Industrial Research And Consultancy Centre

This JEM 2100 ultra HRTEM 200kVwas installed at the IIT Bombay in June 2010 as part of an DST-IRPHA project with partial support from the Institute. This HRTEM facility is equipped with a few useful attachments such as a scanning image observation device that incorporates provisions for obtaing STEM images, an EELS, an EDS, and most importantly, a Cryo mode facility with cryo specimen holder, cryo transfer unit, dry pumping station, and cryo sample preparation equipment like Gatan Cryo Plunger and FEI Vitrobot, and cryo specimen holder. This facility is supported by IRCC as a Central Facility of the Institute. This facility is open for all IIT Bombay internal users to meet their research necessities. Due to the sophisticated nature of the equipment and elaborate set up, this instrument does see periods of downtime.

Configuration :

  • Resolution —point 0.194 nm and lattice 0.14 nm. Actual performance is less for a typical users sample.
  • Focal Length — 1.9 mm
  • Max. Eucentric tilt —±80°
  • Magnification —50x to 1500000x

Electron Source :

  • LaB6 emitter
  • Accelerating Voltage 80, 100, 120, 160, 200 KV

Vacuum :

  • Fully interlocked
  • Differentially pumped column
  • Ultra High Vacuum for contamination free observation of the specimen
  • Specimen chamber and gun area with 2X10 -5 Pa.

Specimen Stage - Compustage :

  • Fully computer controlled, Eucentric side entry, high stability Compustage
  • High tilt and large field of view
  • Choice of variety of Specimen Holders (Cryo, Multiple, Rotation Holders etc.)
  • X, Y movement 2 mm, specimen size 3 mm
  • Specimen position store and recall including magnification and illumination settings.
  • Specimen exchange without switching off High Tension and Emitter
  • High tilt (±80°) and large field of view.

Imaging :

  • High contrast,
  • Automated contrast enhancement function.
  • Focusing aid, adjustable wobbler for all magnification and directions.
  • Rotation free magnification and diffraction series.
  • Magnification aberration corrected lens series.
  • Embedded CCD Camera - Gatan
  • Low dose software.
  • Image acquisition software with wide choice for image manipulation and analysis.

 

 

 

  • TEM analysis of biological as well as soft solids can be done on Cryo mode or normal mode.
  • High contrast helps in imaging biological samples without staining.
  • With ultra-clean high vacuum for contamination free observation
  • Compustage for accurate specimen control.
  • Especially fits the need for Cryo-observation.
  • A key component to the success of low temperature observation is the installation of a specific cold-trap, which is effective for the preventing vapour deposition on Cryo holders and their specimens while in the column.

List Of Publications:

1. In Vivo Analysis of Biodegradable Liposome Gold Nanoparticles as Efficient Agents for Photothermal Therapy of Cancer AK Rengan, AB Bukhari, A Pradhan, R Malhotra, R Banerjee, Nano letters 15 (2), 842-848

2. Mayur K Temgire, Akkihebbal K. Suresh,  Shantaram G. Kane,and Jayesh       Bellare Establishing The Interfacial Nano-structure And Elemental Composition of Homeopathic Medicines Based on Inorganic Salts: A Scientific Approach - September 2015 10.1016/j.homp.2015.09.006

3. Formation of nanoparticles and nanocrystals of mercury by α-lactalbumin Chebrola Pulla Rao. Atul Gajanan Thawari . Vijaya Kumar hinge · Mayur Temgire RSC Advances 09/2014; DOI:10.1039/C4RA07156E

4. Chain length dependence of polyol synthesis of zinc ferrite nanoparticles: Why is diethylene glycol so different? Supriya N Rishikeshi· Satyawati S Joshi · Mayur K Temgire · Jayesh R Bellare, Dalton Transactions 02/2013; 42(15). DOI:10.1039/c2dt32026f 

5. Redox Decomposition of Silver Citrate Complex in Nanoscale Confinement: An Unusual Mechanism of Formation and Growth of Silver Nanoparticles  Sabyasachi Patra†, Ashok K. Pandey†, Debasis Sen‡, Shobha V. Ramagiri§, Jayesh R. Bellare§, S. Mazumder‡, and A. Goswami*†  langmuir, 2014, 30 (9), pp 2460–2469

6. Enhancing cubosome functionality by coating with a single layer of poly-ε-lysine Deshpande S1, Venugopal E, Ramagiri SV, Bellare JR, Kumaraswamy G, Singh N.Volume 6, Issue 19, 8 October 2014, Pages 17126-17133 ACS Appl. Mater. Interfaces 

7. Aravind Kumar Rengan, Amirali B Bukhari, Arpan Pradhan, Renu Malhotra, Rinti Banerjee, Rohit Srivastava, Abhijit De, (2015) Invivo analysis of biodegradable liposomes gold nanoparticles as efficient agents for photothermal therapy of cancer. Nanoletters, 12, 19.

