Microscopy Techniques and History

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Questions and Answers

Which of the following scientists is known for their significant contributions to early microscopy, particularly in the discovery of bacteria?

Robert Hooke

Antony van Leeuwenhoek (correct)

Michael Faraday

Louis Pasteur

What is the formula for calculating the numerical aperture (NA) of a microscope objective?

NA = n / α

NA = n cos(α)

NA = n sin(α) (correct)

NA = n tan(α)

In fluorescence microscopy, what effect describes the difference in wavelength between the absorbed light and the emitted light?

Stokes shift (correct)

Doppler effect

Raman shift

Rayleigh scattering

Which component of a light microscope is responsible for adjusting the light before it reaches the specimen?

Condenser Signup and view all the answers

Which of the following is NOT a characteristic of live-cell fluorescence microscopy?

Uses fixed cells to minimize movement Signup and view all the answers

What does the Jablonski diagram primarily illustrate in the context of fluorescence?

Energy levels and transitions Signup and view all the answers

Which of the following is a key feature of 3D electron microscopy?

It allows the visualization of internal structures in three dimensions. Signup and view all the answers

Which of the following fluorescent dyes is commonly used to stain DNA?

DAPI (4',6-diamidino-2-phenylindole) Signup and view all the answers

What is the advantage of Light-Sheet Fluorescence Microscopy compared to conventional microscopy methods?

Reduced photo damage Signup and view all the answers

Which microscopy technique utilizes a combination of stimulated emission and depletion to achieve super-resolution?

STimulated Emission Depletion (STED) Signup and view all the answers

What is the Abbe diffraction limit formula for resolution?

D = λ/2NA Signup and view all the answers

What is the resolution achievable with Photoactivated Localization Microscopy (PALM)?

30nm Signup and view all the answers

In which microscopy technique is the numerical aperture (NA) particularly crucial for enhancing resolution?

Super Resolution Microscopy Signup and view all the answers

Which of the following aspects makes confocal microscopy advantageous over traditional fluorescence microscopy?

Reduction of out-of-focus light Signup and view all the answers

What is the primary downfall of conventional fluorescence microscopy compared to confocal methods?

Difficulty in 3D imaging Signup and view all the answers

Which microscopy method utilizes a z-stack of individual 2D sections for 3D imaging?

Confocal Laser Scanning Microscopy Signup and view all the answers

What does the numerical aperture (NA) primarily depend on?

The medium refractive index and aperture angle Signup and view all the answers

What is the formula for calculating the radius of the Airy disk?

rAiry = 0.61λ/NA Signup and view all the answers

According to the Rayleigh criterion, when are two adjacent object points considered resolved?

When the central diffraction spot of one coincides with the first minimum of the other Signup and view all the answers

What outcome occurs when utilizing a higher numerical aperture?

Improved point-spread function (PSF) Signup and view all the answers

In fluorescence microscopy, what is one method used to achieve better PSF in the axial direction?

Implementing confocal and light-sheet microscopy Signup and view all the answers

What is the contribution of the wavelength ($ ext{λ}$) in the resolution formula (Abbe's resolution formula)?

Directly impacts the minimum resolvable distance Signup and view all the answers

What happens when the numerical aperture is increased alongside a shorter wavelength?

Resolution improves alongside PSF Signup and view all the answers

Which factor does NOT contribute to the point-spread function improvement?

Longer working distance Signup and view all the answers

Study Notes

Electron Microscopy

Types of Electron Microscopes:
- Raster Electron Microscope (REM, SEM)
- Transmission Electron Microscope (TEM)
  - Scanning Transmission-EM (STEM)

History of Microscopy

Antony van Leeuwenhoek (1632-1723):
- Member of the Royal Society (1680)
- Observed Infusoria (1674)
- Observed Bacteria (1676)
- Observed Sperm cells (1677)
- Observed Striated muscle (1682)

Development of Cell Theory

Theodor Schwann (1838):
- German zoologist
- Described animal cells with a nucleus

Microscopy Parts

Microscope:
- Light source
- Condenser
- Specimen holder
- Objective lens
- Ocular

