Cell Biology and Microscopy Techniques

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Questions and Answers

What are tweezers used for in cell manipulation?

Tweezers are used to position cells without physically touching them.

How can you measure the resistance and elasticity of erythrocytes?

By attaching two beads to the erythrocytes and assessing their response to applied forces.

Explain the process of measuring cell-cell adhesion with different cell types.

Two different cell types are allowed to bind, and the force needed to detach them is measured.

What is the significance of the multi-immersion imaging system?

It enhances visualization by mimicking the light-reflecting properties of the mollusk eye. Signup and view all the answers

How does introducing a mutation in a gene help in studies?

It allows researchers to observe the resulting phenotype and understand gene function. Signup and view all the answers

What role does elasticity play in cell mechanics?

It impacts how cells deform under stress and recover their original shape. Signup and view all the answers

Describe how the tweezers' technique can be applied in vivo.

They can be used to manipulate cells, such as red blood cells, within a living organism's capillaries. Signup and view all the answers

What advantage does manipulating gene expression have in cell engineering?

It allows scientists to control cell behavior and investigate the relationship between genes and their functions. Signup and view all the answers

What distinguishes super-resolution microscopy from regular microscopy?

Super-resolution microscopy integrates advanced mathematical models to reconstruct images, allowing for higher resolution than traditional methods. Signup and view all the answers

Explain how fluorescent markers contribute to the functionality of super-resolution microscopy.

Fluorescent markers allow researchers to visualize changes in samples with precision and can be activated or deactivated based on specific wavelengths of light. Signup and view all the answers

What is the definition of microscope resolution in the context of super-resolution techniques?

Microscope resolution refers to the shortest distance that allows the microscope to distinguish two separate objects rather than a single blurred point. Signup and view all the answers

Describe how the overlap of signals from closely situated fluorophores affects regular microscopy.

In regular microscopy, closely positioned fluorophores lead to signal overlap, resulting in a generalized single signal that obscures individual details. Signup and view all the answers

What role do specific fluorophore labels play in super-resolution microscopy techniques?

Specific fluorophore labels can be activated or switched off, allowing researchers to see different colors and enhance resolution in their samples. Signup and view all the answers

How can the mathematical models used in super-resolution microscopy impact imaging results?

Mathematical models improve the reconstruction of images, enabling clearer visualization of features that would normally be indistinguishable. Signup and view all the answers

What common characteristic is shared by different modalities of super-resolution microscopy?

All modalities of super-resolution microscopy are based on the principle of utilizing fluorescent markers to enhance image resolution. Signup and view all the answers

Why is the ability to shine specific wavelengths of light on fluorophores important in super-resolution microscopy?

It allows for the activation and deactivation of fluorophores, providing control over which labels are visualized and improving sample analysis. Signup and view all the answers

What is required for a gene to be expressed?

A gene needs to be downstream of a promoter, and the promoter's sequence dictates the gene's expression. Signup and view all the answers

What are the two main approaches to engineer gene expression in cells?

The two main approaches are transient and stable gene expression. Signup and view all the answers

When is transient gene expression typically used?

Transient gene expression is used to observe short-term effects of gene modifications. Signup and view all the answers

How does stable gene expression differ from transient expression?

Stable gene expression results in permanent genetic modification, whereas transient expression is temporary. Signup and view all the answers

What advancements in gene modification began in the 1960s?

Modifying bacteria to introduce specific genes for expression and translation into proteins. Signup and view all the answers

What does constitutive expression of a transgene imply?

Constitutive expression means the transgene is always expressed and translated into proteins. Signup and view all the answers

What conditions might affect the inducible expression of a transgene?

Inducible expression occurs under specific conditions that activate gene expression. Signup and view all the answers

Why are bacteria considered a model of choice in genetics?

Bacteria are preferred for genetic studies due to their simplicity and rapid growth. Signup and view all the answers

What is the purpose of a promoter in gene expression?

