MICROSCOPY Human Vision o Important retina populated by rods and cones Cones color sensing Rods sensitivity o There is spatial organization of ligh sensing cells Color vision is lost in low light rods remained saturated in moderate light conditions Fovea is densely populated by cones but sparsely populated by rods Foveola avascular zone Resolution The smallest detectible distance between two objects o o Microscopes Function o Objective lens collects cone of light rays to create image o o Condenser lens focuses a cone of ligh onto each point in the specimen Resolution 0 61 lambda nsin theta Theta half the angular width of the cone of rays collected by the objective max value of 1 Lambda wavelength of light used white light is 0 53 um N refractive index of the medium air or oil usually 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 o Lambda is emission wavelength in fluoresce Properties of light wave nature wavelength polarization Particle nature relflection refraction scattering o o o Utilize 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 emitted o Jablonski Diagram o Stoke s shift 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 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 light o Fluorophores molecules or proteins that absorb at one wavelength and emit at another o Need to remove other intense waves Fluorescent probes fluorophores Barrier and bandpass filters Filters preferentially transmit or reflect discrete wavelengths of light How do we put probes into place 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 Antibodes are favorite protein or amino acid sequence linked to a reporter enzyme 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 Idk o Dynamic 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 complexes o Biosensors 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 stable transformation or transient expression of new genes Using properties of light and how light interacts with matter Absorption phase delaym polarization etc Review o The optical train Resolution Sample prep contast Analysis and Function o Know 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 sample Can resolve sub cellular structures but not protein level structures Phototoxicity Alternatives o TIRF use critical angle excitiation to illuminate only the thin section adjeacent to the coverslip EVANESCENT WAVE excites fluorophores in sample o Actin 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 Problems o Penetration depth o Sperical abberation o Ringing and noise amplification marvin minsky o Needed 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 structures o Pinhole detection a pinhole placed in front of the detector blocks light scattered from structures out of the focal axis CLSM o o o High power light sources laser o High sensitivity detectors PMTs o High speed scanning descanning computers Potential EC Oxford optoelectronics shipped lasersharp SOM Biorad shipped MRC 500 Sarastro shipped phoibos 1000 Confcol maintains intact 3D info for image reconstruction Scanning