Microscopy Techniques in Cell Biology
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What is one primary use of fluorescent microscopy with respect to nucleic acids?

  • To enhance the visibility of live cultured cells
  • To determine the optical density of cells
  • To visualize unstained tissues
  • To localize nucleic acids separately in cells (correct)

Which compound specifically stains DNA and emits blue fluorescence under UV light?

  • GFP
  • Fluorescein
  • Rhodamine
  • Hoechst (correct)

What advantage does phase-contrast microscopy provide for studying cell structures?

  • It requires cells to be dyed with fluorescent compounds
  • It enhances the optical densities of cellular parts
  • It allows observation of transparent, unstained cells (correct)
  • It can visualize stained cells with high detail

How are antibodies utilized in fluorescence microscopy?

<p>They are labeled with fluorescent compounds for staining (B)</p> Signup and view all the answers

What characteristic is true of living cultured cells in phase-contrast microscopy?

<p>They can be observed without the need for staining (B)</p> Signup and view all the answers

What is the primary basis for brightness in bright-field microscopy?

<p>Ordinary light (B)</p> Signup and view all the answers

Which optical component focuses light on the object being examined in bright-field microscopy?

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

How is total magnification calculated in a light microscope?

<p>Multiplying the powers of the objective and ocular lenses (A)</p> Signup and view all the answers

What is the maximal resolving power of a light microscope?

<p>0.2 µm (C)</p> Signup and view all the answers

What happens to two structures separated by less than 0.2 µm in a light microscope?

<p>They are seen as one object (A)</p> Signup and view all the answers

Which of the following cannot be distinctly seen with a light microscope?

<p>Single ribosome (B)</p> Signup and view all the answers

What is a significant factor required to obtain detailed images with a light microscope?

<p>Resolving power (B)</p> Signup and view all the answers

Which type of microscopy uses the interaction of light specifically to study tissue features?

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

What primarily determines the resolving power of a microscope?

<p>The quality of its objective lens (D)</p> Signup and view all the answers

What is the relationship between magnification and resolution in microscopy?

<p>Magnification is valuable only with high resolution (B)</p> Signup and view all the answers

What role does the eyepiece lens have in microscopy?

<p>It only enlarges the image without affecting resolution (D)</p> Signup and view all the answers

What is a significant advantage of virtual microscopy over traditional light microscopy?

<p>It allows for digital access and ease of use (C)</p> Signup and view all the answers

Which light is primarily used to excite tissues in fluorescence microscopy?

<p>Ultraviolet (UV) light (B)</p> Signup and view all the answers

How do fluorescent substances appear in fluorescence microscopy?

<p>Bright on a dark background (D)</p> Signup and view all the answers

What is a common example of a fluorescent stain that binds to nucleic acids?

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

What does virtual microscopy utilize to capture specimens?

<p>Digital slide-scanning microscope (B)</p> Signup and view all the answers

What principle does phase-contrast microscopy rely on?

<p>The change in light speed through different refractive indices (D)</p> Signup and view all the answers

How does differential interference contrast microscopy enhance the visualization of living cells?

<p>By providing a more apparent three-dimensional aspect (A)</p> Signup and view all the answers

What advantage does confocal microscopy have over traditional bright-field microscopy?

<p>It reduces stray light, enhancing contrast and resolving power (D)</p> Signup and view all the answers

What feature of confocal microscopy is critical for its high resolution?

<p>The alignment of a point light source, focal point, and detector's aperture (C)</p> Signup and view all the answers

Which microscopy technique is specifically noted for its use in cell culture without fixation or staining?

<p>Phase-contrast microscopy (C)</p> Signup and view all the answers

What is the primary advantage of confocal microscopy over bright-field microscopy?

<p>It improves resolution and localization precision. (B)</p> Signup and view all the answers

Which component is crucial for the confocal microscope's ability to rapidly move the point of illumination?

<p>The beam splitter (B)</p> Signup and view all the answers

What is the typical resolution limit of a transmission electron microscope (TEM)?

