Mechanics of Cell Signals and Techniques

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Questions and Answers

Which of the following best describes macroscopic mechanical signals?

Stiffness of cell membranes

Blood pressure and lung expansion (correct)

Forces applied by individual cells

Traction forces experienced by cells

What is the purpose of using micropillars in cell mechanics?

To assess overall cellular stiffness

To evaluate lung expansion

To monitor cell traction forces (correct)

To measure blood pressure

What phenomenon does FRET allow researchers to study in the context of cellular behavior?

Changes in mechanical rigidity of tissues

Variations in blood flow during physical activity

Interactions between Src kinases and their substrates (correct)

Responses of cells to external temperature changes

Which technique uses laser tweezers to apply force on a cell membrane?

Integrative Tension Sensor Signup and view all the answers

What type of mechanical signal is characterized by the stiffness of bones and forces during walking?

Macroscopic mechanical signals Signup and view all the answers

Which cellular measurement technique involves imaging cell adhesive force at a single molecule level?

Cell adhesive force imaging Signup and view all the answers

What happens to FRET responses when mechanical stimulation is applied to a cell?

FRET responses indicate directional wave propagation Signup and view all the answers

What best characterizes the microscopic mechanical signals observed in cells?

Stiffness and forces applied by individual cells Signup and view all the answers

What does the force pattern in mechanobiology primarily consist of?

Individual clusters produced by focal adhesions Signup and view all the answers

In Atomic Force Microscopy (AFM), how is cantilever deflection measured?

Through the reflection of a laser beam off the cantilever Signup and view all the answers

What is a primary advantage of confocal microscopy over widefield microscopy?

It produces sharper images with higher resolution. Signup and view all the answers

What significant contribution did Dr. Arthur Ashkin make to mechanobiology?

Development of optical tweezers for biological applications Signup and view all the answers

Which of the following describes the function of a pinhole in confocal microscopy?

It blocks out-of-focus light. Signup and view all the answers

What represents macroscopic mechanical signals?

Blood pressure, lung expansion, and walking forces Signup and view all the answers

Which of the following is NOT a part of the molecular mechanisms of mechanotransduction?

Optical tweezers Signup and view all the answers

In the context of image processing, what does a mean filter primarily accomplish?

It reduces noise within the image. Signup and view all the answers

Which cell behavior is NOT typically associated with mechanobiology?

Cell division Signup and view all the answers

What is the primary cause of diffraction in microscopy?

Interaction of light with small particles or features. Signup and view all the answers

Which imaging technique focuses on capturing sharp images of a specific plane rather than the entire sample depth?

Confocal microscopy Signup and view all the answers

What kind of signals do microscopes typically focus on in studying mechanobiology?

Microscopic mechanical signals from cells Signup and view all the answers

In which organ systems is mechanobiology particularly relevant?

Cardiovascular, bone, cartilage, and liver systems Signup and view all the answers

Which type of filter is used in image processing to remove outliers and excessive noise from images?

Median filter Signup and view all the answers

During image processing, converting an image from grayscale to binary primarily serves what purpose?

To identify key features in the image. Signup and view all the answers

What role does resolution play in microscopy?

It defines the ability to distinguish between two closely spaced objects. Signup and view all the answers

What is the primary mechanism used in STED microscopy to de-excite a fluorescent probe?

Stimulated emission Signup and view all the answers

What must happen in STORM for precise localization of fluorescent molecules?

Molecules must emit enough photons consistently. Signup and view all the answers

What criterion is necessary for the separation of fluorescent molecules in STORM?

More than 250 nanometers apart Signup and view all the answers

Which Nobel Prize category was awarded for the development related to STED microscopy?

Nobel Prize in Chemistry 2014 Signup and view all the answers

In STED microscopy, what is the initial state of a fluorescent probe before excitation?

Ground state Signup and view all the answers

What is a significant challenge in STORM microscopy?

Avoiding fluorescence from non-target molecules Signup and view all the answers

What happens to a photoswitchable molecule in STORM after approximately 250 nanometers separation?

It enters a dark state. Signup and view all the answers

Which aspect distinguishes STED from STORM microscopy?

Emphasis on stimulated emission depletion Signup and view all the answers

What defines the diffraction limit according to the Rayleigh criterion?

Two images are just resolvable when the center of one is over the minimum of the other. Signup and view all the answers

What is the primary purpose of super-resolution microscopy?

To break the diffraction barrier and improve optical resolution. Signup and view all the answers

How is resolution calculated using the formula given in the content?

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

What is the resolution achieved using a 10x objective with NA=0.25 and λ=550nm?

1.34µm Signup and view all the answers

What distinguishes PALM from other microscopy techniques?

It employs photoactivatable fluorophores for resolution of details. Signup and view all the answers

Which microscopy technique specifically resolves adjacent fluorophores?

PALM microscopy Signup and view all the answers

What happens to fluorophores once activated by lasers in PALM microscopy?

They emit for a short time but then bleach. Signup and view all the answers

Which of the following statements best describes the Airy disk?

It consists of a central bright spot and surrounding rings. Signup and view all the answers

Study Notes

Mechanical Signals

Microscopic mechanical signals: Forces applied by cells, stiffness of cells
Macroscopic mechanical signals: Blood pressure, lung expansion/contraction, forces during walking, stiffness of bones, etc.