8. Supriya N Rishikeshi, Satyawati S Joshi, Mayur K Temgire, Jayesh R Bellare,  Chain length dependence of polyol synthesis of zinc ferrite nanoparticles: why is diethylene glycol so different? Dalton Trans 2013 Apr 19;42(15):5430-8.

9. Chhabra, H., Gupta, P., Verma, P.J., Jadhav, S., Bellare, J.R.;             Gelatin-PMVE/MA composite scaffold promotes expansion of embryonic stem cells; (2014) Materials Science and Engineering C, 37 (1), pp. 184-194.

10. Jaiswal, A.K., Dhumal, R.V., Ghosh, S., Chaudhari, P., Nemani, H., Soni, V.P., Vanage, G.R., Bellare, J.R.; Bone healing evaluation of nanofibrous composite scaffolds in rat calvarial defects: A comparative study; (2013) Journal of Biomedical Nanotechnology, 9 (12), pp. 2073-2085.

11. Jaiswal, A.K., Dhumal, R.V., Bellare, J.R., Vanage, G.R.; In vivo biocompatibility evaluation of electrospun composite scaffolds by subcutaneous implantation in rat; (2013) Drug Delivery and Translational Research, 3 (6), pp. 504-517.

12. Singh, R., Wagh, P., Wadhwani, S., Gaidhani, S., Kumbhar, A., Bellare, J., Chopade, B.A.; Synthesis, optimization, and characterization of silver nanoparticles from Acinetobacter calcoaceticus and their enhanced antibacterial activity when combined with antibiotics; (2013) International Journal of Nanomedicine, 8, pp. 4277-4290.

13. Sagar, N., Pandey, A.K., Gurbani, D., Khan, K., Singh, D., Chaudhari, B.P., Soni, V.P., Chattopadhyay, N., Dhawan, A., Bellare, J.R.; In-Vivo Efficacy of Compliant 3D Nano-Composite in Critical-Size Bone Defect Repair: A Six Month Preclinical Study in Rabbit; (2013) PLoS ONE, 8 (10), art. no. e77578, 

14. Kanitkar, M., Jaiswal, A., Deshpande, R., Bellare, J., Kale, V.P.; Enhanced Growth of Endothelial Precursor Cells on PCG-Matrix Facilitates Accelerated, Fibrosis-Free, Wound Healing: A Diabetic Mouse Model; (2013) PLoS ONE, 8 (7), art. no. e69960.

 

2019-2020:

1. Effects of Ethanol Addition on the Size Distribution of Liposome Suspensions in Water, Ankush Pal, P. Sunthar, D. V. Khakhar*, Ind. Eng. Chem. Res. 2019, 58, 18, 7511–7519

2. Efficient separation of biological macromolecular proteins by polyethersulfone hollow fiber ultrafiltration membranes modified with Fe3O4 nanoparticles …, A Modi, J Bellare - International journal of biological macromolecules, 2019 - Elsevier

3. The consequence of silicon additive in isothermal decomposition of hydrides LiH, NaH, CaH2 and TiH2 R Kalamkar, V Yakkundi, A Gangal - … Journal of Hydrogen Energy, 2020

4. Synthesis and properties of amino and thiol functionalized graphene oxide, HP Manwatkar, SD Gedam, CS Bhaskar… - Materials Today …, 2020 - Elsevier

5. Impact of thermal annealing inducing oxidation process on the crystalline powder of In 2 S 3, A Timoumi, W Zayoud, A Sharma, M Kraini… - Journal of Materials …, 2020 - Springer

6. Unique Structure-Induced Magnetic and Electrochemical Activity in Nanostructured Transition Metal Tellurates Co1 – xNixTeO4 (x = 0, 0.5, and 1)copyright © 2020 American Chemical Society

