Lecture 1--microscopy

The infrared illumination also twice as deep

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Unformatted text preview: ght c annot penetrate deeply into specimen two photons have to c oincidentally excite it out of focus light can bleach samples. use double the optimale excitation wavelength Limit of light microscopy: 200-250 nm wavelenght of light puts limit on how much it can dfract apoint is foing to eb spread so much by light, even with best obejctive, that property of light is omething y ou cannot change deconvolution, argorithm to s ee how it would look like without the sdistorter light super-resolution imagong transcends the limits of light microscopy Limit of light microscopy: 200-250 nm Super-resolution imaging transcends the limits of light microscopy What limits light microscopy? --The point spread function (PSF): The size of a single point of light diffracted through a microscope never exact size --Objects smaller than the PSF appear the same size, and objects that are closer than the PSF if you have two 15 cannot be distinguished micrometers appart,objectslook like theyll one blob, not two different things --For most light microscopes PSF is 200-250 nm in X/Y plane, and 450-700 nm in z direction Super-resolution microscopy improves on the PSF by at least two-fold at least 100 nm resolutions Galbraith CG, Galbraith JA. (2011). Super-resolution microscopy at a glance. J Cell Sci.15:1607. How does super-resolution work? --Modulate excitation of the sample either in space or time all are fluorescent microspcopy. Galbraith CG, Galbraith JA. (2011). Super-resolution microscopy at a glance. J Cell Sci.15:1607. How does super-resolution work? --For example, Structured illumination microscopy (SIM) images are generated by illuminating specimen with a striped pattern and recording the interference; the illumination is then rotated and the interference patterns can be computationally reconstructed into an image; allows ~100 nm resolution not illuminating all sample at once Galbraith CG, Galbraith JA. (2011). Super-resolution microscopy at a glance. J Cell Sci.15:1607. SIM microscopy Nikon Technology web page How does super-resolution work? --For example, Structured illumination microscopy (SIM) images SIM Confocal Gustafsson lab, HHMI How does super-resolution work? --In single molecule techniques, points in a sample are illuminated one by one and the centroid of the illuminated sample is determined, by stochastically il...
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