Kusuma School of Biological Sciences houses some of the following facilities, amongst others:
Transmission Electron Microscopes
• Microscope — FEI Tecnai TF20
• Plunge freezing device — Vitrobot Mark IV
• Plasma Cleaner — Model 1020 Plasma Cleaner
• Images of Instruments and Testing Results
Atomic force microscopy
Atomic force microscopy (AFM) is one of the most versatile and powerful imaging technology for studying a variety of sample types at nanoscale. The major advantage of AFM is its capability to produce 3-dimensional topographical information along with other surface associated features of samples. AFM falls in a category of scanning probe microscopes (SPM) where a cantilever/probe scans over a small area of the sample, measuring the topological and surface properties simultaneously. The deflection of the cantilever tip towards the surface due to the close-range, attractive forces between the surface and the tip is derivatized into signal which is later converted into images. Further, by using a feedback loop to control the height of the tip above the surface, an accurate topographic map of the surface features is generated. The AFM imaging has been widely utilized in material sciences to analyze different amalgamating minerals, ceramics as well as comparing their mechanical properties. In biological sciences, besides being a major imaging technique for elucidating structures of various cells, tissues and large protein aggregates such as amyloid fibers, it finds major application in comparative analysis of variation in surface features such as roughness and elasticity of biological samples.
Mass Spectrometry Facility
The LC-MS facility at KSBS, IIT Delhi is a Bruker Electrospray Ionization instrument-amaZon SL Dual Funnel Iontrap bench top with a mass accuracy of 0.1Da. It is connected to a nano liquid chromatography setup- EASY nLC-II with C18 column to fractionate sample containing mixture of peptides. There is a facility of direct probe injection as well as sample injection through nLC. Our instrument is capable of performing analysis of intact proteins, mixtures of proteins and peptides, metabolites like sugars, nucleotides, steroids, biomarker studies, identification of post translational modifications, determination of purity of samples, identification of oligomeric state, chemical structure determination using MSn and many more applications.
Mass spectrometry through MALDI TOF/TOF provides ultrahigh performance and extreme flexibility in bimolecular characterization including clinical and applied proteomics applications. It operates at 2 kHz in TOF mode and at 1 kHz in the TOF/TOF mode. Samples can be analyzed and identified precisely with very high accuracy using MALDI TOF/TOF. The instrument is used specially for the analysis of intact proteins, post translational modifications in proteins and characterization of unknown proteins along with other biological samples. Proteins can be characterized through Peptide Mass Fingerprint in TOF mode to provide MS spectra and peptide fragmentation (LIFT process) in TOF/TOF mode to provide MS/MS spectra with high efficiency and sensitivity. The instrument has an advantage that nearly 380 samples can be spotted at a time on its target plate and only 1µl sample per spot is enough for analysis. High accuracy, sensitivity and efficiency along with less time consumption for sample analysis make MALDI TOF/TOF Mass Spectrometer as an important and necessary instrument in modern day biological research.
Transmission Electron Microscopy Facility
Transmission electron microscopes (TEMs) typically use high energy electron beams transmitted through very thin samples in 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 which produces 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.
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.
The Vitrobot (Mark IV) is an automated device for vitrification 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.
The Model 1020 plasma cleaner from Fischione Instruments is capable of cleaning both TEM support grids and TEM specimen holders.
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.
EPU software from FEI is available for automated data collection. Softwares for single particle reconstruction such as EMAN, RELION, SCIPION and SPIDER are freely available.