Microscopes PDF

❑ Microscope ⮚ Optical Principals of Microscope ⮚Optical Resolution ⮚Numerical Aperture(N.A.) -Resolution(R) ⮚Bright Field Microscopy ⮚Dark Field Microscopy ⮚ Phase Contrast Microscopy A 17th century Compound Microscope ⮚Fluorescence Microscope ⮚Confocal Microscope ⮚Electron Microsco...

❑ Microscope ⮚ Optical Principals of Microscope ⮚Optical Resolution ⮚Numerical Aperture(N.A.) -Resolution(R) ⮚Bright Field Microscopy ⮚Dark Field Microscopy ⮚ Phase Contrast Microscopy A 17th century Compound Microscope ⮚Fluorescence Microscope ⮚Confocal Microscope ⮚Electron Microscope ⮚Scanning Electron Microscope ❑ Microscope ⮚ A microscope is an instrument for viewing objects that are too small to be seen by the naked or unaided eye. ⮚ The name microscope was coined by Johannes Faber (1574-1629) who in 1628 borrowed from the Greek to combined micro-, small with skopein, to view. ⮚ The science of investigating small objects using such an instrument is called microscopy, and the term microscopic means minute or very small, not easily visible with the unaided eye. In other words, requiring a microscope to examine. A 17th century Compound Microscope ⮚ The most common type of microscope is the optical microscope. This is an optical instrument containing one or more lenses that produce an enlarged image of an object placed in the focal plane of the lenses. ⮚ There are two kinds of Optical Microscopes: 1- Simple Optical Microscope 2- Compound Optical Microscope ❑ Optical Principals of Microscope Basic optical principles of a microscope is quite simple: ⮚ The objective lens is a very high powered magnifying glass with a very short focal length. This is brought very close to the specimen being examined so that the light from the specimen comes to a focus about 160 mm inside the microscope tube. This creates a virtual and enlarged image of the subject. This image is inverted and can be seen by removing the eyepiece and placing a piece of tracing paper over the end of the tube. By careful focusing a rather dim image of the specimen, much enlarged can be seen. It is this virtual image that is viewed by the eyepiece lens that provides further enlargement. ⮚ Usually, the eyepiece is a compound lens, which is made of two lenses one near the front and one near the back of the eyepiece tube forming an air separated couplet. So, the virtual image comes to a focus between the two lenses of the eyepiece, the first lens bringing the virtual image to a focus and the second lens enabling the eye to focus on the image. ⮚ In all microscopes the image is viewed with the eyes focused at infinity. Headaches and tired eyes after using a microscope are usually signs that the eye is being forced to focus at a close distance rather than at infinity ❑ Optical Resolution A lens magnifies by bending light. Optical microscopes are restricted in their ability to resolve features by a phenomenon called diffraction. It is based on the numerical aperture (NA or AN) of the optical system and the wavelengths of light used (λ), sets a definite limit to the optical resolution (Rlim). Assuming that optical aberrations are negligible, the resolution (R) is given by: λ of 550 nm is assumed. With air as medium, the highest practical NA is 0.95, Rlim~ N.A. and with oil, up to 1.5. Due to diffraction, even the best optical microscope is limited to a resolution of 0.2 micrometers. Rlim~ Rlim~ They are all approximations nsinθ D  ❑ Parts of a Microscope ⮚ Compound optical microscopes can magnify an image up to 1000× ( or rarely 2000x) and are used to study thin specimens as they have a very limited depth of field. Typically they are used to examine a smear or a thinly sectioned slice of some material. With a few exceptions, they utilize light passing through the sample from below and special techniques are usually necessary to increase the contrast in the image to useful levels. On a standard compound optical microscope, there are three objective lenses: a scanning lens (4×), low power lens (10×), and high power lens (40×). ⮚ Advanced microscopes often have a fourth objective lens, called an oil immersion lens. To use this lens, a drop of oil is placed on top of the cover slip, and the lens moved into place where it is immersed in the oil. They usually has a power of 100×. The actual power or magnification is the product of the powers of the eyepiece, usually about 10×, and the objective lens being used. To study the thin structure of metals and 1- Ocular(eyepiece); 2- Nosepiece 3- Objective Lenses 4- Coarse Adjustement Knob minerals, another type of microscope is used, where