Microscopy Techniques in Cell Biology

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Questions and Answers

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?

They are labeled with fluorescent compounds for staining Signup and view all the answers

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

They can be observed without the need for staining Signup and view all the answers

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

Ordinary light Signup and view all the answers

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

Condenser Signup and view all the answers

How is total magnification calculated in a light microscope?

Multiplying the powers of the objective and ocular lenses Signup and view all the answers

What is the maximal resolving power of a light microscope?

0.2 µm Signup and view all the answers

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

They are seen as one object Signup and view all the answers

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

Single ribosome Signup and view all the answers

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

Resolving power Signup and view all the answers

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

Light microscopy Signup and view all the answers

What primarily determines the resolving power of a microscope?

The quality of its objective lens Signup and view all the answers

What is the relationship between magnification and resolution in microscopy?

Magnification is valuable only with high resolution Signup and view all the answers

What role does the eyepiece lens have in microscopy?

It only enlarges the image without affecting resolution Signup and view all the answers

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

It allows for digital access and ease of use Signup and view all the answers

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

Ultraviolet (UV) light Signup and view all the answers

How do fluorescent substances appear in fluorescence microscopy?

Bright on a dark background Signup and view all the answers

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

Acridine orange Signup and view all the answers

What does virtual microscopy utilize to capture specimens?

Digital slide-scanning microscope Signup and view all the answers

What principle does phase-contrast microscopy rely on?

The change in light speed through different refractive indices Signup and view all the answers

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

By providing a more apparent three-dimensional aspect Signup and view all the answers

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

It reduces stray light, enhancing contrast and resolving power Signup and view all the answers

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

The alignment of a point light source, focal point, and detector's aperture Signup and view all the answers

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

Phase-contrast microscopy Signup and view all the answers

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

It improves resolution and localization precision. Signup and view all the answers

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

The beam splitter Signup and view all the answers

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

3 nm Signup and view all the answers

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

By passing a beam of electrons through the tissue section Signup and view all the answers

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

40-90 nm 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)?

It enhances contrast and visibility of macromolecules. 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?

Cryofracture and freeze etching Signup and view all the answers

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

They are rapidly frozen and fractured. Signup and view all the answers

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

Gold Signup and view all the answers

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

It reflects off the surface of the specimen. Signup and view all the answers

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

Absorption or deflection of electrons Signup and view all the answers

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

It allows visualization of protein components and lipid bilayers. Signup and view all the answers

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

Ethanol Signup and view all the answers

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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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.