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?

<p>Condenser (A)</p> Signup and view all the answers

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

<p>Uses fixed cells to minimize movement (B)</p> Signup and view all the answers

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

<p>Energy levels and transitions (D)</p> Signup and view all the answers

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

<p>It allows the visualization of internal structures in three dimensions. (C)</p> Signup and view all the answers

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

<p>DAPI (4',6-diamidino-2-phenylindole) (A)</p> Signup and view all the answers

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

<p>Reduced photo damage (C)</p> Signup and view all the answers

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

<p>STimulated Emission Depletion (STED) (B)</p> Signup and view all the answers

What is the Abbe diffraction limit formula for resolution?

<p>D = λ/2NA (C)</p> Signup and view all the answers

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

<p>30nm (A)</p> Signup and view all the answers

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

<p>Super Resolution Microscopy (D)</p> Signup and view all the answers

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

<p>Reduction of out-of-focus light (D)</p> Signup and view all the answers

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

<p>Difficulty in 3D imaging (C)</p> Signup and view all the answers

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

<p>Confocal Laser Scanning Microscopy (A)</p> Signup and view all the answers

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

<p>The medium refractive index and aperture angle (C)</p> Signup and view all the answers

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

<p>rAiry = 0.61λ/NA (B)</p> Signup and view all the answers

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

<p>When the central diffraction spot of one coincides with the first minimum of the other (D)</p> Signup and view all the answers

What outcome occurs when utilizing a higher numerical aperture?

<p>Improved point-spread function (PSF) (C)</p> Signup and view all the answers

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

<p>Implementing confocal and light-sheet microscopy (B)</p> Signup and view all the answers

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

<p>Directly impacts the minimum resolvable distance (C)</p> Signup and view all the answers

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

<p>Resolution improves alongside PSF (A)</p> Signup and view all the answers

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

<p>Longer working distance (D)</p> Signup and view all the answers

Flashcards

Numerical Aperture

The ability of a microscope lens to gather light and resolve fine detail. It's influenced by the refractive index of the surrounding medium and the lens's angular aperture.

Brightfield Microscopy

A type of microscopy where light passes through the specimen, creating an image based on light absorption and transmission.

Fluorescence Microscopy

A microscopy technique that utilizes fluorescent dyes to illuminate specific molecules or structures within cells.

Interference Microscopy

A microscopy technique that utilizes light waves' interference patterns to resolve details beyond the limits of traditional light microscopy.

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Immunofluorescence

A technique that uses fluorescent antibodies to label specific proteins or structures within cells, enabling visualization of cellular processes.

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Wide-field Fluorescence Microscopy

A type of microscopy where the specimen is illuminated with a specific wavelength of light, causing fluorescent molecules within the specimen to emit light at a longer wavelength.

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Live-Cell Fluorescence Microscopy

A type of microscopy used to observe live cells and their dynamics over time.

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Electron Microscopy

A microscopy technique that utilizes electrons instead of light to create magnified images of ultra-fine structures.

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Confocal Laser Scanning Microscopy

A type of microscopy that uses a focused laser beam to illuminate a sample, capturing only light emitted from the focal plane, resulting in improved resolution and 3D imaging.

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Z-Stack in Confocal Microscopy

A 3D image reconstruction technique using sequential 2D image slices captured via confocal microscopy, allowing visualization of intricate structures.

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Light-Sheet Fluorescence Microscopy

A microscopy technique that uses a sheet of light to illuminate a sample, reducing photodamage and allowing fast, high-resolution imaging.

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Super-Resolution Microscopy

A technique that overcomes Abbe's diffraction limit, improving microscopy resolution and allowing visualization of previously unresolvable details.

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STED (Stimulated Emission Depletion) Microscopy

A super-resolution technique using a doughnut-shaped laser beam to deplete fluorescence outside of the focal point, leading to higher resolution.

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PALM (Photoactivated Localization Microscopy)

A super-resolution technique that uses photoactivatable fluorescent molecules to pinpoint locations with high precision, allowing reconstruction of high-resolution images.

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Abbe Diffraction Limit

A technique that uses the numerical aperture (NA) of the objective lens and the wavelength of light to determine the smallest resolvable distance, known as the Abbe diffraction limit.

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Numerical Aperture (NA)

The ability of a lens to gather light and focus it, related to its angular aperture and the refractive index of the surrounding medium.

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Spatial Resolution

The ability of a microscope to distinguish between two closely spaced objects. It's determined by the wavelength of light used and the numerical aperture of the objective lens.

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Rayleigh Criterion

The smallest distance between two objects that can be distinguished as separate entities. It's defined by the point where the central diffraction spot of one object overlaps the first minimum of the other.

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Airy Disk

The central bright spot of light produced when light passes through a diffraction-limited aperture. Its size determines the resolution.

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Point-Spread Function (PSF)

The distribution of light intensity from a point source as captured by a microscope objective. It's a way to represent the resolution and image quality.

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Confocal Microscopy

A technique that uses a pinhole to block out-of-focus light, improving resolution and reducing blur in images.

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Light-Sheet Microscopy

A type of microscopy that uses a laser beam to illuminate a thin sheet of the sample, improving resolution and depth penetration.

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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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