Biocompatible Materials and Sensing Principles
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Questions and Answers

In a microscope, how do spherical wave fronts behave in the illuminating ray path?

  • They are converted directly into plane waves.
  • They remain unchanged as they pass through the aperture planes.
  • They converge and focus onto the aperture planes. (correct)
  • They diverge and scatter across the aperture planes.

    • What behavior characterizes light rays when passing between conjugate planes in a microscope?

  • Rays focused in aperture planes remain focused in field planes.
  • Rays focused in aperture planes become focused in field planes.
  • Rays focused in field planes immediately diverge when they reach the next conjugate plane.
  • Rays focused in one set of conjugate planes become nearly parallel when passing through the other set. (correct)

    • How are spherical waves in the imaging ray path transformed in a microscope?

  • They remain as spherical waves through all of the conjugate planes.
  • They are transformed into diffused light at the aperture plane.
  • They converge into focused rays at the field planes. (correct)
  • They are instantly converted to parallel light.

    • What is the primary significance of the reciprocal relationship between the two sets of conjugate planes in a microscope?

    <p>It fundamentally dictates the interaction between ray paths in image formation and has operational consequences. (B)</p> Signup and view all the answers

    If the illumination rays are focused on aperture planes, how will they propagate in the field planes?

    <p>They will become nearly parallel. (A)</p> Signup and view all the answers

    What is the primary limitation on the smallest focused spot of light achievable by a perfect lens with a circular aperture?

    <p>Diffraction of light at the aperture (B)</p> Signup and view all the answers

    In confocal microscopy, what process is integral to the formation of a 3D image from the acquired data?

    <p>Convolution of the true specimen with the point spread function (B)</p> Signup and view all the answers

    Which of the following concepts is most directly related to the 'Airy disc'?

    <p>The practical limitation of resolution in a microscope (C)</p> Signup and view all the answers

    What does the term 'convolution' typically describe in the context of image formation within a confocal microscope?

    <p>The operation combining the true specimen distribution of light sources with the microscope's PSF (B)</p> Signup and view all the answers

    Which phenomenon directly establishes the limit of resolution in a light microscope?

    <p>Diffraction (D)</p> Signup and view all the answers

    What is the maximum number of biotin molecules that a single streptavidin molecule can bind?

    <p>Four (D)</p> Signup and view all the answers

    What is the typical use of biotin in conjunction with streptavidin?

    <p>To conjugate it to other molecules like enzymes, antibodies, or target proteins (B)</p> Signup and view all the answers

    Which of the following is NOT a method used to study protein and cell-biomaterial interactions?

    <p>High-performance liquid chromatography (HPLC) (D)</p> Signup and view all the answers

    Why are combinatorial sensing and chemical mapping techniques important for studying protein and cell interactions on biomaterials?

    <p>They help understand the dynamic arrangement and conformation (B)</p> Signup and view all the answers

    What do in-situ sensing techniques specifically help to understand in the context of biomaterials?

    <p>The impact of surface engineering on the host response (A)</p> Signup and view all the answers

    In light microscopy, what is indicated by the crossover points of the ray traces?

    <p>The focal conjugates of each plane (C)</p> Signup and view all the answers

    What is the primary property measured by Optical Waveguide Lightmode Spectroscopy (OWLS) to determine adsorbed mass?

    <p>Refractive index variation (B)</p> Signup and view all the answers

    What key aspect is illustrated by the diagram of conjugate planes in light microscopy?

    <p>The reciprocal nature of the two sets of conjugate planes (A)</p> Signup and view all the answers

    According to the provided information, which component's refractive index change is NOT directly detected by OWLS?

    <p>Coupled water within the adsorbed layer (A)</p> Signup and view all the answers

    What analytical technique, described in the content is NOT primarily used in the study of protein adsorption?

    <p>Biotin-streptavidin conjugation system (D)</p> Signup and view all the answers

    What is the main purpose of Köhler illumination in microscopy?

    <p>To ensure uniform illumination of the field of view (A)</p> Signup and view all the answers

    The de Feijter's formula, used in connection with OWLS, relies on what measure.

    <p>The difference in refractive indices between the adsorbed layer and the cover medium (B)</p> Signup and view all the answers

    Which of the following is a biospecific interaction that can be used in a Quartz Crystal Microbalance (QCM) immunosensor?

    <p>Antigen-antibody binding (D)</p> Signup and view all the answers

    What is the primary measured parameter in Quartz Crystal Microbalance (QCM) to quantify mass change?