the light is 5- Fine Adjustement Knob 6- Object Holder(Stage 7- Mirror 8-Diaphragm(Condenser) reflected from the examined surface. The actual power (magnification)=power of the eye piece. (times) power of the lens  ❑ Stereo Microscope ⮚ The stereo or dissecting microscope is designed differently from the diagrams above, and serves a different purpose. It uses two separate optical paths with two objectives and two eypieces to provide slightly different viewing angles to the left and right eyes. In this way it produces a three- dimensional(3-D) visualisation of the sample being examined. ⮚ The stereo microscope is often used to study the surfaces of solid specimens or to carry out close work such as sorting, dissection, microsurgery, watch-making, small circuit board manufacture or inspection, and the like. ⮚ Great working distance and depth of field are important qualities for this type of microscope. Both qualities are inversely correlated with numerical angle (NA): the higher the NA, the smaller the depth of field and working distance. NA depth of field working distance ⮚ The stereo-microscope should not be confused with ordinary compound microscopes equipped with a binocular eyepieces. In these microscopes both eyes can see the image but the binocular head provides greater viewing comfort and slightly better appearance of resolution. However the image in such microscopes remains monocular. ❑ Other Medical Devices ❑ Otoscope ⮚ An Otoscope is a medical device which is used to look into the ears. Health care providers use otoscopes to screen for illness and investigate when a symptom involves the ears. With an otoscope, it is possible to visualize the outer and middle ear. ⮚ Otoscopes consist of a handle and a head. The head contains an electric light source and a low power magnifying lens. The front end of the otoscope has an attachment for disposable plastic ear speculums. After inserting the ear Image of an Otoscope and accessories speculum side of the otoscope into the external ear, the examiner looks through a lens on the rear of the instrument to see inside the ear canal. ⮚ Many models have a detachable sliding rear window which allows the examiner to insert instruments through the otoscope into the ear canal, such as for removing earwax (cerumen). Most models also have an insertion point for a bulb capable of pushing air through the speculum. This puff of air allows an examiner to test the mobility of the tympanic membrane.  ❑Bright Field Microscopy ⮚ Some specimens are considered amplitude objects because they absorb light partially or completely, and can thus be readily observed using conventional bright field microscopy ⮚Simple set up with basic equipment ⮚The technique can only image dark or strongly refracting objects effectively. ⮚Low optical resolution. Diffraction limits resolution to approximately 0.2 micron. Diffraction limited ⮚Out of focus light from points outside the focal plane reduces image clarity. ⮚ reducing or inreasing amount of ligth source How to make enhancement? ⮚Use of oil immersion objective ⮚Use of staining methods http://zeiss- ⮚Use of filters on the light source campus.magnet.fsu.edu/articles/basics/res olution.html  ❑Bright Field Microscopy Low optical resolution. Diffraction limits resolution to approximately 0.2 micrometers in the best conditions Use of filters on the light source Resolution of an optical device (eye or microscope) is its ability to distinguish between two very closely placed objects as separate objects. The resolution limit (resolving power) of a microscope depends on wavelength of light numerical aperture (NA) of the lens system used. Abbe Equation Resolution (Limit) ~ λ/NA ~ λ/2n Sinθ ~ λ/n Sinθ Resolution The ability to distinguish two very small and closely spaced objects as separate entities Best when the distance separating is small ❑Bright Field Microscopy N.A. ~ nsin(θ) Rlimit ~ λ/N.A. λ = wavelength of light used; 450-750 nm for visible light used in a compound microscope; Blue light has the shortest wavelength (λ =450nm) gives maximum resolution. Therefore, blue filter blue light is commonly used in microscopy. NA – n Sin θ; where n is the refractive index of the medium (usually air or oil) between the specimen and objective lens. For air n = 1.0 and for immersion oil, n = 1.5. θ or a- half angle of the cone of light entering the objective lens from the specimen. If oil and blue light used, then resolution power is 0.2 micrometers  Sin (θ) θ θ θ θ θ θ Higher NA collects more light NA More light Resolution limit Rlimit ~ λ/N.A. Higher NA, Resolution limit is