<p>3 nm (C)</p> Signup and view all the answers

How are the images created by a transmission electron microscope (TEM) primarily formed?

<p>By passing a beam of electrons through the tissue section (A)</p> Signup and view all the answers

What is the typical thickness of tissue sections studied by a transmission electron microscope?

<p>40-90 nm (C)</p> Signup and view all the answers

What is the primary effect of adding heavy metal ions to the fixative or dehydrating solutions used in Transmission Electron Microscopy (TEM)?

<p>It enhances contrast and visibility of macromolecules. (B)</p> Signup and view all the answers

Which of the following techniques allows for the study of membrane structures without the need for fixation or embedding?

<p>Cryofracture and freeze etching (C)</p> Signup and view all the answers

What occurs to tissue specimens during the cryofracture and freeze etching techniques?

<p>They are rapidly frozen and fractured. (D)</p> Signup and view all the answers

What material is commonly used to coat specimens in scanning electron microscopy to enhance electron reflection?

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

In scanning electron microscopy, how does the electron beam interact with the specimen?

<p>It reflects off the surface of the specimen. (C)</p> Signup and view all the answers

What characteristic do areas of an electron micrograph that appear darker represent?

<p>Absorption or deflection of electrons (B)</p> Signup and view all the answers

What is a significant advantage of cryofracture and freeze etching in studying cell membranes?

<p>It allows visualization of protein components and lipid bilayers. (A)</p> Signup and view all the answers

Which of the following is NOT a heavy metal compound typically used in TEM sample preparation?

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

Flashcards

Resolving Power

The ability to distinguish two closely spaced objects as separate entities.

Bright-Field Microscopy

A specialized microscope that uses visible light to illuminate and view stained specimens.

Objective Lens

The lens that magnifies the image of the specimen before it reaches the eye.

Eyepiece Lens

The lens that further magnifies the image from the objective lens, allowing it to be seen by the eye.

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Total Magnification

The total magnification achieved by a microscope is calculated by multiplying the magnifying power of the objective lens by that of the eyepiece lens.

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Resolving Power

The smallest distance between two structures that can be distinguished as separate objects under a microscope.

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0.2 µm

The limit to which light microscopes can resolve details.

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Unresolvable Structures

Structures smaller than 0.2 µm cannot be seen with a light microscope due to its resolution limits.

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Magnification

The process of enlarging an image. It's valuable only when combined with high resolution.

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Virtual Microscopy

A technique that involves converting stained tissue slides into digital images that can be viewed on a computer or other digital device.

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Fluorescence Microscopy

A technique that uses fluorescent dyes to visualize specific structures or molecules in cells and tissues. These dyes emit light when illuminated with UV light.

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Fluorescent Compounds

Substances that emit light when illuminated with specific wavelengths (usually UV light) in fluorescence microscopy.

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Acridine Orange

A fluorescent dye that binds to DNA and RNA, making it visible under fluorescence microscopy.

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DAPI and Hoechst Staining

A technique that uses specific dyes that bind to DNA and emit blue fluorescence under UV light, allowing visualization of cell nuclei.

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Fluorescent Antibodies

Antibodies that are linked to fluorescent compounds. They are essential tools in immunohistological staining, allowing researchers to identify and visualize specific proteins and antigens within tissues.

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Phase-Contrast Microscopy

A microscopy technique that enhances the contrast of transparent and unstained cells and tissues by converting differences in refractive index into visible changes in brightness. This allows visualization of cellular details without the need for staining.

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Live-cell Imaging

A microscopy approach that allows direct observation of living cells and tissues without the need for staining. This is achieved by using a lens system that converts differences in refractive index into visible changes in brightness.

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Differential Interference Contrast Microscopy

A variation of phase-contrast microscopy that uses Nomarski optics to create a more pronounced three-dimensional (3D) image of living cells.

