Instrumentation-iBRIC

The Department of Biotechnology (DBT) supported National Cryo-EM Facility is located in the inStem building and is jointly managed by the Institute for Stem Cell Science & Regenerative Medicine (DBT-inStem) and the National Centre for Biological Sciences (NCBS-TIFR) on the Bangalore Life Science Campus. This is the first Cryo-EM that was installed in India for the analysis of biological samples. Currently the facility contains two electron microscopes including high-resolution transmission electron microscope (TEM) Tecnai T12 G2 spirit and high-resolution field emission scanning electron microscope (SEM) Merlin Compact VP as well as a sample, purification and preparation lab.

1. Transmission Electron Microscope or TEM (Tecnai G2 Spirit Bio-TWIN )

   

   • High contrast, high resolution for 20 –120 kV operation Accelerating Voltage 20, 40, 60 80 100 120 kV
   • Beam source: LaB6 filament.
   • Vacuum system: Oil Diffusion (ODP) and Rotary for projection chamber; Ion (IGP) and Turbo vacuum pumps for column.
   • +/-80˚ fully computer controlled Goniometer stage
   • Optimized for 2D & 3D imaging of cells, cell organelles and soft matter
   • Smart Tracking Position System for sample navigation Low dose mode available
   • Cryo attachment for imaging of biological specimens and materials samples at sub-zero temperatures
   • 4k x 2k GatanOrius side mount CCD camera.
   • DM3 operation software (Gatan Digital Micrograph)
   • 3D reconstruction software
   • Holders: Standard single tilt; Gatan multiple specimen holder; Standard single tilt +/- 80˚ Tomography specimen holder; Gatancryo holder single tilt +/- 80˚ Tomography Cryotransfer Holder

2. Scanning Electron Microscope or SEM (The MERLIN Compact VP)

   

   • Resolution: 0.8nm @ 15KV; 1.4nm @ 1 KV; 0.8nm @ 30KV (STEM mode)
   • Probe current: 5 pA to 100nA
   • Acceleration voltage: 0.02 V to 30 kV
   • Magnification: 12 – 2,00,000 X
   • Electron Emitter: Thermal (Schottky) field emission gun.
   • Detection Modes
   • Correlative Microscopy with Shuttle and Find.
   • Cryo SEM and freeze fracture for imaging hydrated samples or for beam/vacuum sensitive specimens.
   • EDAX for chemical composition analysis and element mapping.
   • STEM: Low voltage optimized bright field, 4 quadrant dark field, and high angular dark field transmission imaging.
   • Variable Pressure: High efficiency VPSE detector for pressure up to 60 Pa
   • Secondary Ion: High efficiency in-lens SE detector Everhart Thornley Secondary Electron detector
   • In-lens Duo: Switchable between on-axis in-lens secondary electron detection for highest surface sensitivity and energy selective backscattered detection for advanced materials contrasts.
   • Specimen Stage: 5-Axes Motorized Eucentric Specimen Stage:X = 130mm; Y = 130mm; Z = 50mm; T = - 3º to 70º; R = 360º (continuous)
   • Chamber: 330mm(Ø) x 270mm(h), 15 accessory ports for various options including CCD-Camera with IR-illumination
   • Vacuum system: High vacuum mode and variable pressure mode

   The facility began operations in 2018, and more than 66 users (both internal and external) have availed the electron microscopy facility.

Advanced Technology Platform Centre (ATPC) is a DBT-funded technology platform at the NCR Biotech Science Cluster. The centre is equipped with state-of-the-art research facilities and offers a wide range of services such as Electron Microscopy, Mass Spectrometry, Genomics, Optical Microscopy, Protein Purification, and Flow Cytometry. Some of the high-end facilities at ATPC are given below.

Electron Microscopy Facility

The Electron Microscopy Facility houses Cryo-Electron Microscope: JEM2200FS; Transmission Electron Microscope: JEM1400 FLASH; and VolumeScope SEM with serial block-face imaging solution.

1. Transmission Electron Microscope: JEM-1400Flash

   Transmission electron microscope (TEM) is used in a wide range of fields, such as biology and material sciences, to provide morphological and compositional information of samples. JEM1400 Flash is 120 kV TEM equipped with tungsten filament as an electron source and a highly-sensitive sCMOS camera. It can achieve high contrast imaging of biological specimens (including macro-molecular materials, tissue sections and viruses, etc.) and material science samples (polymeric and metallic nanoparticles, etc.) from low magnification (min. mag. 10X) to high magnification (max. mag. 1.2 MX) with resolution up to 0.38 nm.