7. Amoxicillin removal using polyethersulfone hollow fiber membranes blended with ZIF-L nanoflakes and cGO nanosheets: Improved flux and fouling-resistance, A Modi, J Bellare - Journal of Environmental Chemical Engineering, 2020 - Elsevier

8. La/Ce mixed metal oxide supported MWCNTs as a heterogeneous catalytic system for the synthesis of chromeno pyran derivatives and assessment of green …, RA Rather, S Siddiqui, WA Khan, ZN Siddiqui - Molecular Catalysis, 2020 - Elsevier

9. Compositional Control as the Key for Achieving Highly Efficient OER Electrocatalysis with Cobalt Phosphates Decorated Nanocarbon Florets, J Saha, S Verma, R Ball, C Subramaniam… - Small, 2020 - Wiley Online Library

10. Hybrid silver–gold nanoparticles suppress drug resistant polymicrobial biofilm formation and intracellular infection, E Bhatia, R Banerjee - Journal of Materials Chemistry B, 2020 - pubs.rsc.org

11. Development of oxidation resistant and mechanically robust carbon nanotube reinforced ceramic composites, V Verma, SC Galaveen, L Gurnani… - Ceramics …, 2020 - Elsevier

12. Zeolitic imidazolate framework-67/carboxylated graphene oxide nanosheets incorporated polyethersulfone hollow fiber membranes for removal of toxic heavy …, A Modi, J Bellare - Separation and Purification Technology, 2020 - Elsevier

13. Enhanced wettability and photocatalytic activity of seed layer assisted one dimensional ZnO nanorods synthesized by hydrothermal method, D Upadhaya, DD Purkayastha - Ceramics International, 2020 - Elsevier

14. Di-tert-butylphosphate Derived Thermolabile Calcium Organophosphates: Precursors for Ca(H2PO4)2, Ca(HPO4), α-/β-Ca(PO3)2, and Nanocrystalline Ca10(PO4)6 …, S Verma, R Murugavel - Inorganic Chemistry, 2020 - ACS Publications

For Year 2021: 

1. Probing Kinetics and Mechanism of Formation of Mixed Metallic Nanoparticles in a Polymer Membrane by Galvanic Replacement between Two Immiscible Metals ... NG Gaidhani, S Patra, HS Chandwadkar, D Sen... - Langmuir, 2021 - ACS Publication. ...

2. Influence of carbon nanotube type and novel modification on dispersion, melt‐rheology and electrical conductivity of polypropylene/carbon nanotube composites J Banerjee, S Kummara, AS Panwar... - Polymer ..., 2021 - Wiley Online Library

3. Composition uniformity and large degree of strain relaxation in MBE-grown thick GeSn epitaxial layers, containing 16% Sn J Rathore, A Nanwani, S Mukherjee... - Journal of Physics D ..., 2021 - iopscience.iop.org

4. Hydrothermal-assisted synthesis and stability of multifunctional MXene nanobipyramids: structural, chemical, and optical evolution B Singh, R Bahadur, S Neekhra, M Gandhi... - ... Applied Materials & ..., 2021 - ACS Publications

5. Influence of carbon nanotube type and novel modification on dispersion, melt‐rheology and electrical conductivity of polypropylene/carbon nanotube composites J Banerjee, S Kummara, AS Panwar... - Polymer ..., 2021 - Wiley Online Library

6. Interaction of Aromatic Amino Acid-Functionalized Gold Nanoparticles with Lipid Bilayers: Insight into the Emergence of Novel Lipid Corona Formation A Maity, SK De, A Chakraborty - The Journal of Physical Chemistry ..., 2021 - ACS Publications

7. Ultra-fast, chemical-free, mass production of high quality exfoliated graphene A Islam, B Mukherjee, KK Pandey, AK Keshri - ACS nano, 2021 - ACS Publications

8. ]Multifunctional hybrid soret nanoarchitectures for mobile phone-based picomolar Cu2+ ion sensing and dye degradation applications S Bhaskar, P Jha, C Subramaniam... - Physica E: Low ..., 2021 - Elsevier

9. Polyphenol stabilized copper nanoparticle formulations for rapid disinfection of bacteria and virus on diverse surfaces K Sadani, P Nag, L Pisharody, XY Thian, G Bajaj... - ..., 2021 - iopscience.iop.org
...