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Confocal Microscopy

A microscopy technique that uses a focused laser beam to illuminate a specimen, scanning it point-by-point to create a high-resolution image with sharp focus.

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Point light source in Confocal Microscopy

In Confocal microscopy, a small, high-intensity light source, often a laser, is used to illuminate the specimen.

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Pinhole Aperture in Confocal Microscopy

In Confocal microscopy, a plate with a small hole (pinhole) is placed in front of the detector to block unfocused light, improving image clarity and resolution.

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What is confocal microscopy?

Confocal microscopy uses a computer-controlled mirror to scan a specimen with a laser beam, capturing images of very thin slices (optical sections). These sections are then digitally combined to create a 3D image.

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How does confocal microscopy improve resolution?

The advanced focusing technique in confocal microscopy allows for much higher resolution than conventional bright-field microscopes, revealing finer details of biological specimens.

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What is the fundamental difference between electron microscopy and light microscopy?

Electron microscopes use a beam of electrons instead of light to illuminate and view specimens. Electrons have a much shorter wavelength than light, leading to a 1,000-fold increase in resolution.

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What is Transmission Electron Microscopy (TEM)?

Transmission electron microscopy (TEM) uses a beam of electrons that passes through a very thin specimen, creating an image with varying shades of gray. This technology is used to view extremely small structures, such as organelles and proteins.

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What are the capabilities of TEM?

TEM allows scientists to visualize incredible details of biological structures. Its resolving power is 3 nanometers, enabling magnification of up to 400,000 times.

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Electron-Lucent Areas

Regions of an electron micrograph that appear brighter or electron-lucent, indicating areas where electrons passed readily through the tissue.

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Electron-Dense Areas

Regions of an electron micrograph that appear darker or more electron-dense, indicating areas where electrons were absorbed or deflected.

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Heavy Metal Staining

Compounds with heavy metal ions (like osmium tetroxide, lead citrate, and uranyl compounds) used in TEM to enhance contrast and resolution by binding to cellular macromolecules, increasing their electron density.

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Cryofracture

A technique in TEM where very small tissue specimens are rapidly frozen in liquid nitrogen and then fractured with a knife, exposing the internal structure for examination.

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Freeze Etching

A technique used after cryofracture, where a thin coat of metal (like platinum) is vaporized onto the exposed frozen surface, creating a replica that can be examined by TEM.

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Cryofracture and Freeze Etching for Membranes

A TEM technique used to study membrane structure without fixation or embedding, exposing protein components within the lipid bilayers for analysis.

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Scanning Electron Microscopy (SEM)

A type of electron microscope that provides a high-resolution view of the surfaces of cells, tissues, and organs by focusing a narrow beam of electrons onto the specimen.

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Heavy Metal Coating in SEM

A thin layer of heavy metal (often gold) used in SEM to coat the specimen, reflecting electrons in the beam that scans the surface, creating an image.

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Study Notes

Histology I

  • Histology I course by Dr. Mustafa Ghanim & Dr. Fatina Hanbali
  • Delivered by the Faculty of Medicine and Health Sciences
  • Presentation date: 30/01/2021

Light Microscopy

  • Conventional bright-field, fluorescence, phase-contrast, confocal, and polarizing microscopy rely on light interactions with tissue components to study tissue features
  • Bright-field microscopy examines stained tissue with ordinary light passing through the specimen (Figure 1-3)
  • Optical components: condenser focuses light on the specimen, objective lens enlarges and projects the image, and eyepiece (ocular lens) magnifies further and projects onto the viewer's retina or a CCD camera
  • Total magnification is the product of objective and ocular lens magnifications
  • Resolving power is the smallest distance between two structures that can be seen as separate objects (approximately 0.2 µm for light microscopes)
  • A magnified image from 1000 to 1,500 times is possible
  • Objects smaller than 0.2 µm cannot be differentiated
  • Tissues can be observed at low (X4), medium (X10), or high (X40) magnification

