{"id":1552,"date":"2015-05-22T12:18:29","date_gmt":"2015-05-22T12:18:29","guid":{"rendered":"http:\/\/candle.am\/microscopy-2\/"},"modified":"2015-07-03T11:44:50","modified_gmt":"2015-07-03T11:44:50","slug":"microscopy","status":"publish","type":"page","link":"https:\/\/candle.am\/hy\/microscopy\/","title":{"rendered":"\u0544\u0561\u0576\u0580\u0561\u0564\u056b\u057f\u0561\u056f\u056b \u056f\u0561\u0575\u0561\u0576"},"content":{"rendered":"<p><strong><img loading=\"lazy\" decoding=\"async\" class=\"alignnone wp-image-2017 size-thumbnail\" src=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/IMG_78891-129x150.jpg\" alt=\"IMG_7889\" width=\"129\" height=\"150\" srcset=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/IMG_78891-129x150.jpg 129w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/IMG_78891-258x300.jpg 258w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/IMG_78891-768x893.jpg 768w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/IMG_78891-911x1060.jpg 911w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/IMG_78891-280x326.jpg 280w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/IMG_78891-456x530.jpg 456w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/IMG_78891.jpg 1709w\" sizes=\"auto, (max-width: 129px) 100vw, 129px\" \/>Coordinator \u2013 Dr. <a href=\"mailto:tatikyan@asls.candle.am\">Stepan Tatikyan<\/a><\/strong><\/p>\n<p style=\"text-align: justify;\">Our Two-Photon Fluorescence Laser Scanning Microscopy System (MOM Sutter instruments) is available for users from other institutions. For sample excitation Amplitude System infrared pulse laser is used.<\/p>\n<p>Wavelength: 1030 nm<br \/>\nEnergy: up to 25 nJ<br \/>\nPulse length: 280 fs<\/p>\n<p><a href=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/IMG_201-3.jpg\"><img loading=\"lazy\" decoding=\"async\" class=\"  aligncenter wp-image-2252 \" src=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/IMG_201-3-400x300.jpg\" alt=\"IMG_201-3\" width=\"407\" height=\"305\" srcset=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/IMG_201-3-400x300.jpg 400w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/IMG_201-3-150x113.jpg 150w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/IMG_201-3-768x576.jpg 768w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/IMG_201-3-706x530.jpg 706w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/IMG_201-3-280x210.jpg 280w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/IMG_201-3.jpg 945w\" sizes=\"auto, (max-width: 407px) 100vw, 407px\" \/><\/a><\/p>\n<p style=\"text-align: center;\">If you are interested in microscope usage please contact coordinator.<\/p>\n<ul>\n<li><a href=\"#04\">2 photon Fluorescence Microscopy Applications <\/a><\/li>\n<li><a href=\"#01\">Multiphoton Laser Microscopy<\/a><\/li>\n<li><a href=\"#03\">Experiments<\/a><\/li>\n<\/ul>\n<p><a name=\"04\"><\/a><\/p>\n<h3 style=\"text-align: center;\"><span style=\"color: #3366ff;\">Application of 2 photon\u00a0Fluorescence Microscopy<\/span><\/h3>\n<p>2 Photon Fluorescence Microscopy is widely used in many fields of:<\/p>\n<ul>\n<li>medicine<\/li>\n<li>biochemistry<\/li>\n<li>genetics<\/li>\n<li>histology<\/li>\n<li>virusology<\/li>\n<li>microbiology<\/li>\n<li>environmental sciences<\/li>\n<li>etc<\/li>\n<\/ul>\n<table class=\" aligncenter\" width=\"620\">\n<tbody>\n<tr>\n<td style=\"width: 40%;\">\n<p id=\"yui_patched_v3_11_0_1_1435836115547_452\">Atherosclerosis in heart vessels<\/p>\n<\/td>\n<td><a href=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/Atherosclerosis.jpg\"><img decoding=\"async\" class=\"  aligncenter wp-image-2347\" src=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/Atherosclerosis.jpg\" alt=\"Atherosclerosis\" width=\"242\" srcset=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/Atherosclerosis.jpg 457w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/Atherosclerosis-145x150.jpg 145w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/Atherosclerosis-290x300.jpg 290w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/Atherosclerosis-280x289.jpg 280w\" sizes=\"(max-width: 457px) 100vw, 457px\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td>Ostheoarthritis in cartilage.<span id=\"yui_patched_v3_11_0_1_1435836315974_467\" class=\"ntnu-caption\">Middel layer of cartilage. Collagen fibers in magenta (SHG) and chondrocytes residing in their lacuna seenn in green (TPEF).