2. Cryo Electron Microscope: JEM-2200FS

   The JEM-2200FS is a field emission electron microscope that can be employed for biological and material science samples. It is equipped with a 200 kV field emission gun (FEG), a piezo-controlled goniometer, holders for cryo-observation and tomography, an in-column energy filter (Omega filter), and a Gatan camera (K2 summit direct detection camera). This instrument is utilized for high-resolution cryo-electron microscopy, zero-loss imaging, energy-filtered imaging, spectroscopy for elemental analysis, and tomography.

3. Scanning Electron Microscope: ApreoVolumeScope FEI

   VolumeScope is a novel serial block-face imaging solution that enables excellent z-resolution from multi-energy deconvolution SEM combined with the efficiency of high-resolution cryo-electron microscopy in situ sectioning. The system offers the highest contrast and optimal SNR with in-lens and in-column detectors for HiVac, as well as dedicated detectors for optimal resolution in LoVac. The VolumeScope SEM unravels the complex 3D architecture of cells and tissues in their natural context, which is crucial for gaining an understanding of the structure-function correlation in biological systems.

   A total of 187 users (both internal and external) have availed of the Cryo-EM facility at RCB.

Mass Spectrometry Facility

Mass Spectrometry Facility provides services for identification and quantification of small and large molecules like secondary metabolites, peptides and proteins. The facility houses ESI-MS: TripleTOF® 5600+; MALDI TOF/ TOF Mass Spectrometer: SCIEX TOF/TOFTM 5800; ESI-Mass Spectrometer: SCIEX QTRAP® LCMS/MS 6500+ and Perkin Elmer FlexarTM HPLC system.

1. ESI-Mass Spectrometer: SCIEX TripleTOF® 5600+

   Mass Spectrometry enables the determination of the molecular masses of small molecules as well as of large biomolecules such as proteins and peptides. The SCIEX Triple TOF 5600+ mass spectrometer is a high-resolutionLC-MS/MSmass spectrometer with high sensitivity and high mass accuracy. The Triple TOF® 5600+ has both ESI and APPI ionization sources and is hyphenated with an ExionLC for LC-MS analysis of small molecules and also with an EksigentekspertnanoLC 425 for qualitative and quantitative proteomics analysis.

2. MALDI TOF/ TOF Mass Spectrometer: SCIEX TOF/TOFTM 5800

   The facility also houses SCIEX TOF/TOFTM 5800 mass spectrometer with an EKSpot MALDI spotter for qualitative and quantitative proteomics analysis. The system is widely used for the identification of protein/ peptide, characterization of PTM, de novo sequencing, and relative quantification using iTRAQ, SILAC, TMT, etc.

3. ESI-Mass Spectrometer: SCIEX QTRAP® LCMS/MS 6500+

   The facility is equipped with SCIEX QTRAP® LCMS/MS 6500+ system. It is the fastest and most sensitive QTRAP. It provides the best LOD's and LOQs to enable the detection and quantification of the widest scope of chemical compounds in the most challenging matrices. Improved polarity switching and MRM3 speeds allow faster chromatography and better throughput. The built in QTRAP functionality enables quantitative MRMs and qualitative QTRAP scans in the same injection to maximize throughput. It is best utilized for performing applications requiring ultimate sensitivity for ultra-low-level identification and quantitation of both small and large molecules and for confirmatory analysis of metabolites.

4. Perkin Elmer FlexarTM HPLC system

   The facility provides services for cation exchange/high pH fractionation of complex proteomics samples using the Perkin Elmer FlexarTM HPLC system, which is coupled with an automated fraction collector for wide coverage of proteome.

   p style="text-align:justify;">The facility has been used for processing and analyzing more than 3000 samples from more than 360 users since its installation.

Genomics Facility

Genomics Facility currently houses two instruments- DNA Sequencer: ABI 3500 Genetic Analyzer and Droplet Digital PCR (Bio-Rad).

1. DNA Sequencer: ABI 3500 Genetic Analyzer

   3500 Genetic Analyzer is a fluorescence based 8-capillary sequencing instrument which is based on Sanger DNA sequencing. The system is configured with 50 cm array and integrated software for instrument control, data collection, quality control, base-calling, and size-calling of samples. The system has a varied range of applications, like microbial sequencing, targeted DNA sequencing, epigenetic analysis, pathogen analysis, genetic disease research, SNP analysis, and biomarker analysis.

2. Droplet Digital PCR: ddPCRTM (Bio-Rad)

   ddPCRTM is an advanced version of PCR that provides simultaneous clonal amplification and fluorescence-based quantification of nucleic acids. A typical reaction in ddPCRTM is partitioned into several thousands of water-in-oil droplets where individual reactions are carried out, providing absolute, precise, and reliable quantification. ddPCRTM has demonstrated usage in the detection of rare DNA targets, determination of copy number variations and measurement of gene expression levels.

   The Genomics facility is heavily used, and the services have been availed by a total of more than 1300 users for processing and analyzing more than 7800 samples.