    <p>Change in resonant frequency of the crystal (D)</p> Signup and view all the answers

    What is the fundamental interaction used in the Biotin-streptavidin conjugation system?

    <p>High affinity binding between biotin and streptavidin (D)</p> Signup and view all the answers

    Which application, mentioned in the content, uses a Quartz Crystal Microbalance (QCM)?

    <p>Stem cell selection and extraction (A)</p> Signup and view all the answers

    Which of the following microscopy techniques is specifically mentioned as capable of imaging beyond the diffraction limit?

    <p>Structured Illumination Microscopy (D)</p> Signup and view all the answers

    What does PALM/STORM microscopy rely on for localization?

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

    What is a primary application of Focused Ion Beam (FIB) in the context of the provided materials?

    <p>Preparing samples for electron microscopy (C)</p> Signup and view all the answers

    According to the information, what factor modulates the response of soft tissue cells to titanium implants?

    <p>Blood-implant interactions (C)</p> Signup and view all the answers

    What is the main use of (Bio)MEMS in mechanotransduction studies as indicated in the text?

    <p>To investigate cellular responses to mechanical forces (B)</p> Signup and view all the answers

    What is the purpose of using a stretchable transwell chamber platform?

    <p>To apply mechanical stretch to cell layers (C)</p> Signup and view all the answers

    What is the primary role of FRET biosensors in the context of the material discussed?

    <p>To measure force and remodeling processes at cell junctions (D)</p> Signup and view all the answers

    What cellular structure is explicitly mentioned in conjunction with integrated molecular FRET tension sensors?

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

    What is a common method used for spatially patterning cells, as discussed in the material?

    <p>Microcontact printing of proteins (B)</p> Signup and view all the answers

    What is the lift-off technique primarily used for in the context of cell studies?

    <p>To control cell shape through protein patterning (D)</p> Signup and view all the answers

    Flashcards

    Conjugate planes

    Sets of planes where light rays are focused and nearly parallel.

    Illuminating ray path

    The pathway taken by light rays that illuminate the sample.

    Imaging ray path

    The pathway of light rays that create the image after passing through the sample.

    Spherical wave fronts

    Curved surfaces of constant phase used in light propagation.

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    Reciprocal relationship

    The interaction between two sets of conjugate planes that affects image formation.

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    Contrast in Microscopy

    Difference in light intensity between the specimen and the background, enhancing visibility.

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    Fluorescence Microscopy

    A technique using fluorescence to observe the properties of organic and inorganic substances.

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    Point Spread Function (PSF)

    Describes how a point source of light is spread out in an optical system, defining image resolution.

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    Convolution in Microscopy

    Mathematical operation used to combine the real light sources with the PSF in forming images.

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    Biocompatible Materials

    Materials compatible with biological systems for medical applications.

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    Ellipsometry

    Optical technique to measure thin film thickness and refractive index.

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    OWLS

    Optical Waveguide Lightmode Spectroscopy; measures refractive index changes.

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    Refractive Index

    Ratio of the speed of light in a vacuum to its speed in a medium.

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    QCM

    Quartz Crystal Microbalance; measures mass changes at a surface.

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    Biotin-Streptavidin

    Strong, specific interaction used in biological applications.

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    Protein Adsorption

    Process where proteins adhere to surfaces, critical in biocompatibility.

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    BioMEMS

    Micro-electromechanical systems tailored for biological applications.

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    Deconvolution

    A mathematical process to reverse convolution effects in images.

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    Super-resolution microscopy

    Imaging technique that enhances resolution beyond the diffraction limit.

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    Structured Illumination Microscopy

    A method that improves resolution using patterned light.

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    PALM / STORM

    Techniques for super-resolution imaging using fluorescent markers.

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    Quantitative microscopy

    A method to measure fluorescence intensity quantitatively.

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    Electron microscopy

    A high-resolution imaging method using electron beams.

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    Focused ion beam (FIB)

    Technology used for imaging and modifying materials at micro and nanoscale.

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    Mechanotransduction

    Process through which cells sense and respond to mechanical stimuli.

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    Microcontact printing

    Technique to print proteins on surfaces for cell patterning.

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    Lift-off protein patterning

    Method to control cell shape by removing layers of proteins.

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    Multivalent properties of streptavidin

    Streptavidin can bind up to four biotin molecules simultaneously with high affinity.

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    Biotin conjugation

    Biotin is usually attached to an enzyme, antibody, or target protein.

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    Label free analysis

    Techniques studying biomolecule interactions without labels or tags.

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    In situ techniques

    Methods that study biological processes in their natural location.

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    Ex situ techniques

    Methods that analyze biological samples outside their natural environment.

