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Questions and Answers
What technique monitors the diffusion of fluorescent molecules after photobleaching?
What technique monitors the diffusion of fluorescent molecules after photobleaching?
Which fundamental requirement is necessary for Fluorescence Resonance Energy Transfer (FRET)?
Which fundamental requirement is necessary for Fluorescence Resonance Energy Transfer (FRET)?
Which microscopy technique is noted for achieving enhanced resolution while minimizing damage?
Which microscopy technique is noted for achieving enhanced resolution while minimizing damage?
Who developed the electron microscope in the 1930s?
Who developed the electron microscope in the 1930s?
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What type of imaging does standard Transmission Electron Microscopy (TEM) provide?
What type of imaging does standard Transmission Electron Microscopy (TEM) provide?
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Which person was the first to visualize a cell using an electron microscope?
Which person was the first to visualize a cell using an electron microscope?
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What is a primary feature of Volume Electron Microscopy (Volume EM)?
What is a primary feature of Volume Electron Microscopy (Volume EM)?
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What principle does FRET involve rather than the transfer of photons?
What principle does FRET involve rather than the transfer of photons?
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What technique allows for the visualization of 3-dimensional objects in the Transmission Electron microscope?
What technique allows for the visualization of 3-dimensional objects in the Transmission Electron microscope?
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Which microscopy technique combines live light microscopy with the high resolution of electron microscopy?
Which microscopy technique combines live light microscopy with the high resolution of electron microscopy?
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What effect does vitrification have on biological samples in electron microscopy?
What effect does vitrification have on biological samples in electron microscopy?
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What is the purpose of segmenting membranes and gold particles in a tomogram?
What is the purpose of segmenting membranes and gold particles in a tomogram?
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What role does Scanning Electron Microscopy play in 3D imaging?
What role does Scanning Electron Microscopy play in 3D imaging?
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Which process can be described as having both volumetric and structural analysis capabilities?
Which process can be described as having both volumetric and structural analysis capabilities?
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Which of the following is NOT a characteristic of Volume EM?
Which of the following is NOT a characteristic of Volume EM?
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What is the primary advantage of using Electron Tomography?
What is the primary advantage of using Electron Tomography?
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What was recognized with the Nobel Prize in Chemistry in 2008?
What was recognized with the Nobel Prize in Chemistry in 2008?
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Which of the following steps is performed first in the vEM workflow?
Which of the following steps is performed first in the vEM workflow?
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What is the primary purpose of using heavy metal salts during sample preparation?
What is the primary purpose of using heavy metal salts during sample preparation?
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What significant advancement in imaging technologies was recognized with a Nobel Prize in 2008?
What significant advancement in imaging technologies was recognized with a Nobel Prize in 2008?
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What is a consequence of the poor penetration of the electron beam in vEM?
What is a consequence of the poor penetration of the electron beam in vEM?
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What aspect does cryogenic electron microscopy (cryo-EM) significantly enhance?
What aspect does cryogenic electron microscopy (cryo-EM) significantly enhance?
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Which component is NOT part of the general workflow for visualization electron microscopy (vEM)?
Which component is NOT part of the general workflow for visualization electron microscopy (vEM)?
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Which Nobel Prize was awarded for the development of super-resolution light microscopy technologies?
Which Nobel Prize was awarded for the development of super-resolution light microscopy technologies?
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What does the term 'resolution revolution' refer to in the context of cryogenic electron microscopy?
What does the term 'resolution revolution' refer to in the context of cryogenic electron microscopy?
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How are the samples encased during preparation for vEM?
How are the samples encased during preparation for vEM?
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What is the primary purpose of staining samples with heavy metal salts in vEM?
What is the primary purpose of staining samples with heavy metal salts in vEM?
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Which Nobel Prize was awarded for the development of super-resolution light microscopy technologies?
Which Nobel Prize was awarded for the development of super-resolution light microscopy technologies?
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What recent imaging methodology is mentioned as revealing the complexity of cells in three dimensions?
What recent imaging methodology is mentioned as revealing the complexity of cells in three dimensions?
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How is the sample prepared after chemical or cryogenic fixation in vEM?
How is the sample prepared after chemical or cryogenic fixation in vEM?
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What crucial step in the vEM workflow involves slicing the hardened block of sample?
What crucial step in the vEM workflow involves slicing the hardened block of sample?
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Which of the following scale complexities has vEM been applied to study?
Which of the following scale complexities has vEM been applied to study?
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What cell type was treated with LA in the experiment?
What cell type was treated with LA in the experiment?
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What does volume electron microscopy (vEM) primarily reveal?
What does volume electron microscopy (vEM) primarily reveal?
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What effect did LA treatment have on particle shape?
What effect did LA treatment have on particle shape?
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What has driven the development of volume electron microscopy (vEM)?
What has driven the development of volume electron microscopy (vEM)?
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What is indicated by the particle eccentricity graph in relation to LA concentration?