European Synchrotron Radiation Facility (ESRF) Access Program at RCB

RCB and European Synchrotron Radiation Facility (ESRF) entered into a three-year agreement concerning the medium-term use of synchrotron for non-propriety research for the benefit of the Indian scientific community as a whole, and notably the structural biology research groups. The program provides access to Indian investigators to high-intensity beamlines for macromolecular crystallography, small angle X-ray scattering experimental stations and the Cryo-Electron Microscopy Facility located in ESRF, France.

The first agreement was for three years from 1/2/2017 and was renewed for another three years starting February 1, 2020.ESRF provides funds for travel, lodging, and boarding for all visits to ESRF by Indian researchers. Indian researchers with samples can screen them at the RCB in-house X-ray diffraction facility and subsequently submit a proposal for access to ESRF resources at the portal: https://esrf.rcb.res.in.

In the first phase of the agreement (2/2017-1/2020), 108 researchers from 28 different institutions in India used ESRF resources. In the current phase, 64 researchers from 16 different institutions have used ESRF resources. In the current and previous phases, 172 research papers in international peer-reviewed journals have been published using ESRF resources by Indian researchers.

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which maps brain activity by measuring the magnetic fields produced by the naturally occurring electrical currents in the brain. Arrays of SQUIDs (superconducting quantum interference devices) are used as magnetometers to detect magnetic signals from the brain. MEG is generally used to identify the exact location of source of epileptic seizures (epileptogenic focus).

MEG facility has been installed at NBRC under RRSFP-SAHAJ scheme, funded by the DBT.

The objective of the facility is to perform MEG scans and analysis in persons with epilepsy (PWE) and issue the signed report for further procedures. At the MEG Resource facility, 2 PWEs are investigated every day. The scanning duration is 2-3 hours.

Details of infrastructure: ElektaNeuromag TRIUX (MEG)

• Dedicated MEG Lab on ground floor with separate MEG Acquisition Room.
• Patient Data Recording Room.
• HP Server for Patient Data Storage
• UPS backup for MEG Acquisition Room.
• TMS (Transcranial Magnetic Stimulation) Machine Room.
• Both Male and Female wards (3beds in each ward) with basic medical equipment.
• Dedicated computer working station with software for Patient Data Analysis.

MEG scanning has been performed for a total of 1155 epilepsy patients at NBRC in the last 5 years.

National Neuroimaging Facility at NBRC

The DBT funded National Neuroimaging Facility, was established to facilitate/support cutting edge brain imaging research. The facility is equipped with state-of-the-art equipment including 3T Magnetic Resonance Imaging (MRI)Scanner, sensors and computers for Electroencephalography (EEG) and setup for Evoked Response Potential Recording (ERP).

Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) is a non-invasive medical imaging procedure which uses a combination of a large magnet, radiofrequencies and a computer to produce detailed images of organs and structures within the body. A brain MRI can provide highly detailed images of the brain and brain stem and help in locating tumors, inflammation, aneurysms or other such abnormalities.

MRI provides much greater contrast between the different soft tissues of the body compared to computed tomography (CT), making it especially useful for neurological (brain), musculoskeletal and cardiovascular imaging. Various imaging modalities also play an important role in providing crucial information which can aid in diagnosing different disease conditions. Amongst the different imaging modalities used at NBRC are:

1. MR Spectroscopy (MRS) which provides non-invasive estimations of differentneurochemicals in the brain and enables clinical correlation.

2. Functional MRI (fMRI), whichcorrelates functional (haemodynamics) activity with images of brain activation.

The 3 Tesla Phillips whole-body MRI scanner at the National Neuroimaging Facility is equipped with state-of-the-art hardware, software and data processing software required for each imaging modality. The facility is being used for performing structural, metabolic (multinuclear, e.g. proton and phosphorous) and functional MRI.

Equipment details:

• Make- Philips

• Model No. - 3T Achieva

• Version- Release 5.1 version

Data acquisition capabilities:

• fMRI

• MRS (1H,31p and Flourine also)

• DTI with 128 Directions (User Defined also accepted)

• ASL (Arterial Spin Labeling)

• PCA (Phase Contrast Angiography) and Venogram

In the last 5 years, National Neuroimaging facility (using 3T MRI) has been used for the following number of patients: Alzheimer's patients- 9; Early Alzheimer's Patients (MCI)- 115; Parkinson’s Disease patients – 61; Elderly persons age (60-94Y range)- 361.

The National Genomics Core facility has been established by the DBT at CDFD to provide high end genomic solutions and services for the research needs of academic institutes and industry.

