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-MICROSCOPY-Human VisionoImportant: retina populated by rods and cones-Cones - color sensing -Rods - sensitivityoThere is spatial organization of ligh sensing cells -Color-vision is lost in low-light, rods remained saturated in moderate light conditionsoFovea is densely populated by cones but sparsely populated by rods-Foveola = avascular zoneoResolution: The smallest detectible distance between two objects-Microscopes FunctionoObjective lens collects cone of light rays to create imageoCondenser lens focuses a cone of ligh onto each point in the specimenoResolution = 0.61(lambda)/(nsin(theta))-Theta = half the angular width of the cone of rays collected by the objective (max value of 1)-N = refractive index of the medium (air or oil usually)-Lambda = wavelength of light used (white light is 0.53 um)-Nsin(theta) = numerical aperature is function of light collecting ability. Dry lense max is 1, oil is 1.4. Higher the numeircal aperature the greature the resolution and the brighter the image (very short working distance and small depth of field)oLambda is emission wavelength in fluoresce-Properties of lighto wave nature - wavelength polarizationoParticle nature - relflection, refraction, scatteringoUtilize different properties of light for different microscope techniques-Brightfield - look at how light is absorbed-Phase - look at how light is phase shifted-Differential Interference Contrast - look at how light is differentially phase shifted-Fluorescence - look at how light is absorbed and re-emittedoJablonski Diagram-Once a molecule has absorbed energy in the form of electromagnetic raditation,there are a number of routes by which it can return to ground state oStoke's shift-When a photon is released its emission is at a lower energy so than the light absorbed, so it will have a larger wavelength-Fluorescence Microscopy: use of one wavelength of light to "excite" a fluorophore and then detect the emiitted lightoFluorophores: molecules or proteins that absorb at one wavelength and emit at anotheroNeed to remove other intense waves:-Fluorescent probes (fluorophores)-Barrier and bandpass filtersFilters preferentially transmit or reflect discrete wavelengths of light-1 is the first barrier filter, lets through blue light wavelength in this example-2 is beam-splitting mirror: reflects light below a certain value, but transmits light above (ie 510 nm)-3 is second barrier, cuts out unwanted fluorescent signals passing the specific green fluorescein emission between 520 and 560-How do we put probes into place? Antibodes are favorite protein or amino acid sequence linked to a reporterenzyme, particle, flurophore etcRadioisotopes: radioactive forms of metabollic materials including ATP, methionineFluorescent proteins - conjugated to favorite protein or built into bio-sensorSynthetic fluorescent molecules - conjugated to proteins, or reporters of local chemistry of physical propertiesTake immobilized antigen-Primary antibody (against antigen A)-Secondary antibody ( against primary antibody)-Will have marker attached Radioisotopes: pulse chase experiement-Determine the activity of certain cells over a prolonged period of time -Protein kinase etc-First exposed to radioactive lable (pulse) nd then used into metabolic processes, then unlabed chase is introduced (washes out signal)Fluorescnet Proteins allow for the production of genetically encoded contrast agents-Useful for …. IdkoDynamic Processes-Microscopy can be used to see invisible properties such as activity, chemical environment, and strain or elastic modulus-Can be revealed by photo-activation, photo-uncaging, photo-bleachingCalculate diffusion coeffs, and half life of proteins in complexesoBiosensors-Fura Compounds/reporters of ion concentration are chelatorsNeutralize the ion charge and in the process alter their ability to fluorescently absorb/emit light-FRET (fluorescence resonant energy transfer) can reveal when two proteins are nearby or the conformation of an enzyme or multi molecular complex changesRequires two flurophores that quench/transfer photons between the donor and the acceptor. Can use any fluorophore.-Before imaging cells you need to load reporters into the cellsMicropipette containing substance X-Micro injection into the cellCell places in substance X between electrodes followed by shock-Transient pored made in the membrance allow substance to enter cellMembrane-enclosed vesicles contain X-Induced plasma membrane fusion between vesicles and plasma membrane of target cell releases substance into cytoplasmGold particles coated with DNA-DNA coated gold particles shot into cell at high velocity allows stabletransformation or transient expression of new genes-Review oThe optical train (Resolution)-Using properties of light and how light interacts with matterAbsorption, phase delaym polarization etc-Sample prep (contast)-Analysis and FunctionoKnow your eyes! -Fluorescence-Advantages: can follow live events, can interogate local environment (biosensors)-Disadvantages: Sea of fluorescence - excitation light re-enters objective from all layers of sampleoCan resolve sub cellular structures but not protein level structuresoPhototoxicityAlternatives:oTIRF-use critical-angle excitiation to illuminate only the thin section adjeacent tothe coverslip-EVANESCENT WAVE excites fluorophores in sampleoActin filaments, myosin GFP so smallll stuffGreen excitation laser fed into optical axis at rear of objective -Confocal/Deconvolutionuse pinholes/point-spread functions to remove out of plane fluorescence, reduces signal levelsOptical signagture of a single point of fluorescence recovered through numerical decon with point spread functionCCD camera collects z-seriesProblemsoPenetration depthoSperical abberationoRinging and noise amplification**marvin minsky**oNeeded to study cells within the brain"One day it occurred to me that the way to avoid all that scattered light was to never allow any unnecessary light to enter in the first place""Use a pinhole to exclude out-of-focus light"Focused Illumination: focusing incident light to minimize fluorescnece from neighboring structuresoPinhole detection - a pinhole placed in front of the detector blocks light scattered from structures out of the focal axisCLSM:oHigh power light sources (laser)oHigh sensitivity detectors -PMTsoHigh speed scanning/descanning - computers-Potential EC:Oxford optoelectronics shipped


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Pitt BIOSC 1500 - MICROSCOPY

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