What is indicated by the particle eccentricity graph in relation to LA concentration?
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Which of the following organisms has NOT been mentioned as a model for delivering connectomes using vEM?
Which of the following organisms has NOT been mentioned as a model for delivering connectomes using vEM?
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What maximum diameter (nm) was shown in the results?
What maximum diameter (nm) was shown in the results?
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Which of the following best describes Volume EM as mentioned in the document?
Which of the following best describes Volume EM as mentioned in the document?
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What scale does volume electron microscopy (vEM) effectively analyze?
What scale does volume electron microscopy (vEM) effectively analyze?
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What has vEM technology revealed about biological systems?
What has vEM technology revealed about biological systems?
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What does LA stand for in the context of this study?
What does LA stand for in the context of this study?
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In the context of the experiment, what does the term 'untreated' relate to?
In the context of the experiment, what does the term 'untreated' relate to?
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What community effort is contributing to the vEM’s success in the life sciences?
What community effort is contributing to the vEM’s success in the life sciences?
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Which of the following represents a significant factor observed in particle shape post LA treatment?
Which of the following represents a significant factor observed in particle shape post LA treatment?
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What kind of structures does vEM target for visualization?
What kind of structures does vEM target for visualization?
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What aspect of tissue and cell study is highlighted in the context of vEM?
What aspect of tissue and cell study is highlighted in the context of vEM?
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Study Notes
Advanced Molecular Cell Biology
- Advanced imaging techniques are crucial for life science research.
- Topics include Advanced Light Microscopy and Advanced Electron Microscopy.
Learning Objectives
- Understand microscopy resolution, magnification, and detection differences.
- Explain fluorescence microscopy principles and sample labeling.
- Describe how to image samples in 2D and 3D, gaining quantitative data on dynamics using various microscopy techniques.
- Understand super-resolution and light sheet microscopy principles.
- Understand scanning electron microscopy (SEM) and transmission electron microscopy (TEM).
- Explain the benefits of correlative light electron microscopy (CLEM).
- Explain 3-dimensional electron microscopy techniques.
- Appreciate how these techniques apply to other topics in the course.
- Cover basic principles of light microscopy, fluorescence light microscopy, Green Fluorescent Protein (GFP), live cell imaging, and electron microscopy.
- Understand differences between scanning and transmission electron microscopy.
Life science research
- Life science research heavily relies on imaging.
Advanced Light Microscopy
- Topics include super-resolution light microscopy, light sheet microscopy, and lattice light sheet microscopy.
Introduction
- Microscopy scales are presented, ranging from the eye to smaller units like nanometers.
- Light microscopes and electron microscopes are introduced
- Microscopes include phase contrast and fluorescence, and transmission electron microscopy.
What do you notice?
- VSVG tsc45-GFP + tubulin-rhodamin is a sample used for observation and analysis.
- A problem like "hilling fluorophores" and "cell over-illumination" in light sheet microscopy is addressed
Light Sheet Microscopy
- Imaging in 3D with reduced photodamage by using a sheet of light instead of a point.
- Long-term imaging is possible with less photodamage.
- Data analysis and image rendering are important aspects.
Less light = less damage
- Confocal vs. light sheet microscopy is discussed, focusing on the reduction in photodamage using light sheets for long-term imaging.
Imaging Zebrafish Development
- Imaging of zebrasish development using live data provides raw data for analysis.
- Important to quantitatively process this data to extract meaningful information.
20 nm microtubule
- Image lacking microtubules is noted.
Resolution
- Resolution is the ability to separate two objects optically.
- Unresolved, partially resolved, and resolved images are illustrated.
- Resolution depends on wavelength and numerical aperture.
Light Microscopy
- Selective illumination (TIRF) is used to achieve higher resolution, exceeding the diffraction limit.
- Electron microscopy is used as a contrast technique.
Total Internal Reflection Fluorescence (TIRF) Microscopy
- Light comes in at a specific angle to provide highly localized illumination in the evanescent field.
- Vesicles and microtubules are within the field and are visualized.
Super-Resolution Light Microscopy
- The Nobel Prize in Chemistry 2014 was awarded to scientists for super-resolution fluorescence microscopy.
- Methods like STED, PALM/STORM allow finer details with images of cell structures.
Super Resolution Light Micoscopy
- STED, a stimulated emission depletion technique, allows for single molecule imaging.
- PALM/STORM use single molecule fluorescence and stochastic optical reconstruction for high resolution visualization.
PALM
- Statistical solutions predict the origin of a point light in an image.
- The signal to noise ratio needs to be exceptionally high.
- The number of fluorophores needs to be reduced.
Photoactivation of single fluorophores
- Single fluorophores are activated through a process that can be described as photoactivation.
Super Resolution Light Microscopy
- Photoactivation Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) are described.
Light Microscopy - Modes and Properties
- Table summarizes different microscopy technologies, their resolutions, illumination techniques, probes, acquisition/processing times, data size, and possible limitations.