<\/span><\/td>\n<td style=\"text-align: center;\"><a href=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/cartilage_fig2.jpg\"><img decoding=\"async\" class=\"  aligncenter wp-image-2348\" src=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/cartilage_fig2.jpg\" alt=\"cartilage_fig2\" width=\"232\" srcset=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/cartilage_fig2.jpg 512w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/cartilage_fig2-150x150.jpg 150w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/cartilage_fig2-300x300.jpg 300w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/cartilage_fig2-280x280.jpg 280w\" sizes=\"(max-width: 512px) 100vw, 512px\" \/><span id=\"yui_patched_v3_11_0_1_1435836315974_467\" class=\"ntnu-caption\"><\/span><\/a><\/td>\n<\/tr>\n<tr>\n<td>Studying Ca2+ dynamics in live cells using two-photon microscopy.<\/td>\n<td style=\"text-align: center;\"><a href=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/Ca-ion.gif\"><img decoding=\"async\" class=\"alignnone wp-image-2349\" src=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/Ca-ion.gif\" alt=\"Ca+ion\" width=\"211\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td>\u00a0Human metaphase chromosomes<\/td>\n<td style=\"text-align: center;\">\u00a0<a href=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/Chromosom.jpg\"><img decoding=\"async\" class=\"alignnone wp-image-2357\" src=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/Chromosom.jpg\" alt=\"Chromosom\" width=\"223\" srcset=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/Chromosom.jpg 360w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/Chromosom-150x100.jpg 150w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/Chromosom-280x187.jpg 280w\" sizes=\"(max-width: 360px) 100vw, 360px\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td>Herpesvirus infection test<\/td>\n<td style=\"text-align: center;\"><a href=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/herpesvirus.jpg\"><img decoding=\"async\" class=\"alignnone wp-image-2356\" src=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/herpesvirus.jpg\" alt=\"herpesvirus\" width=\"209\" srcset=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/herpesvirus.jpg 360w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/herpesvirus-150x100.jpg 150w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/herpesvirus-280x187.jpg 280w\" sizes=\"(max-width: 360px) 100vw, 360px\" \/><\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<h3 class=\"align-center border4px margin-bottom-50\"><\/h3>\n<h3 style=\"text-align: center;\"><span style=\"color: #3366ff;\">Multiphoton Laser Fluorescence microscopy<\/span><\/h3>\n<p style=\"text-align: justify;\">Multiphoton fluorescence microscopy is a powerful research tool that combines the advanced optical techniques of laser scanning microscopy with long wavelength multiphoton fluorescence excitation to capture high-resolution, three-dimensional images of specimens tagged with specific fluorophores.<\/p>\n<p style=\"text-align: justify;\">The concept of two-photon excitation is based on the idea that two photons of comparably lower energy than needed for one photon excitation can also excite a fluorophore in one<span style=\"color: #000000;\"> quantum event.<\/span> Each photon carries approximately half of the energy necessary to excite the molecule. An excitation results in the subsequent emission of a fluorescence photon, typically at a higher energy than either of the two excitatory photons.<\/p>\n<p>&nbsp;<\/p>\n<p><a href=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/2photon_fluorescence.jpg\"><img loading=\"lazy\" decoding=\"async\" class=\"  wp-image-2214 aligncenter\" src=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/2photon_fluorescence-881x530.jpg\" alt=\"2photon_fluorescence\" width=\"493\" height=\"297\" srcset=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/2photon_fluorescence-881x530.jpg 881w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/2photon_fluorescence-150x90.jpg 150w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/2photon_fluorescence-400x241.jpg 400w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/2photon_fluorescence-768x462.jpg 768w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/2photon_fluorescence-280x168.jpg 280w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/2photon_fluorescence.jpg 924w\" sizes=\"auto, (max-width: 493px) 100vw, 493px\" \/><\/a><\/p>\n<p style=\"text-align: justify;\">Three-photon excitation is a related non-linear optical absorption event that can occur in a manner similar to two-photon excitation. The difference is that three photons must interact simultaneously with the fluorophore to illicit a transition to the excited singlet state. A benefit of three-photon excitation is that successful absorption requires only a tenfold greater concentration of photons than two-photon absorption, making this technique attractive for some experiments.