01. Light Microscopy - Handout.pdf

http://www.smsoptical.com/Accessories%20Images/M5-nosepiece-250.jpg MAIN PARTS OF LIGHT MICROSCOPE https://www.olympus-lifescience.com/en/ 1) MECHANICAL PART 2) OPTICAL PART 3) ILLUMINATING PART  MECHANICAL PART Stand Arm Head (body tube) Micr...

http://www.smsoptical.com/Accessories%20Images/M5-nosepiece-250.jpg MAIN PARTS OF LIGHT MICROSCOPE https://www.olympus-lifescience.com/en/ 1) MECHANICAL PART 2) OPTICAL PART 3) ILLUMINATING PART  MECHANICAL PART Stand Arm Head (body tube) Micro- and macroscrew Stage https://cemeq.ufg.br/n/97536-o-fabuloso-microscopio Revolving nosepiece with objectives Screws for moving the stage, condenser and screw for light regulation Switch https://cemeq.ufg.br/n/97536-o-fabuloso-microscopio  OPTICAL & ILLUMINATING PART OPTICAL PART: Eyepiece (ocular lens) Objective https://cemeq.ufg.br/n/97536-o-fabuloso-microscopio ILLUMINATING PART: Illuminating source Condenser  FUNCTION OF THE OBJECTIVE & THE EYEPIECE OBJECTIVE: → the most important and the most complex part of microscope → it DISTINGUISHES DETAILS and CREATES THE IMAGE of subject EYEPIECE: → magnifies the image created by objective → it works as a MAGNIFYING GLASS and makes the image visible for the human eye → for the creating of image, the SET OF EYEPIECE & OBJECTIVE is needed → if the eyepiece is put away, the object is not visible WORKING DISTANCE: → it is the distance at which an objective is able to focus → it is measured from the cover slip to the lens of the objective → the more the objective is magnifying the smaller is the working distance  TOTAL MAGNIFICATION OF MICROSCOPE (M) M = Mobj. x Meyep. x Kt Mobj. = magnification of objective Meyep. = magnification of eyepiece Kt = magnifying coefficient of the body tube (head of microscope) (usually = 1) the total magnification is the PRODUCT OF MAGNIFICATION OF INDIVIDUAL LENSES if the objective lens magnifies 100-fold and the eyepiece magnifies 10-fold, the final magnification will be 1000-fold  NUMERICAL APERTURE magnification (NA) 10x/0.25 numerical aperture (NA) NA = n x sin α n = THE REFRACTIVE INDEX of the environment between object (specimen) and objective (nair = 1, nimmersion oil = 1.5 (the same as the glass)) α = HALF-ANGLE of the cone of light entering the lens from the specimen objective 10x lens THE BIGGER IS THE VALUE 2α half-angle of the OF NUMERICAL α cone of light entering the lens APERTURE, THE BETTER IS THE RESOLUTION OF THE specimen MICROSCOPE https://micro.magnet.fsu.edu/primer/anatomy/numaperture.html  POWER OF RESOLUTION (d) THE ABILITY TO DISTINGUISH TWO VERY CLOSELY POSITIONED OBJECTS IT IS THE MINIMUM DISTANCE BETWEEN TWO DISTINGUISHABLE OBJECTS THE SMALLER IS THE λ VALUE OF d THE d= BETTER IS THE NAobj.+ NAcon. RESOLUTION OF THE MICROSCOPE d: The value of d depends on the numerical aperture of objective (NAobj.) and condenser (NAcon.) and from the wavelength (λ) of incident light The limit of resolution of a light microscope is about 0.2 μm  OBJECTIVES 1) DRY OBJECTIVE: NA = n x sin α nair = 1.000727 (1) NA = n x sin α αmax = 90° → sin 90°= 1 NAmax = 1 x 1 = 1 2) OIL-IMMERSION OBJECTIVE: - cedar oil is used as the immersion (n = 1.5) or we can use water (n = 1.3) - numerical aperture of the oil-immersion objective NAmax= 1.3 – 1.5 (better resolution).  USEFUL MAGNIFICATION → the value of NA states the RESOLVING POWER of the objective as well as the LIMITS FOR MAGNIFICATION → if the total (final) magnification of the microscope lies at interval from 500 to 1000 multiple of NA, we call this magnification USEFUL MAGNIFICATION → total magnification that is out of this interval is called EMPTY MAGNIFICATION USEFUL MAGNIFICATION: 500 x NAobj. < M < 1000 x NAobj. EMPTY MAGNIFICATION: magnification out of this interval USEFUL MAGNIFICATION https://nanopdf.com/download/optical-aberrations-and-objective-lens_pdf It is possible to Image is more magnified with distinguish the details no corresponding increase in present in an image detail resolution = image degradation  USEFUL MAGNIFICATION TASK Calculate the useful magnification, if we use objective 45x with NA = 0.65 and decide if it is right to use eyepiece magnifying 20x. TOTAL MAGNIFICATION: M = 45 x 20 x 1 = 900x USEFUL MAGNIFICATION: 500 x NAobj. < M < 1000 x NAobj. 