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    Combinatorial sensing

    Using multiple sensing techniques to analyze proteins and cells.

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    Light microscopy

    A technique that uses light to magnify small objects for visual analysis.

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    Köhler illumination

    A microscope illumination technique providing even illumination across the field of view.

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    Study Notes

    Biocompatible Materials

    • The presentation covers biocompatible materials, analytical tools, microscopy, bioMEMS, and patterning techniques.
    • The date of the presentation is 20.11.2024.
    • Leading experts presented, including Prof. Dr. Katharina Maniura, Dr. Markus Rottmar, and Prof. Dr. Marcy Zenobi-Wong.

    Basic Principles in (Bio)Sensing

    • Key components in living organisms are proteins, nucleic acids, cells, and tissues.
    • These are primarily composed of carbon (C), hydrogen (H), oxygen (O), nitrogen (N), phosphorus (P), and sulfur (S).
    • Biosensors rely on biological functions like affinity (key-lock principle) and catalysis, plus physical functions like optical readouts and electrochemical methods.
    • Biomolecules are immobilized onto a transducer surface.

    Analysis Methods

    • Spectroscopy techniques include electron spectroscopy (XPS, AES), ion spectroscopy (SIMS), mass spectroscopy (MALDI-TOF), infrared spectroscopy, fluorescence spectroscopy, UV-Vis spectroscopy, and evanescent field methods SPR, OWLS.
    • Acoustic methods like QCM are also used.
    • Optical techniques encompass AFM, SEM, light microscopy, superresolution microscopy, ellipsometry, and contact angle measurement (label free).
    • Other techniques include ELISA, SDS-PAGE, and chromatography.

    Ellipsometry

    • Advantages of ellipsometry include label-free capability, in situ measurements, and the detection of very small changes (0.1-1 nm sensitivity, 1 ng/µm²).
    • Key limitations include the need for precisely flat surfaces in the sample and difficulties in resolving complex or irregular film thicknesses.

    Optical Waveguide Lightmode Spectroscopy (OWLS)

    • OWLS measures refractive index changes and adsorbed mass changes.
    • The method captures changes in optical path length related to protein adsorption, utilizing evanescent field techniques.
    • A critical part of this technique involves creating easy-to-functionalize optical waveguides with transparent coatings.
    • Refractive index changes are calculated using formulas and enable detailed analysis without the need to image the solvent.

    Quartz Crystal Microbalance (QCM)

    • QCM is an acoustic method that measures mass changes through changes in resonance frequency within a quartz oscillator.
    • It can analyze adsorbed mass and trapped water within protein layers.
    • Mass adsorption kinetics are difficult to interpret through this technique.
    • This method is demonstrated for stem cell selection and extraction with a QCM immunosensor using different techniques including Avidin, Biotinylated thiol and biotynilated antibody.

    Biotin-Streptavidin Conjugation System

    • Streptavidin has multivalent properties allowing it to bind up to four biotin molecules.
    • Biotin is frequently conjugated to enzymes, antibodies, or target proteins.
    • The binding interaction is strong and allows for efficient detection.

    Protein/Biomolecule Adsorption Analysis

    • Various techniques are available to study protein/cell–biomaterial interactions, including label-free methods and in situ or ex situ techniques.
    • Combining sensing with chemical mapping is needed to understand protein arrangements and conformations on biomaterials.
    • In situ sensing helps gain insight into the impact of surface design on how the host system interacts with proteins.

    Light Microscopy

    • Light microscopy is an important technique enabling the imaging and illumination of sample structures through different plane sets.
    • This relationship, allowing for the fundamental interaction of both ray paths, is crucial for image formation.
    • Different types of light microscopy include brightfield, phase contrast, differential interference contrast (DIC), and darkfield microscopy.

    Fluorescence Microscopy

    • Fluorescence microscopy employs different filter sets in the optical system to examine the fluorescence of a specific molecule after excitation.
    • High-numerical-aperture objective lenses are essential for higher brightness.
    • There is an overlap between excitation and emission profiles in the spectra.

    Confocal Microscopy

    • Provides 3D sectioning by focusing light.
    • It uses a pinhole to eliminate out-of-focus light.
    • The ability to optically section specimens creates a three-dimensional view.

    Diffraction and Resolution

    • Rayleigh criterion clarifies the relationship between the first diffraction minimum and the maximum of an adjacent point source.
    • Resolving power (or resolution) is largely dependent on the numerical aperture (NA) and wavelength of light.
    • Higher NA improves resolution, while shorter wavelengths offer greater detail.