<\/p>\n<p style=\"text-align: justify;\">The methodology is particularly useful to cell biologists who endeavor to study dynamic processes in living cells and tissues without inflicting significant, and often lethal damage to the specimen. Although classical <span style=\"color: #000000;\">widefield<\/span> fluorescence microscopy can often provide submicron resolution of biochemical events in living systems, the technique is limited in sensitivity and spatial resolution by background noise caused by secondary fluorescence throughout areas situated above and below the focal plane.<\/p>\n<p style=\"text-align: justify;\">Excitation in multiphoton microscopy occurs only at the focal point of a diffraction-limited microscope, providing the ability to optically section thick biological specimens in order to obtain three-dimensional resolution. Individual <strong>optical sections<\/strong> are acquired by <span style=\"color: #000000;\">raster scanning the<\/span> specimen in the x-y plane, and a full three-dimensional image is composed by serially scanning the specimen at sequential z positions. Because the position of the focal point can be accurately determined and controlled, multiphoton fluorescence is useful for probing selected regions beneath the specimen surface.<\/p>\n<h4 style=\"text-align: center;\">Comparison of the Fluorescence Microscopy Techniques<\/h4>\n<p><a href=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/WF-con-2ph_2.jpg\"><img loading=\"lazy\" decoding=\"async\" class=\"  aligncenter wp-image-2216 size-medium\" src=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/WF-con-2ph_2-365x300.jpg\" alt=\"WF-con-2ph_2\" width=\"365\" height=\"300\" srcset=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/WF-con-2ph_2-365x300.jpg 365w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/WF-con-2ph_2-150x123.jpg 150w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/WF-con-2ph_2-768x632.jpg 768w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/WF-con-2ph_2-644x530.jpg 644w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/WF-con-2ph_2-280x230.jpg 280w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/WF-con-2ph_2.jpg 957w\" sizes=\"auto, (max-width: 365px) 100vw, 365px\" \/><\/a><\/p>\n<table class=\" aligncenter\" width=\"420\">\n<tbody>\n<tr>\n<td><a href=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/confocal-2photon-2.jpg\"><img loading=\"lazy\" decoding=\"async\" class=\"wp-image-2215 aligncenter\" src=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/confocal-2photon-2-500x530.jpg\" alt=\"confocal-2photon-2\" width=\"377\" height=\"399\" srcset=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/confocal-2photon-2-500x530.jpg 500w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/confocal-2photon-2-142x150.jpg 142w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/confocal-2photon-2-283x300.jpg 283w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/confocal-2photon-2-768x814.jpg 768w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/confocal-2photon-2-280x297.jpg 280w, https:\/\/candle.am\/wp-content\/uploads\/2015\/06\/confocal-2photon-2.jpg 816w\" sizes=\"auto, (max-width: 377px) 100vw, 377px\" \/><\/a><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: justify;\">Fluorescent pollen grain was imaged at three depths below its surface (7, 16, and 26 \u03bcm) by widefiled, confocal and 2 photon technique. Two-photon imaging improves image sharpness for deep optical sections by reducing scattering of excitation light and by eliminating fluorescence excitation outside the plane of focus.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>&nbsp;<\/p>\n<h3 style=\"text-align: center;\"><span style=\"color: #993366; font-size: 16px;\"><strong>Advantages of 2 Photon Laser\u00a0Scanning Microscopy (2P-LSM)<\/strong><\/span><\/h3>\n<ul>\n<li style=\"text-align: justify;\"><strong>2P-LSM is more sensitive<span style=\"color: #000000;\"> than C<\/span>onfocal Laser Scanning Microscopy (CLSM) because all the light generated to make an image is sent directly to the photon multiplier tube.