500 x 0.65 - 1000 x 0.65 325x 650x If we use objective 45x in combination with eyepiece 20x, the total magnification will be 900x (higher than upper limit) = EMPTY MAGNIFICATION The objective must be combined with the eyepiece with a lower value of magnification, for example 10x – the total magnification is 450x and it is inside the interval of USEFUL MAGNIFICATION  IMMUNOFLUORESCENCE MICROSCOPE → FLUORESCENT MOLECULES absorb light at one wavelength and emit it at another, longer wavelength → FLUORESCENCE MICROSCOPE is similar to an ordinary light microscope except the illuminating light, from a very powerful source, is passed through two sets of filters 1. FILTER → filters the light before it reaches the specimen, only the wavelengths that excite the particular fluorescent dye can pass 2. FILTER → filters the light obtained from the specimen, it passes only those wavelengths emitted when the dye fluoresces  IMMUNOFLUORESCENCE MICROSCOPE Molecular Biology of the Cell, Sixth Edition; ISBN: 9780815344643  IMMUNOFLUORESCENCE MICROSCOPE → immunofluorescence microscopy is most often used to DETECT SPECIFIC PROTEINS OR OTHER MOLECULES in cells and tissues → a widely used technique is to COUPLE FLUORESCENT DYES TO ANTIBODY MOLECULES, which then serve as reagents that bind selectively to the particular macromolecules → 2 commonly used fluorescent dyes: FLUORESCEIN (FITC) (green) RHODAMINE (TRITC) (red) MARKED PRIMARY ANTIBODY  endothelial cells https://www.microscopemaster.com /fluorescent-dyes.html https://en.wikipedia.org/wiki/Spindle_apparatus https://commons.wikimedia.org/wiki/File:Yeast_membrane_proteins.jpg mitotic spindle yeasts neuron stromatolite https://www.researchgate.net/figure/Figure-C-4-Kryton-Argon-confocal- https://frontal-cortex.tumblr.com/post/34502613123/fixed-neuron- images-on-fluorescence-in-situ-hybridisation-FISH-on_fig42_267851913 a-multi-wavelength-three  ELECTRON MICROSCOPE → the source of illumination is the BEAM OF ELECTRONS → air must be pumped out of the column to create a VACUUM → the electron microscope uses ELECTROSTATIC & ELECTROMAGNETIC LENSES in forming the image → electron microscopes have much greater resolving power than light microscopes and can obtain much higher magnifications of up to 2 million times, while the best light microscopes are limited to magnifications of 2,000 times  ELECTRON MICROSCOPE TYPES OF ELECTRON MICROSCOPES: TRANSMISSION ELECTRON MICROSCOPY (TEM): → is principally quite similar to the compound light microscope, by sending an electron beam through a very thin slice of the specimen. SCANNING ELECTRON MICROSCOPY (SEM): → visualizes details on the surfaces of cells and particles and gives a very nice 3D view. Specimen is coated with an ultrathin metal coating (gold, platinum, osmium). SEM images the sample surface by scanning it with a high-energy beam of electrons in a raster scan pattern.  thylakoids https://ucmp.berkeley.edu/glossary/gloss3/photosyn/pigments.html GA in plant cell https://www.sciencephoto.com/media/546891/view/golgi-apparatus-tem flagellum fhttps://www.researchgate.net/figure/A-D- Chlamydomonas Electron-micrographs-of-C-reinhardtii-flagella- the atomic structure of a diamond A-and-C-Wild-type-g1-B-and- https://ja.m.wikipedia.org/wiki/%E3%83%95%E3%82%A1%E3%82 https://www.britannica.com/technology/electron-microscope D_fig3_5690895lagellum %A4%E3%83%AB:Chlamydomonas_TEM_04.jpg  a section of a cell of Bacillus subtilis https://commons.wikimedia.org/wiki/File:Bacillus_subtilis.jpg transmission electron micrograph of a myelinated axon https://sk.m.wikipedia.org/wiki/S%C3%BAbor:Myelinated_neuron.jpg electron micrograph of an ultra-thin section of a dividing pair of group A streptococci (20,000 x) http://textbookofbacteriology.net/structure_1  an image of a house fly compound eye surface (450x) http://archive.boston.com/bigpicture/2008/11/peering _into_the_micro_world.html an image of a fruit fly's compound eye https://commons.wikimedia.org/wiki/File:Drosophilidae _compound_eye_edit1.jpg https://en.wikipedia.org/wiki/Scanning_electron_microscope#/media/File:Misc_pollen.jpg  hematite https://upload.wikimedia.org/wikipedia/commons/9/99/Hematite_in_Scanning_Electron_Microsco pe%2C_magnification_100x.JPG https://commons.wikimedia.org/wiki/File:LT-SEM_snow_crystal_magnification_series-1.jpg Gills of a fish, the mudskipper (Periophthalmus argentilineatus) https://slideplayer.com/slide/14401790/ http://www2.odn.ne.jp/~umisuzume/yaima001/2002/tonton_s ebire.html the fibrous configuration of a dry macrofoam sponge swab snow crystal http://www.publicdomainfiles.com/show_file.php?id=13531896611989  Juglans nigra (Black Walnut tree) lower leaf Lower surface of Arabidopsis thaliana leaf, surface, showing a variety of trichomes showing a trichome http://archive.boston.com/bigpicture/2008/11/peering_into_the_micro_world.html http://archive.boston.com/bigpicture/2008/11/peering_into_the_micro_world.html tobacco seed https://mvac.uwlax.edu/ProcessArch/lab_floral.html  close-up of a weevil (Curculionidae family) image of pyralidae moth, side view of head http://archive.boston.com/bigpicture/2008/11/peering_into_the_micro_world.html http://archive.boston.com/bigpicture/2008/11/peering_into_the_micro_world.html close-up of an ant a breast cancer cell http://archive.boston.com/bigpicture/2008/11/peering_into_the_micro_world.html http://archive.boston.com/bigpicture/2008/11/peering_into_the_micro_world.html  leukemia cells (left) shown in comparison with bone marrow cells (right) https://pt.slideshare.net/mcasade8/microscopes-7241414 Staphylococcus epidermidis cluster https://owlcation.com/stem/Staphylococcus-epidermidis-Biofilms-and-Antibiotic-Resistance Mite feeding on a mystacocarid https://www.sciencesource.com/Doc/TR7/3/d/1/a/SS2619827.jpg?d63643046111