat Methods 20, 777–782 (2023). at the membrane and organelle scales in three dimensions — volume electron microscopy (vEM), which was recently highlighted as one of Slicing or sectioning may be performed manually on an ultramicrotome using a diamond knife, and the ultrathin sections collected onto metal https://doi.org/10.1038/s41592-023- the ‘seven technologies to watch in 2023’ by Nature. grids, tape or wafers for imaging. Alternatively, the sections may be removed and discarded, and the block surface imaged after each cut. 01861-8 Volume electron microscopy In both cases, the result is a set of sequential images that represent the vEM is a group of techniques that are used to image the structure of volume of the original sample. cells and tissues through continuous depths of at least 1 micrometer at In the second step, vEM imaging is performed using TEM or SEM  volume EM connections in using brain to determine neuron the.  Electron Tomography hilted in sample the microscope images -140 shows computational software thewater all  SBF-SEM scan surface of sample. block raised + top sliced off again scan repeat · data of high a lot res · but 10 much data Serial Block Face - SEM  Volume EM Membranes and gold particles within a specific region of the tomogram are manually outlined and visualised as 3-dimensional model wo usebrain · modeled afterwards (see rid)  Cool technology right! But let’s take it a step further Correlative Light Electron Microscopy Imaging revolution by use of Green Fluorescent Protein Only fluorescent labels are visualised! Correlative Light Electron Microscopy We combine the strengths of (live) light microscopy with the high resolution of electron microscopy  “simple” CLEM ESCRT-III encircles forming nuclei during telophase ESCRT-III components, CHMP2a and 2b localise around the decondensing nuclei during telophase. Localisation is transient, lasting ~ 96 ± 8.9 you'dhavea 100030... seconds with a cell cycle length of 22hours = 0.1%  DAP1 - nucleus stain “simple” CLEM how exactly where 1. Identify cell in telophase with CHMP2a staining -we want to DAPI CHMP2a-FNG Merge 2. Map position of cell on finder grid dish ↑ Id localises To  Membranes and gold particles within a specific region of the tomogram are manually outlined and visualised as 3-dimensional model every time b old eliee ↳ apart from tiny gaps zegt fixed Olmos et al., 2015, Nature  Volume CLEM The needle in the In vivo imaging haystack! Rob Lees Transgenic targeted Transgenic targeted fluorescence fluorescence (z-stack) Filled cell with fluorescent dye (z-stack) (max projection)  Volume CLEM Making NIRB Marks 910 nm Ti-Saph laser light at high power with a high numerical aperture and magnification Autofluorescent marks ↳ can not like jameand NOTON identify around # to sample sample. , - Visualise marks in brightfield and widefield fluorescence at low mag. Must test settings in tissue first interest. Like showing of an arrow newon  Volume CLEM Volume Information of NIRB ↳ Near infrared branding marks Post-brand filled cell (max projection) Post-brand filled cell (max projection)  Volume CLEM Membranes and gold particles within a specific region of the tomogram are manually outlined and visualised as 3-dimensional model Lees, R. M., Peddie, C. J., Collinson, L. M., Ashby, M. C., & Verkade, P. (2017). Methods in Cell Biology Correlative Light and Electron Microscopy III (140, pp. 245–276).  Volume CLEM Max projection from fluorescence microscopy Segmentation from SBFSEM Pre-NIRB Post-NIRB Lees, R. M., Peddie, C. J., Collinson, L. M., Ashby, M. C., & Verkade, P. (2017). Methods in Cell Biology Correlative Light and Electron Microscopy III (140, pp. 245–276). Structural EM: Cryo TEM  Structural EM: Cryo TEM Spoiler alert! (sharp Lecture) ↳ Single particle analysis and in situ structural Biology making it vitrification = glass-like.  Electron Microscopy summary: Volume EM captures the 3-dimensional ultrastructure of samples. Electron Tomography allows for the visualisation of 3- dimensional objects in the Transmission Electron microscope Sectioning inside a Scanning Electron Microscope can provide 3D of large samples Correlative Light Electron Microscopy combines the advantages of Live Light Microscopy and High Resolution of Electron Microscopy the most time C Y need Do i really very complicate  Workshop task The coronavirus SARS-CoV-2 is extensively covered on the news using a lot of microscopy images. When it is covered on the BBC news there is always a specific image in the background. Task 1: Find the image and show it during the workshop Task 2: What technology do you think they have used for this image? Task 3: What other ways can you think of to visualise the virus?  