10. Bicomponent Coassembled Hydrogel as a Template for Selective Enzymatic Generation of DOPA
S Biswas, T Ghosh, DKK Kori, AK Das - Langmuir, 2021 - ACS Publications

11. Improved non-enzymatic H2O2 sensors using highly electroactive cobalt hexacyanoferrate nanostructures prepared through EDTA chelation route R Banavath, R Srivastava, P Bhargava - Materials Chemistry and Physics, 2021 - Elsevier

12. Enhanced antibacterial activity of decahedral silver nanoparticles S Bharti, S Mukherji, S Mukherji - Journal of Nanoparticle Research, 2021 - Springer

13. Non-Enzymatic H2O2 Sensor Using Liquid Phase High-Pressure Exfoliated Graphene R Banavath, SS Nemala, R Srivastava... - Journal of The ..., 2021 - iopscience.iop.org

14. Spontaneous Ion Migration via Mechanochemical Ultrasonication in Mixed Halide Perovskite Phase Formation:
Experimental and Theoretical Insights M Roy, Vikram, Bhawna, U Dedhia... - The Journal of ..., 2021 - ACS Publications

15. Zeolitic imidazolate framework-8 nanoparticles coated composite hollow fiber membranes for CO2/CH4 separation K Sainath, P Kumari, J Bellare - Journal of Environmental Chemical ..., 2021 - Elsevier

16. Non-stoichiometry induced exsolution of metal oxide nanoparticles via formation of wavy surfaces and their enhanced electrocatalytic activity: case of misfit calcium ... KS Roy, C Subramaniam... - ACS Applied Materials & ..., 2021 - ACS Publications

17. Ion-Induced Bending with Applications for High-Resolution Electron Imaging of Nanometer-Sized Samples
S Zhang, V Garg, G Gervinskas... - ACS Applied Nano ..., 2021 - ACS Publications

18. Synthesis, Characterization, and Hydrogen Gas Sensing of ZnO/g-C3N4 Nanocomposite A Ibrahim, UB Memon, SP Duttagupta... - Engineering ..., 2021 - mdpi.com

19. Underlying mechanisms for the modulation of self-assembly and the intrinsic fluorescent properties of amino acid- functionalized gold nanoparticles SK De, A Maity, A Chakraborty - Langmuir, 2021 - ACS Publications

20. [HTML] Novel combination of bioactive agents in bilayered dermal patches provides superior wound healing
MM Pillai, H Dandia, R Checker, S Rokade... - ... , Biology and Medicine, 2022 - Elsevier... Murine

21. Probing Kinetics and Mechanism of Formation of Mixed Metallic Nanoparticles in a Polymer Membrane by Galvanic Replacement between Two Immiscible Metals ... ..., D Sen, C Majumder, SV Ramagiri, JR Bellare - Langmuir, 2021 - ACS Publications

22. Concomitant Effect of Quercetin-and Magnesium-Doped Calcium Silicate on the Osteogenic and Antibacterial Activity of Scaffolds for Bone Regeneration AM Preethi, JR Bellare - Antibiotics, 2021 - mdpi.com

23. “Viscotaxis”-directed migration of mesenchymal stem cells in response to loss modulus gradient
..., V Kumar, D Shah, S Beri, S Das, J Bellare... - Acta biomaterialia, 2021 - Elsevier

24. Matrix dependent spatial distributions of in situ formed rhodium nanostructures in ion-exchange membranes
..., AK Pandey, SV Ramagiri, JR Bellare - Materials Today Chemistry, 2021 - Elsevier

25. pH-driven enhancement of anti-tubercular drug loading on iron oxide nanoparticles for drug delivery in macrophages KB Cotta, S Mehra... - Beilstein journal of ..., 2021 - beilstein-journals.org

26. A large area flexible p-type transparent conducting CuS ultrathin films generated at liquid-liquid interface
SS Pathak, LS Panchakarla - Applied Materials Today, 2021 - Elsevier

What is an electron microscope (EM)?

The electron microscope (EM) uses a beam of electrons to form an image of a specimen. An EM is capable of much higher magnifications and has a greater resolving power than a light microscope, allowing it to visualize much smaller objects in finer detail. 

There are two basic types of electron microscopes: the transmission electron microscope (TEM) produces high resolution two dimensional images, while a scanning electron microscope (SEM) scans surfaces of specimens to produce three-dimensional images.

EMs operates under vacuum conditions and are generally large pieces of equipment. They mostly stand alone in a small, specially-designed room and require trained personnel to operate them.