The promoter regulates the expression of a gene by facilitating the binding of transcription machinery. Signup and view all the answers

How does transient expression differ from stable expression in genetic studies?

Transient expression involves delivering a plasmid that doesn't integrate into the cell's DNA, leading to temporary gene expression. Signup and view all the answers

What significant event in 1990 marked the application of gene therapy in humans?

In 1990, gene therapy was applied to modify gene expression for a specific immunodeficiency disorder. Signup and view all the answers

What was the significance of the genetically modified tomato developed in 1994?

The genetically modified tomato was significant for its ability to resist rotting, demonstrating commercial application of genetic modifications. Signup and view all the answers

What major breakthrough in cloning occurred in 1996?

The cloning of Dolly the sheep was a major breakthrough, achieved by transferring the nucleus from one cell to another. Signup and view all the answers

In the context of gene expression, what happens to expression levels after delivering a gene?

Gene expression levels initially peak after delivery but then gradually decrease over time. Signup and view all the answers

What role do viruses play in gene delivery methods developed by 1980?

Viruses were used as delivery methods to forcibly express foreign genes within host cells. Signup and view all the answers

What mechanism is used to promote the integration of foreign DNA into a cell?

DNA recombination mechanisms are used to promote the integration of foreign DNA into the cell's genome. Signup and view all the answers

What is one way to down-regulate gene expression?

RNA interference (RNAi) is one way to down-regulate gene expression. Signup and view all the answers

What role does synthetic small interfering RNA (siRNA) play in gene silencing?

siRNA introduces a short molecule of double-stranded RNA that matches the target gene's sequence to induce silencing. Signup and view all the answers

What is the function of the RISC complex in RNA interference?

The RISC complex separates the strands of siRNA and uses the complementary strand to degrade the target gene's mRNA. Signup and view all the answers

Why is targeting longer gene sequences more difficult in gene silencing?

The longer the gene sequence, the more complex and challenging it is to effectively target and silence it. Signup and view all the answers

How does dsRNA relate to the cellular response mechanism in RNA interference?

dsRNA typically originates from viruses and triggers the cellular response through the RISC complex during RNA interference. Signup and view all the answers

What happens to the target gene's mRNA once it is hybridized with the complementary siRNA?

Once hybridized, the target gene's mRNA is degraded, leading to reduced expression of the gene. Signup and view all the answers

What challenges might arise when attempting to silence a whole gene?

Silencing a whole gene is challenging due to the longer sequence and potential off-target effects that may occur. Signup and view all the answers

How can the efficiency of RNA interference be increased?

The efficiency of RNA interference can be increased through optimizing the delivery of siRNA into cells and enhancing the binding affinity. Signup and view all the answers

What is the primary role of the strong universal promoter in the experiment?

The strong universal promoter provides a consistent signal that allows for the expression of the GFP across various cell types. Signup and view all the answers

How does the masking strategy aid in the experimental design?

The masking strategy allows light to penetrate only areas where there are gaps, controlling where the GFP expression is activated. Signup and view all the answers

Why is it significant that the promoter works in all cell types?

It enables researchers to study gene effects across different tissues and ensures consistent gene expression throughout the experiment. Signup and view all the answers

What happens to the cells that are illuminated but do not show GFP expression?

These cells have the construct but do not express the GFP protein, indicating that activation requires light exposure. Signup and view all the answers

What is the main focus of the study mentioned regarding the brain?

The study investigates the effects of specific genes in particular cell types within the brain. Signup and view all the answers

In what way does this experimental strategy appear simplistic, yet is effective?

While it may look trivial, it systematically controls gene expression to test the activation mechanisms reliably in vitro. Signup and view all the answers

What does the presence of GFP in illuminated cells indicate?

It indicates successful activation of the construct in those cells as a response to the specific wavelength of light. Signup and view all the answers

Why is the study considered more refined for in vivo research?