Traction Force Microscopy

A technique using fluorescent marker beads to measure cell traction forces.
A thin PAA gel is loaded with fluorescent beads.
Proteins are used to facilitate cell adherence to the gel.
Forces exerted by a cell cause displacement of the gel, which is quantified by imaging bead displacement.

Micropillars

Used to measure cell traction.
A method that involves using micropillars to apply controlled forces to cells.
Includes a glass weight to provide forces in a controlled manner.
A 60 µm spacer is strategically positioned between the two glass components to ensure an appropriate distance that allows for accurate measurements while maintaining structural stability during experiments involving cell traction assessment.

FRET

FRET (Förster Resonance Energy Transfer) is used to monitor Src kinase activity during mechanical stimuli.
Measurement involves the presence of Src kinase or phosphatase and monitoring the FRET change.
This variation in fluorescence intensity, which is meticulously measured over time, serves as an essential indicator not only of the presence but also of the magnitude of a mechanical stimulus impacting the cellular environment. The fluctuations in fluorescence can reveal how cells respond to mechanical forces, which plays a significant role in understanding cellular behaviors such as movement, adhesion, and proliferation under different physical conditions. Consequently, these measurements facilitate valuable insights into the dynamic interactions and biochemical pathways that influence how cells communicate and adapt to their surrounding microenvironment.

Integrative Tension Sensor (ITS)

A biophysical tool that measures cell adhesive forces at a single-molecule level.
Utilizes 18 base pair dsDNA labeled with specific components.
- An integrin ligand that enhances adhesion to specific receptors, promoting interactions with target cells.
- A biotin tag, which facilitates surface immobilization for effective binding and detection.
- A quencher that plays a crucial role in fluorescence measurements by suppressing the signal when in proximity to the fluorescent probe, enabling precise monitoring of molecular interactions.
- An integrin ligand
- A biotin tag, for surface immobilization.
- A quencher (blackhole quencher 2)
- And a fluorophore (Cy3 or Cy5)
The dsDNA is in an inactivated state without fluorescence when the quencher is present.
When the dsDNA is mechanically disassociated by the transmitted force, it becomes fluorescent.
The tension tolerance (Ttol) can be modified by adjusting the anchoring site.

Microindentation using AFM

A technique using a laser beam to detect cantilever deflections.
Cantilever deflections are measured to determine force-displacement curves of a sample.
The method tracks and quantifies how much the surface is moving when an external force is applied.

Profile Microindentation

A technique used to quantify mechanical properties of cells.
A micropipette and cytodex bead system are involved in the method.
The method is used to determine the force applied to cells and is used for cell mechanics testing.

Optical Trap/Tweezer/Levitation

Uses lasers to trap, manipulate, and levitate microscopic objects
Light momentum is used to trap, move, or levitate particles
Involves the transfer of momentum of light to the particle in question

Bioimaging and Image Analysis

Includes image processing: Loading and displaying images, Converting between image types [binary, grayscale, RGB, multi-channel], Counting objects;
Includes pseudo-colour.

Course Outline for Mechanobiology

Covers topics including molecular mechanisms of mechanosensation.
Includes organ system mechanics, disease mechanics, technological innovations in mechanobiology.
Examines the relationship between mechanics and biological systems.

Grayscale to Binary

Converting grayscale images into binary images for analysis
Counting objects in grayscale images
Method involves converting grayscale images to a binary image by choosing a threshold on grayscale values. Images of objects become ones, and the background become zeroes.
Counting objects in the binary image thus corresponds to the number of original objects.

Smoothing (Mean Filter)

A technique for smoothing noisy data by averaging multiple data points.
Used to eliminate the effects of noise in 1d signals.
A 3-point mean filter and 11-point mean filter are examples.

Image Filters (Mean)

Used to reduce noise in images through averaging pixel neighborhood values.
Includes 'imfilter' MATLAB function for applying mean, gaussian, median masks.

Image Filters (Median, Laplace)

Median filter technique removes noise from images by replacing a pixel with the median of its immediate surrounding pixels.
Laplace filter is used to perform sharpening by calculating second-order derivatives.

Confocal Imaging

Combines optical sectioning and 3D reconstruction to generate 3d images.
Uses a pinhole to block out-of-focus light for improved resolution in images.

When to Use Confocal

Confocal microscopy is best suited for:
- Fixed, thin samples & high magnification details
- Samples thicker than 10 µm
Wide-field microscopy is better for:
- Thin samples & low magnification.
- Live, thin, and or low magnification samples for microscopy

Resolution

The Rayleigh criterion is one way to measure resolution
Explains the minimum resolvable distance between two point sources of light.
Resolution limits are due to diffraction, and the Airy disk is a diffraction limited result

Super-Resolution Microscopy

Techniques like PALM, STED, and STORM enable resolution beyond the diffraction limit.
PALM: Uses photoactivatable fluorophores to precisely determine their spatial position
STED: Stimulated Emission Depletion is used to enhance resolution
STORM: Stochastis Optical Reconstruction Microscopy precisely locates individual molecules

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Description

Explore the fascinating world of mechanical signals at both microscopic and macroscopic levels in this quiz. Delve into techniques like Traction Force Microscopy and the use of micropillars for measuring cell traction. Additionally, discover how FRET is utilized to monitor Src kinase activity during mechanical stimuli.