Virtual Microscopy

  • Digital conversion of stained tissue preparations into high-resolution images
  • Tissues are visualized using a computer or other digital device, without a physical slide or microscope
  • Tissue regions are captured digitally in a grid-like pattern at various magnifications.
  • Software processes the data for storage, allowing access, visualization, and navigation of the original slide using web browsers.
  • Replacing traditional light microscopes and glass slide collections in histology

Fluorescence Microscopy

  • Certain cellular substances emit light (fluorescence) when exposed to specific wavelengths.
  • Fluorescence microscopy uses ultraviolet (UV) light to excite fluorescent substances, producing visible light output.
  • Fluorescent substances appear bright against a dark background.
  • The instrument is equipped with a UV or other light source and filters selecting the emitted wavelengths for visualization
  • Fluorescent compounds with an affinity for specific macromolecules are used as stains
  • Example: acridine orange stains DNA and RNA
  • Different fluorescent emissions allowing for independent localization in cells.
  • Other stains include DAPI and Hoechst, which directly stain DNA and emit blue fluorescence under UV light
  • Antibodies labeled with fluorescent compounds are key for immunohistologic staining

Phase-Contrast Microscopy

  • Unstained, transparent cells and tissue sections can be studied.
  • Cellular detail is often difficult to see in unstained tissues because all parts have similar optical densities
  • Phase-contrast microscopy uses a lens system to produce visible images from transparent objects
  • Importantly, it can be used for living, cultured cells (Figure 1-5).
  • Phase contrast microscopy is based on light speed changes when traversing structures with varying refractive indices
  • Structures appear lighter or darker relative to one another
  • Used in cell cultures when fixation or staining are irrelevant.

Differential Interference Contrast Microscopy

  • A phase-contrast modification using Nomarski optics
  • Creates a more three-dimensional image of living cells (Figure 1-5c)

Confocal Microscopy

  • Solves bright-field resolution limitations that arise from stray light by using a small, high-intensity light source (often a laser beam).
  • Focused point and detector pinhole allow for sharp focus at a particular plane in the specimen (confocal).
  • Digital images from many focal planes create a detailed "optical section" of the specimen.
  • 3D reconstruction of the specimen is possible.

Electron Microscopy

  • Transmission and scanning electron microscopes use electron beams instead of light.
  • Electron wavelength is significantly shorter than light wavelength.
  • Providing a significantly higher resolution (approximately 1000x better than light microscopy)
  • Transmission Electron Microscopy (TEM):
    • Allows resolution around 3 nm
    • Enables high magnification visualization of isolated particles (up to 400,000x)
    • Typically utilizes very thin (40-90 nm) resin-embedded tissue sections.
    • Electrons passing through the specimen create black/white/gray regions indicating electron-lucent/absorbed/deflected areas
    • Heavy metal compounds (e.g., osmium tetroxide, lead citrate, uranyl compounds) are added to improve tissue contrast and resolution.

Cryofracture and Freeze Etching

  • Techniques allowing TEM study of cells without fixation or embedding
  • Especially useful for membrane structure analysis
  • Very small tissue samples are quickly frozen in liquid nitrogen and fractured with a knife,
  • Replica of the surface is produced in a vacuum by applying thin coats of vaporized platinum or other metal atoms
  • Random fracture planes often split lipid bilayers, exposing protein components

Scanning Electron Microscopy (SEM)

  • Provides high-resolution views of cell, tissue, and organ surfaces
  • A narrow beam of electrons scans the specimen's surface
  • Images using a spray-coated thin metal layer (e.g., gold) that reflects electrons
  • Reflected electrons are processed to generate black and white images offering a 3D view from the specimen's surface

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Histology I - 30/01/2021 PDF

Description

Explore the fundamental principles and applications of various microscopy techniques such as fluorescent and phase-contrast microscopy. This quiz focuses on their uses in studying nucleic acids, cell structures, and imaging techniques. Test your knowledge on important concepts related to light microscopy and its advantages.

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