smaller, improved resolution (better resolution) ❑ Working Distance light microscope can never resolve two closer particles less than about 0.2 µm apart, no matter how many times the image is magnified. The resolution of electron microscope is about 0.0001 µm (0.1 nm) whereas the human eye is about l00 µm. Bright-field microscopy is a standard light microscopy technique, and therefore magnificationis a standard light microscopy technique, and therefore magnification is limited by the resolving poweris a standard light microscopy technique, and therefore magnification is limited by the resolving power possible with ❑Dark Field Microscopy ⮚ Dark field microscopy produces an image with a dark background ⮚In dark-field microscopy, the non-diffracted rays are removed altogether so that the image is composed solely of diffracted wave components. ⮚Live and unstained samples, increase the contrast ⮚Sample must be strongly illuminated which can damage the sample  http://www.nobelprize.org/educational/physics/microscopes/phas ❑Phase Contrast Microscopy phase shifts in light passing through a transparent specimen to brightness changes in the image. the human eye is only sensitive to amplitude The same cells imaged with traditional bright variations. Phase changes are invisible. field microscopy (left) and with phase contras phase contrast microscopy makes phase microscopy (right). changes visible. ⮚No need staining the change in phase can be increased to half a wavelength by a transparent phase-plate in the microscope and thereby causing a difference in brightness. This makes the transparent object shine out in contrast to its surroundings Comparison of transillumination techniques used to generate contrast in a sample of tissue paper. 1.559 μm/pixel. Bright field Dark field illumination, Phase contrast illumination, sample sample contrast illumination, sample contrast comes comes from contrast comes from from absorbance of light scattered by the interference of different light in the sample. sample. path lengths of light through the sample.  ❑Fluorescence Microscopy ⮚ A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence ⮚Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic ⮚Fluorescent dyes, also known as fluorophores or fluorochromes, radiation are molecules that absorb excitation light at a given wavelength (generally UV), and after a short delay emit light at a longer wavelength. Excitation Emission Spectrum Spectrum Live Cells Fixed Cells Localize/measure enzyme activity  Fluorescence Photoluminescence, light energy, or photons, stimulate the emission of a photon. Fluorescence, specifically, is a type of photoluminescence where light raises an electron to an excited state. The excited state undergoes rapid thermal energy loss to the environment through vibrations, and then a photon is emitted from the lowest- Jablonski lying singlet excited state. diagram  Confocal Microscopy ⮚Light can certainly penetrate through tissue ⮚The confocal microscope is designed to accept light only from a thin slice within the tissue and to reject light reflected and scattered from other regions. ⮚parallel beam of light for illumination of the object (Laser source)  Confocal Microscopy Laser Scanning Confocal Microscope Laser beam used to illuminate spots on specimen we must scan the region point by point a very thin section within the tissue A photomultiplier computer compiles images created from each point to generate a 3- dimensional image Live Cells Fixed Cells Localize/measure enzyme activity TGs in atherosclerotic remodeling 18.75 μm FXIIIA colocalizes with CD68+ cells as shown by immunofluorescent doublestaining; nuclei stained with DAPI Thrombin induces stress fiber formation and TG2 re-distribution in HUVECs 50 μm Rendering SMC in 3D Confocal Microscopy  Electron Microscope Transmission Electron Microscope (TEM) magnify up to 250,000x TEM is electron illuminated. This gives a 2-D view. The samples must be dry, thin, and in some cases coated. Thin slices of specimen are obtained. The electron beams pass through this. It has high magnification and high resolution.  Electron Microscope Transmission Electron Microscope mitochondrion  Scanning Electron Microscope (SEM) magnify up to 100,000x SEM use electron illumination. The image is seen in 3-D. It has high magnification and high resolution. The specimen is coated in gold and the electrons bounce off to give you and exterior view of the specimen. The pictures are in black and white. scanning probe microscopy (SEM) have extremely high resolution can be used to observe individual atoms pigeon blood