How things work ?What is Cryo-elctronmicroscope?How does it work ? 

Chemistry Nobel for Cryo-Electron Microscopes,

 Machines That Can ‘See’ the Atoms of Life

Jacquet Dubochet, Joachim Frank and Richard Henderson optimised the electron microscope through simple workarounds, preserving it as the instrument of choice among structural biologists.

This year’s Nobel Prize for chemistry has been awarded to Jacques Dubochet, Joachim Frank and Richard Henderson for “developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution”.

Cryo-electron microscopy is an optimised form of electron microscopy. In our daily lives, our eyes use photons, the particles of light, to ‘see’ objects around us when they reflect those photons. This is because the wavelength of visible light is comparable in length to that of the smallest deformations we need to be able to see. However, objects that are less wide than this distance are not visible to us because the photons won’t be reflected by them. This is also why radio telescopes are so big: their antennae, or receiver dishes, have to have width comparable to the wavelengths of electromagnetic radiation they’re trying to intercept.

Electrons have a lower wavelength when imagined as a wave than do photons, so they can resolve smaller objects. An electron microscope is a machine that ‘sees’ objects using electrons, and then magnifies those images so humans can study them. It was first invented in the 1930s. However, the beam of electrons produced by the machine is bad for biological tissues it is studying.

The beam is produced and applied to the tissue in a vacuum because, otherwise, molecules in the air would scatter the beam. But in a vacuum, all the water in the tissue evaporates, causing the tissue’s biochemical infrastructure to physically collapse, disrupting what scientists are actually trying to ‘see’. Moreover, the beam also damages the tissue and breaks chemical bonds (especially if it’s of a few hundred kilovolts).

This year’s chemistry laureates fixed these issues by finding simple workarounds, restoring the electron microscope as the instrument of choice among structural biologists.

In the early 1980s, Jacques Dubochet found a way to freeze samples so rapidly that they froze at exactly the moment scientists wanted to look at through a microscope. Dubochet achieved this by transferring the sample onto a metallic mesh using something like a water-dropper. There, parts of the sample would form meniscuses across the mesh. Suddenly, the mesh would be shot into liquid ethane at -90º C, flash-freezing the meniscuses. This prevented the water in tissues from evaporating.

The abstract for a March 2009 paper coauthored by Dubochet read, “Water, which was once the arch enemy of all electron-microscopists, became what it always was in nature – an integral part of biological matter and a beautiful substance.”

Second: in the decade since 1975, Joachim Frank developed a way for the multiple two-dimensional images produced by an electron microscope to be combined into a three-dimensional image – more useful for scientists. In 1990, Richard Henderson used these techniques, and his own improvisations, to produce a three-dimensional image of a protein (called bacteriorhodopsin) down to its atoms.

Atomic resolution typically means a distance of 3.3 angstrom (Å) – more than 250-times smaller than the HIV virus (1 Å is one-tenth of a nanometre). In 2015, a group of biologists from the US pushed the laureate trio’s techniques to the hilt, determining the arrangement of a biochemical structure at a resolution of 2.8 Å. They wrote in their paper, “Reaching resolutions higher than 3 Å is a prerequisite for structure-based drug design and for cryoEM to become widely interesting to pharmaceutical industries. … The results described in this manuscript demonstrate that single particle cryoEM is capable of competing with X-ray crystallography for determination of protein structures of suitable quality for rational drug design.”

Two months later, another group hit 2.2 Å.

Sub-3 Å was a resolution previously reserved only for X-ray crystallography, which made the achievement doubly important. X-ray crystallography requires specimens under study to be crystallised, which is often not possible, and studied in environments inimical to the specimen’s natural ones.

Frank’s contribution is particularly interesting because it augured two prevalent, and convergent, trends in modern chemistry – and in modern science for that matter: machine-learning and computational studies. By 1981, he had developed a computer algorithm that could look at thousands of images of the same proteins generated by an electron microscope and identify similarities. Next, it could produce high-resolution 2D images of the proteins: if one part of one image was fuzzy, the algorithm would identify the same part from another image and fill in the blanks.

While the data-processing machines and algorithms powering 21st century science are far more sophisticated, they are essentially equivalent to what Frank did in the 1970s. His method allows scientists to ‘sample’ specimens with small doses of electron radiation over and over – which means even sensitive matter could be studied – and piece all the images together to get a high-resolution version.

(However, it must be noted that Frank did not pioneer the use of computers to enhance current knowledge. In fact, the ability to assemble a reliable 3D image of a specimen from 2D images was first demonstrated in the 1970s by Anthony Crowther and David DeRosier.)

Since 2010, newer techniques have continued to optimise cryo-electron microscopy. Case in point: when the electrons are shot at the frozen meniscuses, the latter assume a more curved shape because they’re accumulating negative charge. This problem was solved in 2012 with the advent of direct electron detectors, which ‘image’ the doming effect at about 400 frames per second.