What is transmission electron microscopy (TEM)?

Transmission electron microscopy (TEM) is a technique whereby a beam of electrons is partially transmitted through an ultra thin specimen, interacting with the specimen as it passes through. 

An image is formed from the interaction of the electrons transmitted through the specimen. This image is magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film or, increasingly, it is detected by a sensor such as a CCD camera.

TEMs are capable of imaging at a significantly higher resolution than light microscopes, owing to the small wavelength of electrons, in comparison to light. This enables the examination of fine specimen detail - even as small as a single column of atoms, which is tens of thousands times smaller than the smallest resolvable object in a light microscope. The highest resolution achieved on an aberration-corrected TEM is in the region of 0.5 Angstroms.

The TEM is a major analytical tool in a wide range of scientific fields, in both physical and biological sciences.


 

What information does the TEM provide? Why would I use it?

  1. The transmission electron microscope (TEM) is used to examine the structure, composition, and properties of specimens in submicron detail. It can be used for

  2. Image morphology of samples, e.g. view sections of material, fine powders suspended on a thin film, small whole organisms such as viruses or bacteria, and frozen solutions.

  3. Tilt a sample and collect a series of images to construct a 3-dimensional image.

  4. View frozen material in a TEM with a cryostage.

  5. TEM cannot take colour images. Colour is sometimes added artificially to TEM images.

  6. TEM cannot image through thick samples: the usual sample thickness is less than 100nm. Electrons cannot readily penetrate sections much thicker than 200nm.

  7. The TEM cannot reliably image charged molecules that are mobile in a matrix. For example, some species (e.g. Na+) are volatile under the electron beam because the negative electron beam exerts a force on charged material.

What is electron microscopy (EM) specimen preparation?

In most cases, materials to be viewed under a transmission electron microscope (TEM) or scanning electron microscope (SEM) require processing to produce a suitable specimen. The technique, or techniques, required varies depending on the specimen and the analysis required.

What is transmission electron microscopy (HRTEM) specimen preparation?

A vast range of processing methods and technologies are available for both materials and biological specimens. These processes and associated instrumentation include chemical fixation, embedding and ultra-thin sectioning of biological material, and the thinning of metal specimens prior to TEM examination. The resultant specimens are typically held during observation on TEM support grids. 

How long does it take to prepare typical samples for viewing in the electron microscope? 

For the TEM, upon receiving a live sample, a typical turn-around time is ~1-2 weeks for fixation, embedding, sectioning and contrast staining grids before they can be imaged. If immuno-labelling is required, it will be at least two weeks.

Why does the sample have to be so small?

 For the TEM, samples must ideally be less than 1 mm3 to begin with for good preservation of morphology during the fixation stage.
The chemicals used to fix tissues for high resolution do not penetrate very fast.

Why sample preparation is required?

  1. To increase contrast: heavy atoms interact stronger with e- than bio molecules (C, N, O, S, P)

  2. Positive Staining:

treat sample with solution of salt like uranyl acetate, lead citrate, osmium tetraoxide – object is black on light background

  1. Negative Staining:

place sample on dried film of heavy metal salt – object is light spot on black background

Why Cryo mode of operation?

  1. To avoid harsh staining which may change the structure of your sample.

  2. Stabilization of sample by rapid freezing of sample in liquid ethane to form vitreous ice.

  3. into electron microscope at low temperatures to keep sample stable in hydrated state in vacuum

  4. Thickness of ice layer as small as possible!

  • Advantage: 1) sample structure unchanged

2) Inner structure of molecule is accessible

What is difference between Negative staining and cryo mode?

  • Negative stain (usually 0.5% uranyl acetate)

  1. Easy to prepare

  2. Good contrast

  3. Preservation

  4. Sample distortion

  5. Resolution limited to about 20 angstroms

  • Cryo.

  1. Difficult sample prep.

  2. Low contrast.

  3. Best preservation and therefore High resolution capability.

  4. Specimen viewed in fully hydrated state. close to native state

  5. Little or no mechanical damage

  6. Rapid process

  7. Extra information obtained by low-temperature fracturing

I need to use the Cryo-HRTEM. Do I need to train as a TA?

If your usage is infrequent (less than twice per month), one of the operators or the existing TAs can do the imaging for you. If your research project heavily depends on the use of the Cryo-TEM facility, it would be better if you trained as a TA. Do remember, training as a TA comes with certain duties, such as, imaging other people’s samples..