Advanced Cell Biology Dynamic Cell Biology Topic ACB1 Cellular Imaging Workshop Paul Verkade School of Biochemistry  The plan – Attendance – Workshop Mentimeter – Volume EM – Q&A Padlet School of Biochemistry  Advanced Cell Biology Topic ACB1 Cellular Imaging Workshop The coronavirus SARS-CoV-2 is still extensively covered on the news using a lot of microscopy images. When it was covered on the BBC news there were always a few specific images in the background. Attached are a couple of screenshots Task 1: Using the lectures and doing some research: What technology do you think they have used for these images? Task 2: What other ways can you think of to visualise the virus? School of Biochemistry  Advanced Cell Biology School of Biochemistry  Advanced Cell Biology School of Biochemistry  Advanced Cell Biology GFP-SARS-CoV-2 + 50 M LA GFP-SARS-CoV-2 24hpi 36hpi School of Biochemistry a Caco-2/ACE2 cells, infected with GFP-SARS-CoV-2 b Caco-2/ACE2 cells treated with 50 M LA and infected with GFP-SARS-CoV-2 School of Biochemistry  Advanced Cell Biology a GFP SARS-CoV-2/ Caco-2-ACE2 cells b GFP SARS-CoV-2/ Caco-2-ACE2 cells/ LA treated School of Biochemistry  Advanced Cell Biology Much less virus wit Lindeic acid. b GFP SARS-CoV-2/ Caco-2-ACE2 cells/ LA treated LA LA LA c Caco-2-ACE2 cells/ LA treated d School of Biochemistry LA  LA Advanced Cell Biology LA E2 cells/ LA treated d LA LA 1.0 **** **** 200 0.8 Particle eccentricity Max. diameter (nm) 150 0.6 100 0.4 50 0.2 200 nm 0 0.0 ↳ untreated 50 M LA untreated 50 M LA much rounder shape School of Biochemistry volume EM Comment What is volume EM? https://doi.org/10.1038/s41592-023-01861-8 Volume EM: a quiet revolution takes shape Lucy M. Collinson, Carles Bosch, Anwen Bullen, Jemima J. Burden, Raffaella Carzaniga, Cheng Cheng, Michele C. Darrow, Georgina Fletcher, Errin Johnson, Kedar Narayan, Christopher J. Peddie, Martyn Winn, Charles Wood, Ardan Patwardhan, Gerard J. Kleywegt & Paul Verkade Check for updates Volume electron microscopy (vEM) is a group of organelles within cells, to the communities of cells that make up tissues techniques that reveal the 3D ultrastructure of and the architecture of tissues that make up organisms. This makes vEM a critical tool for understanding biological complexity across scales. cells and tissues through continuous depths of Indeed, the development of vEM was originally driven by the quest to at least 1 micrometer. A burgeoning grassroots understand the connections in the brain, from individual vesicles that release neurotransmitters at synapses to entire neurons that make community effort is fast building the profile connections across different brain regions. Since the 1980s, vEM has and revealing the impact of vEM technology in delivered connectomes from model organisms including Caenorhab- the life sciences and clinical research. ditis elegans2, Drosophila melanogaster3–5 and Danio rerio6. However, complexity across scales is present in every organism and vEM is now being used throughout the life sciences, revealing the structural com- The awarding of three Nobel Prizes for imaging technologies between plexity of fertilization7 (Fig. 2), blood vessels8,9, muscles10, sensory 2008 and 2017 has highlighted the key importance of imaging in organs11,12, tumors13,14, pathogen-infected cells and tissues15,16, plants17,18 present-day life science research. The expression of proteins tagged and marine organisms19,20, to name but a few. with green fluorescent protein in living cells and organisms trans- formed the way in which life science research was conducted. The vEM workflows ability to visualize the dynamic nature of proteins in cells and tissues There are three main components that are common to all vEM work- was recognized with the Nobel Prize in Chemistry in 2008. This was fol- flows: (1) sample preparation, (2) imaging and (3) data. lowed by another Nobel Prize in Chemistry in 2014 for the development In the first step, the sample is prepared by chemical or cryogenic of super-resolution light microscopy technologies, in which Abbe’s fixation, followed by staining using heavy metal salts of osmium, lead resolution limit was finally broken to allow localization of fluorescently and uranium to add electron contrast to the membranes and make tagged molecules with a precision of tens of nanometers. Most recently, the sample more conductive. The sample is then dehydrated using the ‘resolution revolution’ in cryogenic electron microscopy (cryo-EM) a solvent, infiltrated with a liquid resin and the resin is polymerized has enabled the determination of the molecular structure of isolated using heat or UV light. This encases the cells and tissues like a mosquito Collinson, L.M., Bosch, C., Bullen, A. et proteins and protein complexes, and was recognized with the 2017 in amber, resulting in a hard block that can be sliced using a diamond Nobel Prize in Chemistry. There is now another imaging revolution knife or an ion beam. Slicing is an essential part of the vEM workflow al. Volume EM: a quiet revolution takes underway that reveals the exquisite complexity of cells and tissues because of the poor penetration of the electron beam into the samples. shape.Nat Methods 20, 777–782 (2023). at the membrane and organelle scales in three dimensions — volume electron microscopy (vEM), which was recently highlighted as one of Slicing or sectioning may be performed manually on an ultramicrotome using a diamond knife, and the ultrathin sections collected onto metal https://doi.org/10.1038/s41592-023- the ‘seven technologies to watch in 2023’ by Nature. grids, tape or wafers for imaging. Alternatively, the sections may be removed and discarded, and the block surface imaged after each cut. 01861-8 Volume electron microscopy In both cases, the result is a set of sequential images that represent the vEM is a group of techniques that are used to image the structure of volume of the original sample. cells and tissues through continuous depths of at least 1 micrometer at In the second step, vEM imaging is performed using TEM or SEM