But we’re not done yet. Just like powerful telescopes have been able to work together to identify features of the cosmos they could not have seen by themselves, advancements in electronics and chemical engineering are keeping cryo-electron microscopy up to date as well as easily usable. For example, the technique is best suited for meniscuses 1 micrometer thick or less, and can’t achieve high contrasts. To solve these problems, engineers invented ion-beam milling and, subsequently, cryo-electron tomography: an imaging technique where samples are simply tilted or shifted while being studied.

शोध की खिड़की से :कैसे काम करता है और 

क्या है क्रायो -इलेक्ट्रॉन माइक्रोस्कोपी (दूसरा भाग ,समापन क़िस्त )?

उन्नीस सौ अस्सी के दशक के आरम्भिक बरसों में (Early 1980s)जेक्स डुबोशे ने जैविक अणुओं (ऊतकों आदि )के सैंपलों को द्रुत गति से प्रशीतित करने की एक ऐसी विधि विकसित की जिसके तहत साम्पिल्स  ठीक उसी समय हिमीकृत हो उठते जब उन्हें इलेक्ट्रॉन सूक्ष्मदर्शी से देखने की चाह एवं   तैयारी  होती। तात्कालिक प्रशीतली - करण था यह एक किस्म का जो पलक झपकते ही संपन्न हो जाता था। और विधि भी इतनी सरल एक धातु के तार की जाली पे लिक्विड - ड्रॉपर जैसी एक युक्ति से साम्पिल को स्प्रेड भर करने की देर -बस साम्पिल जाली पे अनेक घुमावदार (Thin meniscuses)महीन सतह बुन लेते थे।
A meniscus is a rubbery C-shaped disc,a curved surface .

बस यकायक मेनस्क्सिज समेत जाली  को इधर तरल इथेन (-90 C)में दागा उधर वे हिमीकृत भी हो गईं तत्काल प्रभाव के साथ । वही जल जो जैविक ऊतकों का एक आवश्यक अंग रहता है और इलेक्ट्रॉन सूक्ष्म -दर्शी  के लिए अभी तक दुर्जेय शत्रु की तरह  बना रहा था ,अब मित्रवत था। यानी अब माइक्रोस्कोप फ्रेंडली हो गये थे जल -महोदय। 
इधर १९७५ के बाद के बरसों में ही इनके सहकर्मी जो-आकिम फ्रेंक ने सूक्ष्मदर्शी से ली गई दो -आयामीय अनेकानेक छवियों(Images ) को त्रिआयामी छवियों में  संयुक्त करने का काम संपन्न कर लिया था। 
१९९० में इनके तीसरे साथी रिचर्ड हेंडरसन ने इसी काम को और परिष्कृत कर एक प्रोटीन(bacteriorhodospin ) की त्रिआयामी इमेज बनाके दिखला दी.कहाँ कौन सा परमाणु इस प्रोटीन में विराजमान है यह भलीभांति देखा जा सका। यही परमाणुविक स्तर का आरम्भिक  विभेदन था।आगे इसी विभेदन का (resolving power of microscope )और परिष्करण किया गया। 

Atomic resolution typically means a distance of 3.3 angstrom (Å) – more than 250-times smaller than the HIV virus (1 Å is one-tenth of a nanometre).

यह एचआईवी -एड्स वायरस से ढाईसौ गुना छोटा आकार है। 

१ एंगस्ट्रोम =एक नैनोमीटर का भी  दसवां भाग (टेन टू दी पावर टेन मीटर्स ),एंगस्ट्रोम सूक्ष्मतर स्तर पर लम्बाई नापने की एक इकाई है। सूक्ष्मतर तरंगों की लम्बाई के मापन के  लिए इसे स्तेमाल किया जाता है। 
२०१५  में अमरीकी जीवन विज्ञानों के माहिरों  की एक टोली ने इस विभेदन को नै परवाज़ देकर और भी परिष्कृत किया। ३. ३ एंगस्ट्रोम से विज्ञानी अब २. ८ एंगस्ट्रोम रिज़ॉल्यूशन पर आ गए थे। बस देखते ही देखते औषध निगमों के लिए Cryo -TEM का महत्व बढ़ गया ,अब हिमीकृत ट्रॅन्समिशन इलेक्ट्रॉन माई - क्रोस्कोप 'संरचना -आधारित -दवाओं 'के निर्माण की ओर  एक कदम और आगे की ओर रख चुके थे  ।  

इस प्रकार जीवन के लिए ज़रूरी कच्चे माल प्रोटीनों के अंदर तांक - झाँक करने का एक रास्ता खुला।जीवन के रसायन को अब थोड़ा और आगे निकलके देखा जा सकेगा ऐसी उम्मीद बँध  चली है। लाइलाज रहे आये बुढ़ापे के रोग एल्जाइमर्स के लिए उत्तरदाई एम्लोयड प्रोटीन की संरचना का अब और  भी सूक्ष्म -तर स्तर पर खुलासा हो सकेगा।  ( समाप्त )

सन्दर्भ -सामिग्री :
https://thewire.in/184432/nobel-chemistry-cryo-electron-microscopy-dubochet-henderson-proteins-biology/