Histology 5 - Microscopy in Medicine and Research PDF

The function of a microscope is that of magnifying and resolving images. Microscopes increase the resolving power of our eyes Basic elements of a microscope: Light source to illuminate the samples (es: laser or ltered light) Lens (increase the resolving power of the object and magnifying it)...

The function of a microscope is that of magnifying and resolving images. Microscopes increase the resolving power of our eyes Basic elements of a microscope: Light source to illuminate the samples (es: laser or ltered light) Lens (increase the resolving power of the object and magnifying it) Detector (es: our eyes or a camera) This is exactly how our sight works (light+lens+detector) Resolving power (or resolution) —> the minimum distance needed between two objects in order for the microscope to distinguish them as two separate objects The resolving power of a light microscope is 200nm —> if we have two objects closer than 200nm we can’t see them under the microscope To go beyond the 200nm limit we have to use a TEM (0.17nm) or the super resolution technique The Abbe’s formula determines the resolving power of a microscope aught lightwave go halfangleofaperture go ofthe objective refractiveindex theof mediumbetweentheobject andthe objective Refractive index (n) —> dimensionless quantity that expresses how light goes through a medium. Light changes its path when travelling through two media with different refractive index The light of the microscope has to pass through different media (air, glass, tissue…). The refractive index should be as similar as possible Numerical aperture (NA) —> a mix of the refractive index and the angle —> dimension of the part of the objective that is able to collect light. Higher numerical aperture means higher resolution  By using oil we can reduce the refraction as its refractive index is closer to that of the glass. theref index native is if between different t media he theresolutionislower opticalmicroscope Light sources —> lamp, laser, arc lamps… Condenser —> converges the light beams in a single focal point. It provides a homogeneous illumination of the sample and it increases the resolving power and the contrast Objectives —> they are the core of every microscope. The objective barrel contains magni cation, numerical Gent aperture (the bigger it is the more light is collected) ses and working distance (distance between the lens and the onthe the specimen) seize Detectors —> eyepieces (magnify the image and prepare it for the eye, they usually have a collar for myopia), scienti c cameras (can record the whole image of very fast processes) and photomultiplier tubes (PMTs) (transform a photon into an electron which is then ampli ed to a measurable current Bright- eld microscope —> you can see the objects by contrast with the white light The Köhler illumination —> method of illumination with an extremely even illumination of the whole eld of view. To do this you have to have a condenser focus, a eld diaphragm (optical element to control the amount of light) and an aperture diaphragm Field diaphragms regulate the amount and positioning of light that arrives at the condenser and delimitate the microscopic visible eld Aperture diaphragm optimises the contrast of the image by controlling the amount of light that goes to the objective Observation methods: Phase contrast —> takes advantage of the difference between the refractive indexes of the medium Dark eld —> a speci c condenser creates an oblique illumination instead of a perpendicular —> lights up particles on a dark background Differential interference contrast (DIC) —> creates an image where steeper regions are lighter and thus highlighting surface morphology Fluorescent microscopy —> uses light with different wavelengths to selectively excite speci c molecules ( uorophores) Fluorophores — > organic or inorganic dyes with some proteins able to absorb energy from a speci c wavelength of light and emit light at a longer wavelength. Every uorophore has an excitation peak (where it absorbs most energy) and an emission peak (where the most light is emitted). The distance between the excitation peak and the emission peak is called stokes shift the antibodiesstainedwith npohoneCys geo emaeonleughtofthe aught wave to backtolaver goenergy Fluorescence microscopes are composed of an excitation lter to light up the specimen around the excitation peak and an emission lter to collect the light around the emission peak. A dichroic mirror re ects excitation light but allows emission light to pass thus separating the two. By selectively marking speci c protein with uorophores-conjugated antibodies the uorescence microscope allows the study of multiple proteins/functions at the same time Confocal microscope uses a pinhole before the detector to cut the light coming to from the out-of-focus planes. This results in an optical sectioning of the specimen. This de nition was rst given by Marvin Minsky. Colin Shepard built the rst working confocal microscope Laser scanning confocal microscope scans the specimen with laser and scanning mirrors. Emitted uorescence goes through a pinhole before reaching the detectors. This results in an optical sectioning on Z-axis, cutting out the light coming from the out-of -focus planes. Dynamic processes are usually seen with a bright- eld microscope. You have to choose your system according to what you’re trying to see Two-photon microscopy —> like the confocal microscopy but uses pulsed-infrared laser to provide optical sectioning. Infrareds can go deeper into tissues (utile for 3D reconstruction and imaging of dynamic processes) Grim onlyspotof excitation The probability of two photons exciting the same object at the same time is very low (Heisenberg principle). Where the amount of photon is higher (focal point, where we have focused the light) there is a higher chance of two photons exciting the same uorophores. We use two-photon microscopies to visualise biological properties of alive animals —> animal is anesthetised and the organs are exposed. Additional optical microscopy techniques: TIRF  FRAP FRET FLIM Spinning disk Light Sheet SUPER RESOLUTION MICROSCOPY (!) Helped by uorescent molecules the Nobel Laureates in chemistry 2014 ingeniously circumvented the 200nm limitation —> optical microscopy was brought in the nanodimension. If too much energy is given to the uorophores they get bleached —> nothing can be seen anymore as the uorophore is destroyed. We can’t increase too much the power of the depletion laser (or it will go prom depletion to bleaching) thisexcites terisbeocke from illumination resulting spot Y comm genergyofthe depeerioneaser Digital image analysis—> digital images are the numeric representation of an optical image. They provide information that can be processed and quanti ed thus allowing not only to magnify and visualise biological processes but also to measure them —> digital images are a tool to test scienti c hypothesis Starting from microscopic analysis we can reconstruct large portions of tissue to a mesoscopic scale