Mechanical Signals & Bioimaging PDF

Mechanical signals Microscopic mechanical signals -Forces applied by cells, Stiffness of cells Macroscopic mechanical signals -Blood pressure, lung expansion and contraction, forces during walking, stiffness of bones etc.  Mechanical signals Microscopic mechanical si...

Mechanical signals Microscopic mechanical signals -Forces applied by cells, Stiffness of cells Macroscopic mechanical signals -Blood pressure, lung expansion and contraction, forces during walking, stiffness of bones etc.  Mechanical signals Microscopic mechanical signals -Forces applied by cells, Stiffness of cells Macroscopic mechanical signals -Blood pressure, lung expansion and contraction, forces during walking, stiffness of bones etc. Traction Force Microscopy Colin-York et al Current Opinion in Biomedical Engineering 2018 Micropillars – to measure cell traction Makhija et al. BBRC 2015  FRET FRET change of a Src kinase biosensor at the presence of Src kinase or phosphatase Liu et al. Frontiers in Bioengineering and Biotechnology 2020 FRET responses of a cell with clear directional wave propagation away from the site of mechanical stimulation. In these images, a pulling force was applied via laser tweezers on a bead coated with fibronectin on the cell membrane.  Integrative Tension Sensor Imaging cell adhesive force at single molecule level Cell adhesive force imaging of HeLa cells on IT surface. Cells were incubated on the ITS surface for 1.5 hours. The force pattern consists of individual clusters produced by focal adhesions. Source: Mechanobiology: Methods and Protocols – Characterizing Cell’s Mechanosensing Response  Microindentation using AFM Principle of AFM: A laser beam is used to detect cantilever deflections towards or away from the surface. By reflecting an incident beam off the flat top of the cantilever, any cantilever deflection will cause slight changes in the direction of the reflected beam. Radmacher et al Frontiers in Neuroscience 2019  Profile Microindentation Source: Mechanobiology: Methods and Protocols – Quantifying Mechanical Properties of Cells Guillou et al Scientific Reports 2016  Optical Trap / Tweezer / Levitation 2018 Nobel Prize in Physics Dr. Arthur Ashkin for invention of the optical tweezers & their application to biological systems! https://en.wikipedia.org/wiki/Optical_tweezers Optical trapping in vivo: theory, practice, and applications. Favre-Bulle et al 2019  Mechanical signals Microscopic mechanical signals -Forces applied by cells, Stiffness of cells Bioimaging and Image Analysis Macroscopic mechanical signals Blood pressure, lung expansion and contraction, forces during walking, stiffness of bones etc.  Course Outline 0. Introduction to Mechanobiology (Why study mechanobiology, pioneering experiments in mechanobiology) 1. Molecular Mechanisms of Mechanotransduction (Mechanosensory molecules in cytoskeleton, focal adhesions, cell-cell junctions, nucleus, and ion channels) 2. Mechanobiology of cell behavior (migration, epithelial extrusion, dorsal closure) 3. Mechanobiology of organ systems (Cardiovascular, Bone, Cartilage, Liver, Nervous system) 4. Mechanobiology of disease (Muscular dystrophy, cancer, laminopathy) 5. Technology innovation for mechanobiology (Bioimaging, Image Processing, microfluidics, organoids,) 6. Mechanobiology in medical diagnostics and therapeutics (Cell therapy, Cancer diagnostics, Immune profiling) Image Processing Types of Images Pseudo-colour Pseudo-colour Grayscale to Binary Smoothening (Mean Filter) Smoothening (Mean Filter) Image Filter (Mean) Image Filter (Mean) Image Filter (Median, Laplace) Confocal imaging Reference: iBiology.org Confocal imaging Reference: iBiology.org Confocal imaging Confocal microscope uses a pinhole to block out-of-focus light Reference: iBiology.org Confocal imaging Reference: iBiology.org Detector When to use confocal Reference: iBiology.org Widefield microscopy captures images of the entire thickness of the sample, including out-of-focus structures, whereas confocal microscopy only captures images of the in-focus plane. Confocal microscopy produces sharper images with higher resolution than widefield microscopy When to use confocal Reference: iBiology.org Resolution Reference: iBiology.org  Resolution: Diffraction & Airy disk Diffraction is the spreading out of waves as they pass through an aperture or around objects. In the microscope, diffraction of light can occur at the specimen plane due to interaction of the light with small particles or features, and again at the https://semesters.in/diffraction/ margins of the objective front lens or at the edges of a circular aperture within or near the rear of the objective. For an object consisting of a single bright point object in a dark background, the image produced by a perfect microscope, according to diffraction theory, is the so-called Airy disk, consisting of a bright central spot, surrounded by a series of https://semesters.in/diffraction/ rings. Resolution: Diffraction limit Rayleigh criterion for the diffraction limit to resolution states that two images are just resolvable when the centre of the diffraction pattern of one is directly over the first minimum of the diffraction pattern of the other r = 0.61λ / NA, r is resolution, λ is the imaging wavelength NA is numerical aperture of the objective lens. For 10x objective of NA=0.25, the resolution (0.61 x 550nm) / 0.25=1.34µm. Super-resolution Microscopy Super-resolution microscopy breaks the diffraction barrier, enabling "nanoscopy" with substantially improved optical resolution of down to 5–20nm. This method uses the physical or chemical properties of adjacent fluorophores to resolve them from each other. PALM: photo-activated localization microscopy (Eric Betzig) STED: stimulated emission depletion microscopy (Stefan Hell), single-molecule spectroscopy (Moerner).  Super-resolution: PALM Kanchanawong et al, Nature, 2010 PALM microscopy uses photoactivatable fluorophores to resolve spatial details of tightly packed molecules. Once activated by lasers, fluorophores emit for a short period but eventually bleach. The laser stochastically activates fluorophores until all have emitted. Super-resolution: STED Stimulated Emission Depletion  STED In STED microscopy, a fluorescent probe is first excited by light from the ground state (OFF state) to an excited-state (ON state), and then it is de-excited either (i) by light, via stimulated emission, or (ii) spontaneously, via fluorescence emission. Nobel Prize in Chemistry 2014 https://en.wikipedia.org/wiki/STED_microscopy Vividomini et al, Nature Methods, 2018  Super-resolution: STORM STochastic Optical Reconstruction Microscopy (STORM): The activated state of a photoswitchable molecule must lead to the consecutive emission of sufficient photons to enable precise localization before it enters a dark state or becomes deactivated by photobleaching. The sparsely activated fluorescent molecules must be separated by a distance that exceeds the diffraction limit (in effect, greater than approximately 250 nanometers) to enable the parallel recording of many https://www.microscopyu.com/tutorials/stochastic-optical- reconstruction-microscopy-storm-imaging individual emitters.