What does being a TA involve?

The job of a TA is to help us run the facility smoothly and image other people’s samples. You will have a do a minimum of 6 hours of TA duty per week just like the TAs allotted to other central facility equipment. This is non-negotiable. If you are a first year PhD student with loads of coursework, we suggest that you come back after a year. The upside is that you will get really proficient in using a stateof-the-art confocal microscope. You will also be able to book slots during ‘off’ hours (between 6pm and 9am) to run samples for yourself or your research group. On the whole it should be a very useful learning experience for you.

I think I need to train as a TA. What should I do?

The first thing you should do is to check with your advisor on whether both of you agree with the time commitment. You need to contact the TA coordinator of your department to see if you could be assigned as a TA in the central facility. If the answer to both questions is ‘yes’, send an email to the convener of the microscope where you want to train. We will take over from there.

I booked a slot, but my sample is not ready. What should I do?

This can happen once in a while, so don’t worry. Send an email to “ctem@iitb.ac.in” and call the operator on his mobile phone as soon as you realize that you cannot make it to your slot. This is a matter of courtesy to ensure that other people can use your slot. If this happens too many times, clearly you are not planning your experiments very well and we will take a strict view of it.

I need to book more than one consecutive slot. Can I get it?

Sure, if you can justify why. Having ~20 odd samples to image at one go is not a good enough reason. We need to be fair to every user while assigning slots. We will give you as many slots as you need to image all your samples, but they will be distributed over several days.

Can I request a particular TA to image my sample?

No. All TAs have done the same training and it should not matter who images your sample. The conveners have framed this policy to ensure that no single lab/TA monopolizes the use of the facility. If you have any apprehensions about any TA, feel free bring it to the notice of the convener immediately.

 

To achieve the best results during examination in the Electron Microscope (EM), perfect EM Sample Preparation (for TEM, SEM) is require before.

The required techniques depend on the type of samples (biological samples,material samples) as well as on the application.

EM Sample Preparation includes all methods of preparations from embedding, tissue processing, coating, immunogold labeling through ultrathin sectioning with ultramicrotomes, cryo-ultramicrotomy, cryosectioning, critical point drying, plunge freezing, freeze fracturing, freeze drying, contrasting, cryofixation, high pressure freezing, cryo transfer, freeze etching, freeze fracture to ion beam milling, ion beam etching, and target preparation - mechanical grinding and polishing.

Only if each step of sample preparation is of the highest quality, one can get optimum results from a high resolution electron microscope.

  • Preferably sample must be ready while coming for the slot.
  • User must collect sample grids 48 hours before slot.
  • User should know that what kind of sample preparation is required for his/her samples.


A. Dispersion:

  • Which solvent (Ethanol/Methanol/Water/Isopropyl alcohol/ Acetone/Toluene water) is suitable for users samples.


B. Staining:

  • The most widely used stains in electron microscopy are the heavy metals, uranium and lead. The double contrast method of ultrathin sections with uranyl acetate (UA) and lead citrate is the standard contrasting technique for electron microscopy.
  • User must know which type of stain & staining protocol (Negative staining, Positive staining, Fume hood staining) is suitable for his/her samples.


C. Embedding blocks:

  • Use gloves, mask & goggles during sample fixation, dehydration and embedding. All washing and work related to Osmium Tetroxide and glutaraldehyde must be done in Fume hood. Osmium Tetroxide is toxic.
  • Wear gloves while handling Cacodylate salt and buffer as it contains Arsenic and is carcinogenic. Please read the MSDS (Material Safety Data Sheet) properly before using.


D. Sectioning:

  • This work is done by instrument operator only.


E. Vitrification for Cryo Imaging:

  • Sample preparation techniques such as staining and plastic embedding involve the dehydration of biological specimens, which fundamentally removes them from their native, aqueous environment. So Vitrification is done. Imaging of cryogenically immobilized samples by EM is known as cryo-EM. The sample must be frozen extremely rapidly, at a rate of ~106oC/s, so that the water in, and surrounding the specimen is fixed in a vitreous state, done in cryo plunger, plunge-freeze the grid into liquid ethane at the temperature of liquid nitrogen, so that it is suitable for cryo-EM visualization. Once the frozen-hydrated grid is prepared, it is placed in the microscope and kept at approximately -170oC throughout the Imaging.