Advanced Microscopy Techniques Quiz

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.

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.