Imaging Flashcards
Contrast
The ability to distinguish stuff (quantitative)
Contrast-enhancing techniques
Brightfield, Phase contrast, DiC, Hoffman modulation, darkfield, polarized light
Transforms differences in the cell into differences in brightness
Bright-field illumination
Background very bright, specimen dark. Refractive index of cells/biological material just slightly higher than water
Dark-field illumination
Only light scattered by specimen visible. Dark background, bright specimen.
Resolution
How fine you can see (qualitative)
Phase-constrast illumination
Thick part of cells appear dark cause higher viscosity. Restricted to thin specimens
DIC
Better resolution than bright field and PC.
Fluorescence
Photon excites electron with some energy P, electron drops down to ground state and emits some energy E which can be detected
Absorption-Emission fluorescence spectra
Absorption graph show higher energy –> shorter wavelength than the emission graph.
Stokes shift
Overlap of adsorption and emission graphs
Why fluorescence?
High contrast, easy labeling, imaging of living cells
Green fluorescent protein
Link to protein you want to express. If protein is created so is GFP and you can see if it worked.
Widefield microscopy
Fast and efficient, but low contrast compared to confocal point microscopy
Brownian motion
Random nanoparticle motion caused by temperature fluctuation of surrounding molecules
Confocal microscopy
Focusing of different points of the specimen reducing background noise, allowing for high resultion images that can be reconstructed into 3d visualizations.
Gaussian fit function
Intensifies bright pixels and darkens dark pixels, smoothening up pixelated images allowing for specific characterization and localization.
FLIM
Fluorescence Life-time Imaging Microscopy
High resolution cell fluorescent imaging. Image contrast is based on excited state life-time, t, variations across the sample. The higher t, the fewer photons that can be detected from that point.
TIRF
Total Internal Reflection Fluorescence
Fluorescence of samples close to a surface. Ideal for studying molecules/cells/bacteria attached to a surface or membrane.
QCM-D
Quartz Crystal Microbalance with Dissipation
Mechanical technique that measures the mass change at the quartz sensor including the water coupled to the oscillation.
Gold nanoparticles for cancer treatment
Nucleic acid delivery, drug delivery, photothermal theraphy (proposal A)
Plasmons
In simpler terms, plasmons are like synchronized movements of electrons in metals when they interact with light,
Key features of nanoplasmic structures
Field confinement - Plasmons allows light to be focused down to molecular dimensions
Field enhancement - enhancement of optical fields near metal nanostructures
Tunability of the enhancment effects in space and wavelength
FRET
Förster Resonance Energy Transfer
Measures distance between flourophores in molecules. Emission energy from one flourophore becomes absorption energy to another and so on. Used to study protein dynamic, folding, interactions.
SMLM
Single Molecule Localization Microscopy
Obtains information beyond diffraction limit (200 nm) by pinpointing positions of single fluorophores. Resolutions down to a few nanometers
SMLM: Blinking
Fluorophores in samples emit fluorescence briefly and spontaneously. At any given time, most fluorophores are dark and only a few of them are blinking, allowing to localize them by PSF:ing their position. The blinking of fluorophores is aquired as a long sequence of thousands of frames, in order to localize millions of fluorophores.
Blinking techniques are, PALM, STORM