<\/strong><\/li>\n<li style=\"text-align: justify;\"><strong>This contrasts with CLSM where a pin hole is required to select the light from the focal plane. In CLSM there is considerable loss of signal in the optics required to direct the light to the pin hole. <\/strong><\/li>\n<li style=\"text-align: justify;\"><strong>2P-LSM gives a sharper image than CLSM because of the lack of extraneous light and improved geometry of detection. <\/strong><\/li>\n<li style=\"text-align: justify;\"><strong>2P-LSM uses solid-state lasers which are more sta<span style=\"color: #000000;\">ble than the ga<\/span>s lasers used in CLSM. <\/strong><\/li>\n<li style=\"text-align: justify;\"><strong>The longer wavelength light used in 2P-LSM is more penetrating than the shorter wavelength used in CLSM (5 times deeper into biological material &#8211; up to 1 mm). <\/strong><\/li>\n<li style=\"text-align: justify;\"><strong>The tissue above and below the plane of focus is merely subjected to infrared light and 2-photon excitation is restricted to a small focal volume. <\/strong><\/li>\n<li style=\"text-align: justify;\"><strong>2P-LSM microscopy separates excitation and emission light more effectively and has better signal\/background ratio. A wider gap between excitation and emission makes it easier to reject excitation light, with minimal loss of emission photons. <\/strong><\/li>\n<li><strong>Due to<span style=\"color: #000000;\"> reduced <\/span>phototoxicity of IR beam two-photon microscopes are less damaging the sample than a single-photon confocal microscope. <\/strong><\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h2 style=\"text-align: center;\"><span style=\"color: #3366ff;\">Experiments<\/span><\/h2>\n<h3 style=\"text-align: center;\"><span style=\"color: #3366ff;\">Institute of Molecular Biology<\/span><\/h3>\n<p><a href=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/04\/a5.jpg\"><img loading=\"lazy\" decoding=\"async\" class=\"  aligncenter wp-image-41 size-medium\" src=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/04\/a5-400x300.jpg\" alt=\"a5\" width=\"400\" height=\"300\" srcset=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/04\/a5-400x300.jpg 400w, https:\/\/candle.am\/wp-content\/uploads\/2015\/04\/a5-150x113.jpg 150w, https:\/\/candle.am\/wp-content\/uploads\/2015\/04\/a5-768x576.jpg 768w, https:\/\/candle.am\/wp-content\/uploads\/2015\/04\/a5-1413x1060.jpg 1413w, https:\/\/candle.am\/wp-content\/uploads\/2015\/04\/a5-280x210.jpg 280w, https:\/\/candle.am\/wp-content\/uploads\/2015\/04\/a5-707x530.jpg 707w\" sizes=\"auto, (max-width: 400px) 100vw, 400px\" \/><\/a><\/p>\n<p style=\"text-align: center;\">Investigation of red blood cells shock.<\/p>\n<p><a href=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/16-06.jpg\"><img loading=\"lazy\" decoding=\"async\" class=\"  aligncenter wp-image-2285 \" src=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/16-06-941x421.jpg\" alt=\"16-06\" width=\"636\" height=\"285\" srcset=\"https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/16-06-941x421.jpg 941w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/16-06-150x67.jpg 150w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/16-06-400x179.jpg 400w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/16-06-768x344.jpg 768w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/16-06-280x125.jpg 280w, https:\/\/candle.am\/wp-content\/uploads\/2015\/05\/16-06.jpg 1081w\" sizes=\"auto, (max-width: 636px) 100vw, 636px\" \/><\/a><\/p>\n<p style=\"text-align: justify;\">Native (intact) red blood cells do not possess fluorescence (left). After the shock caused by the hydrogen peroxide, damaged cells interact with the fluorescence marker (right). The fluorescence intensity is directly proportional to the degree of damage.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Coordinator \u2013 Dr. Stepan Tatikyan Our Two-Photon Fluorescence Laser Scanning Microscopy System (MOM Sutter instruments) is available for users from other institutions. For sample excitation Amplitude System infrared pulse laser is used. Wavelength: 1030 nm [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"latest-microlab.php","meta":{"_acf_changed":false,"footnotes":""},"class_list":["post-1552","page","type-page","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"https:\/\/candle.am\/hy\/wp-json\/wp\/v2\/pages\/1552","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/candle.am\/hy\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/candle.am\/hy\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/candle.am\/hy\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/candle.am\/hy\/wp-json\/wp\/v2\/comments?post=1552"}],"version-history":[{"count":3,"href":"https:\/\/candle.am\/hy\/wp-json\/wp\/v2\/pages\/1552\/revisions"}],"predecessor-version":[{"id":2451,"href":"https:\/\/candle.am\/hy\/wp-json\/wp\/v2\/pages\/1552\/revisions\/2451"}],"wp:attachment":[{"href":"https:\/\/candle.am\/hy\/wp-json\/wp\/v2\/media?parent=1552"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}