    Point Spread Function

    • The PSF is the intensity distribution of an airy disk in 3D, also known as a diffraction-limited image.
    • This function describes how a point source of light is modified through the microscope.
    • Key observations are captured by the XY maximum intensity projection and the XZ maximum intensity projection.

    Convolution

    • A diffraction-limited image is essentially a "convolution" of the individual PSFs of the components of a specimen.
    • This process clarifies that image formation in a confocal microscope leads to a 3D distribution through the convolution of light from sources with the PSF.

    Deconvolution

    • Deconvolving involves correcting distortions in PSF, such as chromatic aberrations and refractive index mismatches.
    • To enhance the quality of the image, researchers may introduce strategies for obtaining better PSF acquisitions.

    Super-resolution Microscopy

    • Super-resolution techniques exceed the diffraction limit to improve image resolution.
    • PALM/STORM, SIM, and STED are important strategies in these studies.
    • Advanced microscopy methods offer an enhanced level of analysis exceeding the resolution limit associated with light microscopy.

    PALM/STORM Examples (2D and 3D)

    • Examples showcasing various biological systems, including nuclear pore complexes (Xenopus oocyte), and voltage-gated ion channels (neurites in PC12 neurons), and paxillin focal adhesion.
    • The use of these techniques for imaging is shown in multiple examples including cells and biological structures in different states.

    Quantitative (Fluorescence) Microscopy - Word of Caution

    • For accurate quantitation, rigorous optimization of fluorescence microscopy is essential.
    • Criteria like using appropriate fluorophores, maintaining clean optical components, and employing precise imaging conditions are important.
    • Image artifacts, especially those caused by non-uniform illumination and chromatic aberration, must be avoided or carefully controlled.

    Histological Analysis

    • Information on methods for preserving tissue, fixing and dehydrating tissue, infiltration, embedding, cutting, and staining procedures are detailed.
    • Strategies involving undecalcified and decalcified tissues are provided along with details of various solutions.

    Electron Microscopy

    • Provides high-resolution imaging by using electrons instead of light.
    • It is categorized into transmission electron microscopy (TEM) and scanning electron microscopy (SEM).
    • The methods differ in sample preparation and the types of visualizations they provide.
    • It demonstrates high resolution imaging of cells without requiring the preparation of live specimens, requiring the preparation of dead specimens.

    Focused Ion Beam(FIB)-SEM

    • FIB-SEM tools for 3D imaging of biological specimens using ion beams to create thin slices and then image with a scanning electron microscopy.
    • This allows for high-resolution structure visualization.
    • Examples are shown for imaging cellular and tissue interactions using biomaterials.

    (Bio)MEMS for Mechanotransduction Studies

    • MEMS (microelectromechanical systems) are used to study cell responses to mechanical stimuli such as shear stress, interstitial flow, stretch, or compression in different cell types.
    • In the study mechanotransduction, the stimulus, like mechanical input, causes a change inside the cells.
    • The feedback response includes responses such as ECM degradation and rearrangement within the microenvironment.

    Microfabricated Strain Array

    • Microfabricated strain arrays that offer multiple magnitudes, 96-well format for reduced cell number requirements, and precise focal plane control for paracrine signaling prevention are detailed.
    • Array designs for cells and tissues are described.

    Integrin-specific Molecular FRET Tension Sensors

    • Integrin-specific molecular FRET tension sensors are used for measuring changes in force exerted on integrins and cellular response to biomaterials.
    • Specific examples and figures are provided to clarify the tension measurement strategy.

    Spatial Patterning of Cells and Tissues

    • The control of cell and tissue activity is shown through the impact of spreading area and adherence area.
    • The mechanisms of how cells respond to these influences are detailed.

    Microcontact Printing

    • Microcontact printing of proteins on substrata, including patterns for cell control, is described.
    • This surface patterning technique enables the control of cell shapes and the modeling of the behaviour of cell types on a surface.
    • Limitations of this method, such as inconsistencies in pattern repeatability, are acknowledged.

    Lift-off Protein Patterning

    • A lift-off protein patterning method for controlling cell shapes through localized protein patterns is described, illustrating the technique's flexibility.
    • It presents specific diagrams and examples.

    Summary of Microscopy, BioMEMS, Patterning

    • A summary of microscopy techniques, bioMEMS, and patterning methods for biomaterials science is provided.
    • This technique is useful for live cell studies and enabling the analysis of structures beyond the diffraction limit.
    • Useful information is provided about how to improve the efficiency of studies across different setups including the use of biological materials and appropriate tools.

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