Microscopy Methods and Concepts

Light Transmitting Through the Sample: Brightfield:
- Shows images of Adrenal gland and leaf section
Fluorescence Microscopy:
- Uses fluorescent dyes for sensitive and specific labeling
- Includes Jablonski diagrams and fluorescence spectra
Fluoreszenzmikroskopie (Weitfeld) mit dichroitischen Filtern:
- Uses excitation filter, dichroic mirror, emission filter, light source, prism and camera
Fluorescent Molecules:
- Includes Chlorophyll, GFP, DAPI, Fluorescein, Eosin, Rhodamin B
Immunohistochemistrie mit fluoreszierenden Antikörpern:
- Uses primary and secondary antibodies for labeling
- Includes spectral properties of DyLight fluorescent dyes
Multi-labelling:
- Shows excitation and emission spectra of fluorescent dyes
Live-Cell Fluorescence Microscopy:
- Equipment is shown and labelled
Light-wave Interference:
- Light waves interfering and causing optical diffraction effects
- High Magnification, edge as lines and point of light as concentric patterns
Spatial Resolution:
- Numerical aperture (NA)
- Radius Airy disk
Abbe's Resolution Formula:
- nx,y = λ/2NA
- n = refractive index of medium, 0 = aperture angle
Point-Spread Function (PSF) Measurement: Used 175nm beads
PSF Improvement: Higher numerical aperture, shorter wavelengths, higher refractive index
Confocal Microscopy:
- Uses pinholes to eliminate out-of-focus light
Laser Scanning Confocal Microscopy:
- Shows the components of equipment and diagrams
3D Rendering, Reconstruction, and Analysis:
- Used to analyze data
Light-Sheet Fluorescence Microscopy: Diagram demonstrating the technique
Photo-Damage Comparison: Diagram contrasting light-sheet and epifluorescent illumination
3D-EM for High Resolution: Projection of object in EM where 3D information is reduced to 2D and back projection with computer for 3D structure from projections
Cryo-electron tomography (CET): A method for acquiring tilt-series, aligning, determining CTF, and reconstructing a 3D image.
Cryo-electron microscopy(Cryo-EM): Explains the techniques involved in cryo-EM and single-particle analysis.
Vitrification: Describes how samples are frozen for EM imaging.
Holey carbon film: Details the process of creating a suitable layer for sample embedding.
Image Formation: Describes the theory behind image generation in microscopy.
Collecting low-dose data: Describes the radiation dose range required for proteins
Contrast and Image Formation: Explains how contrast limits resolution in biological specimens
EM Development advancements: Shows how FEGs and direct detector cameras reduce the resolution limit
Field Emission Guns(FEG): Details of FEGs showing specifications
Falcon II direct detector: Shows diagram detailing device design and signal degradation prevention.
Fast CMOS DDsensor: Describes the methods behind fast CMOS direct detection sensors in microscopy
Alignment.: Shows results/comparison of Falcon DQE, Spatial freq (cycles/mm)
Advanced Electron Microscopy Setup (e.g., Titan Krios): Description of the electron microscope setup
Automatic Data Acquisition: Diagrams showing the process involved
Particle Picking: Method for extracting particles from images for analysis and creating 2D classes
Projection Matching: Method for refining/reconstructing 3D images from projections
Single-particle cryo-electron microscopy (e.g. ribosome): Explains process for analyzing and reconstructing the 3D macromolecular complex, including modeling interpretation, refinement, and 3D classification steps.
Ribosome: Showing Atomic resolution images, indicating the use of ribosome for study
Amino Acid Structures: Displays different amino acids with diagrams
2020: Real atomic resolution of Apoferritin: Details advancements in technology enabling this resolution
Subtomogram Averaging: Combining cryo-electron tomography (CET) and single-particle analysis (SPA).
Detailed analysis of HIV-1 CA-SP1 lattice: Shows 3.9 A resolution structure.
Correlative light and electron microscopy: Combining light and electron microscopy.
Photo Conversion: Technique for converting fluorescent dyes into electron-dense markers
Photo conversion in vesicular membranes within neuromuscular junction: Shows the process for this special case
3D-EM for High Resolution: Projection of object in EM where 3D information is reduced to 2D and back projection with computer for 3D structure from projections

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Description

Explore the fascinating world of microscopy, from the various types of electron microscopes including SEM and TEM, to the historical contributions of figures like Antony van Leeuwenhoek and Theodor Schwann. This quiz will cover essential components, methods, and concepts in microscopy, as well as the development of cell theory.