The facility houses the following instruments:

Illumina MiseqFGx

MiSeqFGx solution delivers a complete DNA-to-Data workflow for the analysis of forensic DNA samples. The solution begins with the ForenSeq™ DNA Signature Prep Kit, which includes all reagents required to prepare DNA libraries for sequencing. ForenSeq Universal Analysis Software delivers a powerful suite of forensic analysis capabilities including automatic detection of mixed DNA samples, generation of population statistics.

The Illumina MiSeqFGx is also a cost-effective solution for smaller sequencing applications such as Amplicon sequencing, 16s rRNA metagenomics, Small RNA sequencing, Small Genome sequencing. The platform enables up to 15 Gb of output with 25 million sequencing reads and 2 × 300 bp read lengths.

Illumina Nextseq 2000

NGC hosts the first NextSeq 2000 system in the country.

The NextSeq 1000 and NextSeq 2000 Systems support emerging and mid-throughput sequencing applications as well as a broad range of methods such as exome sequencing, target enrichment, single-cell profiling, transcriptome sequencing, and more.

The system features a novel super resolution optics system that yields highly accurate imaging data with greater resolution and higher sensitivity than traditional benchtop systems Capable of producing upto 300Gb data generating 1billion reads with 2x150bp read lengths.

The facility also has Oxford NanoporeGridION X5 (a compact benchtop system designed to run and analyse up to five MinION Flow Cells), Agilent Tapestation 4200 (automated electrophoresis tool for DNA and RNA sample quality control) and Agilent 2100 Bioanalyzer system (automated electrophoresis solution for the sample quality control of biomolecules).

The facility has been used for sequencing and genotyping more than 15000 samples. A total of 126 services have been provided to both internal and externalusers. Additionally, 11692 samples of SARS-Cov-2 have been sequenced under Indian SARS-CoV-2 Consortium on Genomics (INSACOG).

NIBMG is the first institution in India explicitly devoted to research, training, translation & service and capacity-building in Biomedical Genomics. The laboratories are equipped with state of the art instruments. The National Genomics Facility hosts a number of high-end equipment.

• Massively parallel sequencing platforms of GS-FLX (Roche-454), HiSeq-2000 & 2500 (Illumina), Ion Proton & Ion Torrent PGM (Life Technologies) for whole genome sequencing, exome sequencing, targeted resequencing, metagenomics, amplicon sequencing, RNA-Seq& small RNA-Seq, ChIP-Seq
• ABI3500XL Genetic Analyzer (Life Technologies) for Sanger/CE sequencing and fragment analysis
• ABI7900 HT (Life Technologies) and Lightcycler LC480 (Roche) for high throughput real time PCR
• 2100 Bioanalyzer (Agilent) for sequencing library QA/QC

The facility also has Microarray setup for processing Illumina beads, a 300 TB Grid Scalar Central Data Storage platform (DDN), High performance parallel computing Clusters (HPPC Lab), computational labs for data analysis and also has a central Digital Library for accessing high impact journals through the DBT-Electronic LibraryConsortium (DeLCON) network. Flow cytometry, confocal imaging and tissue and bacterial culture are other basic facilities available

A total of 68 users have accessed and utilized the various services and instruments available at the National Genomics Facility at NIBMG.

The NGGF acts as a nodal centre for DNA finger printing of lines and varieties for certification purpose in coordination with Plant Variety Protection Authority. It provides consultancy as well as improved genomic tools to plant breeders and plant biotechnologists to enhance the quality of research and product development.

The facility houses three major platforms:

1. Fluorescent dye-labelled SNP genotyping platforms (LGC Array Tape/KASP)

   This platform can genotype limited number of SNPs with large number of samples at low cost. This has major applications in foreground and background analysis in MAS, genetic fingerprinting and genetic purity testing, diversity analysis and CRISPR/Cas9 genome editing validation.

   

   

   KASP genotypingIntelliCube/ LGC Array

2. Array, Mass and Expression based SNP genotyping platforms (Affymetrix Gene Titan, Agena and Nanostring)

   This platform can genotype large number of SNPs at a genome-wide scale. It is cost effective and rapid. Major applications of this platform are: Germplasm characterization to assists in core & mini-core selection, background analysis for MAS, GWAS and e-QTL mapping to identify loci for traits of interest and genomic selection.

   
   Affymetrix Gene Titan Machine

   
   Nanostringn Counter SPRINT Profiler

   
   AgenaMassARRAYAnalyzer 4

3. NGS-based sequencing and genotyping platforms (NovaSeq 6000 and NextSeq 550)

   This platform is used for medium to large number of SNPs at a genome-wide scale. It is cost effective and high throughput. Its major applications are: Genome sequencing, identification of novel SNPs/structural variants (SNPs, InDels, CNVs, etc) and genomic selection, for genome assembly and germplasm characterization.

   
   NovaSeq 6000

   
   NextSeq 550

   The NGGF has provided genotyping services to a total of 21 users, both internal and external, since its inception in 2019.