Light Sheet Microscopy
- Latest developments in light sheet microscopy, like lattice light sheet, are enabling high-resolution and high-speed live imaging.
Protein Dynamics
- Tools to monitor protein and cell dynamics include dynamic activation and selective photobleaching.
- "Normal" GFP is easily photobleached, contrasted with engineered forms for switching properties.
Photoactivation
- Expressing photoactivatable probes, specifically engineered GFP variants, allows for selectively activating proteins for dynamic imaging.
Fluorescence Recovery After Photobleaching (FRAP)
- FRAP uses photobleaching to quantitatively measure protein diffusion and dynamics.
Protein Dynamics
- FRET (Fluorescence Resonance Energy Transfer) measures protein interactions using spectral overlap and dipole coupling.
Summary: Super-resolution and Dynamics
- The resolution barrier in light microscopy is overcome using different methods.
- Emerging methods combine enhanced resolution with reduced photodamage.
- Protein dynamics are readily studied by switching fluorophores.
Advanced Electron Microscopy
- Topics in advanced electron microscopy include volume electron microscopy and correlative microscopy.
Electron Microscopy
- Wavelength of useful radiation for electron microscopy is presented
- Ernst Ruska developed the electron microscope and received the Nobel prize.
- Keith Porter visualized a cell using an electron microscope.
Cell Organelles
- Cell organelles are illustrated, covering structures like microtubules, centrosomes, chromatin, nuclear pores, nuclear envelope, vesicles, lysosomes, actin filaments, peroxisomes, ribosomes, Golgi apparatus, intermediate filaments, plasma membrane, nucleolus, nucleus, endoplasmic reticulum, and mitochondria.
3D EM
- Standard transmission electron microscopy (TEM) produces 2D images
- Volume electron microscopy (vEM) is used to achieve 3D imaging of cells and tissues.
Correlative Light Electron Microscopy (CLEM)
- Combining the strengths of light and electron microscopy to visualize fluorescent labels and other components of structures and processes.
Simple CLEM
- Methods for identifying and mapping cells for correlative light electron microscopy studies are described.
Volume CLEM
- In-vivo imaging techniques are discussed, highlighting the challenge of locating specific structures within a sample.
Volume CLEM - Making NIRB Marks and Visualisation.
- Methods for creating marks with NIR light for subsequent electron microscopy localization are explained.
Volume CLEM - Post-brand information
- Visualising labeled cells post-marking using various microscopy information is described.
Structural EM: Cryo TEM
- Cryo-electron microscopy, especially cryo-TEM, enables single-particle analysis and in-situ structural biology studies.
Electron Microscopy (Summary)
- Volume EM captures the 3D ultrastructure of samples.
- Electron Tomography visualizes 3D objects.
- Scanning electron microscopy can image 3D structures in large samples.
- Correlative Light Electron Microscopy (CLEM) combines the advantages of light and electron microscopy.
Workshop Task
- SARS-CoV-2 microscopy images are frequently used in news reports.
- Tasks involve identifying a specific image, determining the microscopy techniques used, and suggesting alternative visualization methods.
Advanced Cell Biology - Dynamic Cell Biology
- Topic ACB1 and workshop focused on Cellular Imaging.
The Plan for the Workshop
- The meeting plan encompasses various elements, including attendance verification, the workshop itself, interactive sessions using a tool known as Mentimeter, volume electron microscopy (vEM) demonstrations, question-and-answer (Q&A) sessions, and interactive tools like Padlet.
Advanced Cell Biology-Cellular Imaging Workshop
- Information about the coronavirus SARS-CoV-2 is presented, along with tasks requiring identifying microscopy images used during news reports, discussing microscopy techniques, and generating additional visualization options.
Advanced Cell Biology - Cell Biology Imaging
- Instructions and a QR code for accessing an interactive session using menti.com are provided.
Advanced Cellular Biology
- Information about the coronavirus SARS-CoV-2's free fatty acid binding pocket in its spike protein structure is presented.
Advanced Cell Biology (GFP-SARS-CoV-2)
- Time-lapse imaging of GFP-SARS-CoV-2 in cells (Caco-2 and ACE2) is illustrated, examining the relationship between the virus and host cells over time.
Advanced Cell Biology - Images of CoV-2 and Cells
- Transmission electron microscopy images (TEM) are presented, showcasing the virus in infected cells.
- Quantitative data analyses, like particle counts, area measurements, and eccentricity, are presented to explain results and provide data.
Volume EM
- Electron microscopy for visualizing the complete 3D structure of biological samples using electron tomography and other methods is discussed.
- The challenge associated with 3D biological samples and the methods used to address those issues are covered.
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Description
Test your knowledge on advanced microscopy techniques including FRET, TEM, and electron microscopy. This quiz covers fundamental principles, historical developments, and imaging requirements in the field of microscopy. Challenge yourself to see how well you understand these critical imaging methods.