Because it enables focused examination of gene functions in specific cell types under live conditions compared to in vitro setups. Signup and view all the answers

Study Notes

Super Resolution Microscopy

A different method for collecting light from fluorescent markers
Offers higher resolution than regular techniques
Microscope resolution refers to the shortest distance between two objects that can be seen distinctly, not just a single blurred point
Regular techniques involve exciting multiple fluorophores, resulting in overlapping signals and a general signal
Super resolution microscopes illuminate specific points in a sample, collect data, and then illuminate other points, minimizing overlap
This allows for a clearer view of each individual marker
It is more reliable than regular microscopy due to the lack of signal overlap and avoids artifacts
Useful for examining very close structures like thin fibers

Raman Microscopy

A combined method merging microscopy and Raman spectroscopy
Provides information on the spatial and spectral characteristics of the sample
Label-free technique, meaning it doesn't require labeling
Provides a chemical profile of the sample
Enables creation of a chemical map to study different cell types
Can be used on live and fixed cells
Different chemical compounds react differently when hit by specific wavelengths of a laser
This generates a specific "identity card" for each type of molecule in the sample, useful in identifying cell type differences based on chemical composition
Can be useful in determining cellular components like DNA, lipids, and collagen.
Combining microscopy with Raman spectroscopy allows visualization and chemical composition reading simultaneously

Optical Tweezers Technology

A technique that uses light as a force, a focused light beam, to physically trap and manipulate objects or cells
Useful in vitro and in vivo applications,
Allows manipulation of cell position and measurement of forces inside or outside the cells
Techniques such as cell resistance and cell-cell adhesion can be studied
Used to manipulate cells without having physical contact

Cell Engineering Technologies

Techniques for manipulating gene expression in cells
Techniques to deliver constructs for modification
Modifying bacteria to introduce specific genes for expression and protein production
Developing methods for efficient DNA recombination in cells
Using viruses as delivery vehicles for specific constructs
Techniques for gene therapy in humans
Commercial genetic modified tomato resisting rotting
Cloning of Dolly the sheep, demonstrating nucleus transfer
Fine gene editing with high precision
CRISPR/Cas system for precise gene editing

Gene Expression Manipulation

Transient expression: gene expression for a short period
Stable expression: permanent gene modification or genome integration.
Constitutive expression: always expressed for the duration.
Inducible expression: expression only in specific conditions (e.g., with a drug or a light signal)
Examples: GFP (green fluorescent protein), dsRed (red fluorescent protein).

Repressing Gene Expression

Gene silencing (knockdown) : decreasing gene expression by degrading mRNA.
RNA interference (RNAi) and siRNA molecules – using short RNA molecules to target and degrade specific mRNA
Gene knockout: completely inactivating a gene.
Homologous recombination - a natural DNA repair mechanism that can be used to replace or remove parts of a gene intentionally. Also known as targeted gene editing.
Modifying gene sequences: Introducing mutations in the genome to study their effects on cellular function or to understand disease mechanisms. Types of mutations include "point mutations" (changes in single base pairs), "insertions" or "deletions" .

General Cell Culture Techniques

Aseptic techniques: maintain a sterile environment to prevent contamination
Primary cell cultures: derived directly from a tissue sample, have limited lifespan
Cell lines: established cell cultures, can be maintained indefinitely
Culture media: provide nutrients, hormones, and other factors essential for cell growth.
Biological safety cabinets (BSCs): maintain sterility
CO2 incubators: Maintain temperature and gas conditions for optimal cell growth
Cell detachment: methods to separate cells from culture surfaces (e.g., trypsinization, EDTA treatment)

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Description

This quiz explores various concepts in cell manipulation, elasticity, and advanced microscopy techniques, including super-resolution microscopy. It discusses the use of tweezers in cell studies, the importance of gene mutations, and the role of fluorescent markers in enhancing imaging capabilities. Ideal for students and professionals in cell biology and biomedical engineering.