Transmission electron microscopes typically use high energy electron beams transmitted through very thin samplesin order to analyze the microstructure of materials. Electrons are accelerated at several hundred KV, resulting in wavelengths much smaller than that of light, and are focused with electromagnetic lenses for imaging. Images of samples generated are recorded on digital camera. The TEM facility at IIT-Delhi consists of a 200 KV TEM, with a high brightness field-emission gun (FEG) source whichproduces improved sensitivity and resolution compared to more traditional thermionic sources like LaB6 or Tungsten filaments. Collected images provide detailed information about the size, shape and morphological details of materials. This facility can also be utilized for high resolution analysis of the structure and organization of large biological molecules such as protein complexes, viruses etc through cryoelectron microscopy. This technique involves freezing biological material quickly in vitreous ice and imaging at temperatures less than -180°C. Accessory equipment for plunge freezing samples, plasma cleaning grids and holders are available.
Equipments in Facility :
i) Microscope: FEI TecnaiTF20: The FEI Tecnai TF20 is a high resolution Transmission Electron Microscope (TEM) equipped with a FEG source. It has a maximum accelerating voltage of 200 KV, a +/-70 degrees tilted computer controlled stage, and is equipped with a 4K x 4K Eagle CCD Camera with a 4-port readout and 15μm pixel size.Images may be collected in low dose mode for cryo samples. The microscope is suitable for collecting data on macromolecules, cryoelectron microscopy or tomography of single particles as well as semi-thick frozen sections.
ii) Plunge freezing device:
Make : FEI and Model : Vitrobot Mark IV The Vitrobot (Mark IV) is an automated device forvitrification of aqueous samples for cryo-TEM. Accessories such as liquid ethane bath and ethane temperature controller for plunge-freezing of grids, and liquid nitrogen workstation for cryotransfer are available.
iii) Plasma Cleaner: Model 1020 plasma cleaner (Fischione Instruments Inc) The Model 1020 plasma cleaner from Fischione Instruments is capable of cleaning both TEM support grids and TEM specimen holders.
iv) Others:Cryoholder (Gatan), Turbo pumping station (Gatan), Smartset cold stage controller (Gatan) Single-tilt holder for regular samples and cryotransfer holder for cryo samples are available. The latter is supplied with temperature controller and dry pumping station.
v) Softwares: EPU software from FEI is available for automated data collection. Softwares for single particle reconstruction such as EMAN and SPIDER are freely available
Sample Sample Conc. Sample State Pretreatment Conditions for Cryo-EM Sample SizeHuman Rhinoviruses1.0-2.0 mg/mlDissolve in 50mM Tris buffer, pH~7.2Glow Discharge/Plasm a Cleaning 30 secT=25°C, H=80%, Vol.=4µl, B=0.7- 0.9s 30 nmFlock House Virus1.0-3.0 mg/ml50mM Hepes, 5mM Cacl2 pH~7.4Glow Discharge/Plasm a Cleaning60 secT=11°C, H=99%, Vol.=3µl, B=3s 30 nmPOPC Liposomes2.5 mg/mlUnbufferedGlow Discharge/Plasm a Cleaning60 secT=30°C, H=99%, Vol.=4µl, B=0.8- 1.0s 10-300 nmMicrotubules10 µM1mM EDTA, 1mM Mgcl2,80mM Pipes KOH, pH~6.8Glow Discharge/Plasm a Cleaning30 secNot Establised ~50 micrometerRibosomes1.0-2.0 mg/ml10mM Tris-Hcl pH~7.5, 1mM Mgcl2Plasma Cleaning 30 secT=4°C, H=100%, Vol.=4µl, B=6s 20nmRNA PolymeraseOptimum10 mM potassium phosphate,pH 7.9,1O mM 2mercaptoetha nol, 0.1 M EDTA, and 10% glycerolPlasma Cleaning 60 secT=18°C, H=90%, Vol.=4µl, B=2.5s 400-500 kDaProteins0.5-1 mg/ml20mM Sodium phosphate, 20mM NaclGlow Discharge/Plasm a Cleaning30 secNot Established 18.7 kDa MonomerAntibody0.1-1 mg/ml100mM Citrate pH~3.0, 100mM NaclGlow Discharge/Plasm a Cleaning30 secT=30° 6-10 nm MonomerMaterial ScienceVaries from sample to sampleWell dispersed in a solvent like ethanol or waterUltraSonication for about 30min. to 1 hr. as per sampleFor observation under EM, mount sample amount 5- 10 uL on 3mm Cu grid then dry it overnight 2µm-5nm
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Dr. Manidipa Banerjee
Assistant Professor, Kusuma School of Biological sciences
IIT-Delhi, HauzKhas, New Delhi – 110016
Phone number: 011-2659-7538
Dr. Mohit Kumar
Kusuma School of Biological sciences IIT-Delhi, Hauz Khas,
New Delhi – 110016
Phone number: 011-2659-7514
Operator: Ms. Riti Rawat
EM Laboratory, Ground Floor, Kusuma School of Biological sciences
IIT-Delhi, HauzKhas, New Delhi – 110016