Moleküler Hücre Biyolojisi I. Hafta 11: Hücrelerin görüntülenmesi. Yrd Doç Dr Arzu ATALAY

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1 Moleküler Hücre Biyolojisi I Hafta 11: Hücrelerin görüntülenmesi Yrd Doç Dr Arzu ATALAY

2 Işık mikroskopu ile birbirinden 2 mikron uzaktaki ayrıntılar gözlenebilir Figure 9-1 Molecular Biology of the Cell ( Garland Science 2008)

3 Figure 9-2 Molecular Biology of the Cell ( Garland Science 2008)

4 Figure 9-3 Molecular Biology of the Cell ( Garland Science 2008) Işık mikroskopu

6 Canlılar en iyi faz kontrast veya ayırdedici girişim kontrast mikroskopunda izlenebilir Figure 9-7 Molecular Biology of the Cell ( Garland Science 2008)

7 Işık mikroskopisinin 4 türü: (A) Aydınlık alan, (B) faz-kontrast (C) Nomarski ayırdedici girişim kontrast mikroskopisi (D) karanlık alan Figure 9-8 Molecular Biology of the Cell ( Garland Science 2008)

8 Görüntüler elektronik yöntemlerle düzeltilerek incelenebilir Figure 9-9 Molecular Biology of the Cell ( Garland Science 2008)

9 Doku örnekleri mikroskopta incelenmek üzere sabitlenir ve kesitler alınır Figure 9-10 Molecular Biology of the Cell ( Garland Science 2008)

10 Hücrenin farklı bileşenleri seçici biçimde boyanabilir Figure 9-11 Molecular Biology of the Cell ( Garland Science 2008)

11 Hücrenin farklı bileşenleri seçici biçimde boyanabilir Figure 9-12 Molecular Biology of the Cell ( Garland Science 2008)

12 Hücre içindeki belirli moleküller floresans mikroskopisi ile saptabilir Örnekte filtre floresans veren fluorescein molekülünü saptayacak biçimde ayarlanmıştır UYARILMA SALIM Figure 9-13 Molecular Biology of the Cell ( Garland Science 2008) Boya molekülü tarafından yayınlanan foton soğurulan fotondan mutlaka daha düşük enerjilidir. Aradaki fark soğurma ve salım eğrilerinin tepe noktaları arasındaki farka eşittir.

13 Belirli molekülleri saptamak için antikorlar kullanılabilir Figure 9-15 Molecular Biology of the Cell ( Garland Science 2008)

16 Optik mikroskop ile karmaşık üç boyutlu cisimler gözenebilir Figure 9-19 Molecular Biology of the Cell ( Garland Science 2008)

17 Konfokal floresans mikroskopu odaklanmamış ışığı eleyerek optik kesitler oluşturur Figure 9-20 Molecular Biology of the Cell ( Garland Science 2008)

18 Normal ve konfokal mikroskopinin kıyaslanması Figure 9-21 Molecular Biology of the Cell ( Garland Science 2008)

34 Table 9-1 Molecular Biology of the Cell ( Garland Science 2008)

37 Elektron mikroskopu ile hücrenin ince yapısı görülebilir Figure 9-41 Molecular Biology of the Cell ( Garland Science 2008)

38 Işık ve elektron mikroskopinin kıyaslanması Figure 9-42 Molecular Biology of the Cell ( Garland Science 2008)

39 Elektron mikroskopisi için örneklerin özel olarak hazırlanması gerekir Figure 9-43 Molecular Biology of the Cell ( Garland Science 2008)

40 Elektron mikroskopisi için örneklerin özel olarak hazırlanması gerekir. TEM de (iletim EM) ince örnek kesitleri bakır grid üzerinde bulunur Figure 9-44 Molecular Biology of the Cell ( Garland Science 2008)

42 Bazı makromoleküller bağışıksal altın mikroskopisi ile saptanabilir Figure 9-46 Molecular Biology of the Cell ( Garland Science 2008)

43 Yüzeylerin görüntüsü tarama elektron mikroskopisi (SEM) ile elde edilir Figure 9-47 Molecular Biology of the Cell ( Garland Science 2008)

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56 Surpassing the limitations of the light microscope For a long time optical microscopy was held back by a presumed limitation: that it would never obtain a better resolution than half the wavelength of light. Helped by fluorescent molecules the Nobel Laureates in Chemistry 2014 ingeniously circumvented this limitation. Their ground-breaking work has brought optical microscopy into the nanodimension. In what has become known as nanoscopy, scientists visualize the pathways of individual molecules inside living cells. They can see how molecules create synapses between nerve cells in the brain; they can track proteins involved in Parkinson's, Alzheimer's and Huntington's diseases as they aggregate; they follow individual proteins in fertilized eggs as these divide into embryos. It was all but obvious that scientists should ever be able to study living cells in the tiniest molecular detail. In 1873, the microscopist Ernst Abbe stipulated a physical limit for the maximum resolution of traditional optical microscopy: it could never become better than 0.2 micrometres. Eric Betzig, Stefan W. Helland William E. Moerner are awarded the Nobel Prize in Chemistry 2014 for having bypassed this limit. Due to their achievements the optical microscope can now peer into the nanoworld. Two separate principles are rewarded. One enables the method stimulated emission depletion (STED) microscopy, developed by Stefan Hell in Two laser beams are utilized; one stimulates fluorescent molecules to glow, another cancels out all fluorescence except for that in a nanometre-sized volume. Scanning over the sample, nanometre for nanometre, yields an image with a resolution better than Abbe's stipulated limit. Eric Betzig and William Moerner, working separately, laid the foundation for the second method, singlemolecule microscopy. The method relies upon the possibility to turn the fluorescence of individual molecules on and off. Scientists image the same area multiple times, letting just a few interspersed molecules glow each time. Superimposing these images yields a dense super-image resolved at the nanolevel. In 2006 Eric Betzig utilized this method for the first time.

57 Focal adhesion proteins paxillin (green) and vinculin (red) assemble as interdigitated clusters in several adhesions at periphery of a human foreskin fibroblast cell, as seen at three different magnifications, using dual color PALM in conjunction with the photoactivatable fluorescent proteins Dronpa (green) and tdeos (red). CREDIT: From H. Shroff, et al., Proc. Natl. Acad. Sci. 104, (2007).

58 Post-synaptic densities from a subset of synapses in a transfected mouse brain expressing PSD95::mEos2, as seen in a 70 µm thick resin-embedded section over a 14 x 14 µm field of view (left), with two PSDs shown at higher magnification at right. CREDIT: Sample and image by Haining Zhong.

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62 Glowing proteins a guiding star for biochemistry The remarkable brightly glowing green fluorescent protein, GFP, was first observed in the beautiful jellyfish, Aequorea victoria in Since then, this protein has become one of the most important tools used in contemporary bioscience. With the aid of GFP, researchers have developed ways to watch processes that were previously invisible, such as the development of nerve cells in the brain or how cancer cells spread. Tens of thousands of different proteins reside in a living organism, controlling important chemical processes in minute detail. If this protein machinery malfunctions, illness and disease often follow. That is why it has been imperative for bioscience to map the role of different proteins in the body. This year's Nobel Prize in Chemistry rewards the initial discovery of GFP and a series of important developments which have led to its use as a tagging tool in bioscience. By using DNA technology, researchers can now connect GFP to other interesting, but otherwise invisible, proteins. This glowing marker allows them to watch the movements, positions and interactions of the tagged proteins. Researchers can also follow the fate of various cells with the help of GFP: nerve cell damage during Alzheimer's disease or how insulin-producing beta cells are created in the pancreas of a growing embryo. In one spectacular experiment, researchers succeeded in tagging different nerve cells in the brain of a mouse with a kaleidoscope of colours. The story behind the discovery of GFP is one with the three Nobel Prize Laureates in the leading roles: Osamu Shimomura first isolated GFP from the jellyfish Aequorea victoria,which drifts with the currents off the west coast of North America. He discovered that this protein glowed bright green under ultraviolet light. Martin Chalfie demonstrated the value of GFP as a luminous genetic tag for various biological phenomena. In one of his first experiments, he coloured six individual cells in the transparent roundworm Caenorhabditis elegans with the aid of GFP. Roger Y. Tsien contributed to our general understanding of how GFP fluoresces. He also extended the colour palette beyond green allowing researchers to give various proteins and cells different colours. This enables scientists to follow several different biological processes at the same time

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64 Brain stem, tissue from Mus musculus, Brainbow expression with GFP constructs, Confocal imaging by Lichtman Laboratory published, Nature article