Services

Bio-Imaging Facility

Confocal microscopy is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures within an object. At the NCCS - Bio-Imaging Facility, graduate and postdoctoral research fellows can be trained in microscope research techniques including advanced light microscopy, confocal microscopy, digital image processing of microscope images, and related laboratory techniques. Computer image processing and analysis is taught individually. In addition, the Facility offers workshops related to Microscopy and training programs are designed to train the students in modern and classical methods of preparing microscope slides from time to time. Staff members demonstrate the correct use of the instruments, train students in the microscopic techniques required for successful cell biological research and computer image processing and analysis.

 

Instruments available at Bio-imaging Facility

Point Scanning Microscopes:

1.   Leica Stellaris 8 STED Microscope

 

 

  • This microscope is a tool for advanced imaging applications, offering high-resolution, multicolor, and live-cell imaging capabilities.

  • This imaging system combines confocal microscopy with Stimulated Emission Depletion (STED) super- resolution capabilities with sub-30 nm resolution, having Tunable excitation (440–790 nm).

  • STED microscopy can achieve a lateral resolution of ~30–50 nm and axial resolution of ~120–150 nm.

  • Some unique features are Power HyD detectors with high photon detection efficiency (HyD S & HyD X), real-time analysis of molecular dynamics by Fluorescence Correlation Spectroscopy (FCS) and TauSense.

  • It also has FAst Lifetime CONtrast (FALCON), a FLIM solution for fast acquisition which combines STED with fluorescence lifetime imaging (FLIM) to separate fluorophores based on their lifetimes.

  • LIGHTNING Provides enhanced resolution and contrast in real- time imaging, capturing fine structural details.

  • Objectives: 10x-100x and a special 93x STED objective.

  • Lasers: 405nm laser, Unable White Light Laser (WLL) 440-790 nm, 589nm and 775nm Depletion Lasers.

 

2.    Zeiss LSM880 Confocal Microscope with AiryScan and ELYRA P.1

 

 

  • This imaging system comprises of Axio Observer 7 microscope with AiryScan and Elyra P.1.

  • Airyscan is a 32-channel GaAsP-PMT area detector that collects a pinhole-plane image at every scan position. Each detector element functions as a single&very small pinhole.

  • ELYRA P.1 localizes small structures and even single molecules achieving resolutions of 20nm laterally and 50nm axially.

  • Objectives: 10x, 20x, 40x (oil), 63x (oil), 100x (oil)Alpha PlanApochromat

  • Lasers:For confocal- 405nm, 488nm, 458nm, 514nm, 561nm, 594nm,633nm

  • For Elyra P.1- 405nm, 488nm, 561nm, 640nm

 

3.    Olympus FV3000 Confocal Microscope

 

 

  • This is a high speed spectral confocal microscope for macro to microscopic imaging with 3D FRET, FRAP, Photoactivation applications.

  • It can attain high resolution nearly 140 nm or better (XY) and 350 nm in Z direction for at least two colours simultaneously with OSR (Olympus Super resolution- software based).

  • Z Drift Compensator (ZDC) avoids minor vibrations during live imaging and retains the same focus position in real time for long term time lapse imaging. Images can be acquired at high speed or high scan format.

  • The system has 2 PMT detectors and 2 High sensitivity detectors (GaAsP) with possibility of 8 to 16 channels and dedicated transmitted light detector for DIC imaging.

  • Objectives are 1.25X to 60X, with 30x Silicon

  • Laser: 405nm, 488nm, 561nm, 640nm, 514nm

 

4.  Olympus Multiphoton microscope

 

 

  • Olympus FVMPE-RS multiphoton laser scanning microscope is available for deep tissue imaging of fixed samples and live whole animals.

  • This system features a tunable infrared laser (MaiTai, 680~1040nm) to excite multiple fluorescence dyes optimally for precise multispectral imaging.

  • It also provides resonance scanning mode for high speed imaging and laser light stimulation for photomanipulation of fluorophores such as FRAP and optogenetics.

  • With dedicated multiphoton-specific objectives (10x and 25x water dipping), these features allow users to image the large samples (up to 8 mm thickness) in multicolor 4D.

  • Multicolor Imaging filter cubes: FVG (violet & green), FGR (green & red), FRCY5 (red &Cy5) are available.

  • The system is equipped with two GaAsP PMTs and also has 3D deconvolution and rendering functions.

 

5.    Leica SP5 II Confocal Microscope

 

 

  • This is a high-end Broadband Confocal Laser Scanning Microscope with 3 PMTs, 2 Hybrid detectors and AOBS technology.

  • The microscope is equipped with CO2 incubation chamber for live cell experiments as well as FRET, FRAP experiments and routine multi dye imaging.

  • This is fully motorized, automated and computer controlled microscope Leica DMI 6000.

  • Objectives : 10x, 20x, 20x multi-immersion, 40x oil, 63x oil, 100x oil.

  • Laser: Diode laser 405nm, Multi Argon ion Laser with 458/476/488/496/514nm lines, DPSS 561nm, He-Ne 594nm, He-Ne 633nm

 

 

Spinning Disc Microscopes:

 

1.    Nikon Structured Illumination Microscope (SIM) / X-Light V3 and DeepSIM system

 

 

  • X-Light V3 system is a spinning disk confocal system offering precise 3D sectioning at the highest speed and largest field of view maintaining uniformillumination.

  • The DeepSIM module allows researchers to seamlessly enhance their imaging capabilities from widefield to confocal to super-resolution.

  • The 2D Lattice SIM technology behind DeepSIM enhances the illumination efficiency and homogeneity with minimal phototoxicity in live cell imaging experiments.

  • Objectives:10x, 20x, 40x, 40x Silicon, 60x oil and SR HP Apo 100x oil.

  • Lasers: 405nm, 488nm, 555nm, 640nm

 

2.   Olympus SPIN SR 10 - Spinning Disk High Resolution Microscope

 

 

  • This Spinning disc confocal microscope is equipped with YOKOGAWA W1- SoRa scan-unit.

  • It can attain high resolution minimum 250 nm (XY) and 800 nm in Z. Super-resolution mode gives XY resolution of 120nm and Z of 540nm approximately.

  • The system has 2 high-speed cameras for simultaneous imaging of at least two dyes.

  • Objectives: 10X to 100X.

  • Lasers: 405 nm, 488nm, 514nm, 561nm, 640nm

 

3.    Thermo CellInsight CX7 LZR - High Content Screening System

 

 

  • This is a fast, laser-based, automated cellular imaging and analysis platform for quantitative microscopy and phenotypic screening.

  • It has a 7-channel, laser-based illumination system with Software and laser-based autofocus for consistent scan times.

  • Major applications are fluorescent, confocal and 5-channel brightfieldimaging.

  • It is highly compatible with standard microplates (6, 24, 48, 96, 384, 1536 wells). It is loaded with HCS Studio software for integrated data collection and analysis.

  • The system provides powerful statistical method to carryout various bio applications like- Autophagy assay, Cell viability assay, Kinetic assays, Apoptosis, DNA damage, Transfection efficiency, Cell toxicity and colony formation assays etc

  • Objectives: 2x, 10x, 20x, 40x to 60x.

  • Laser: 405nm, 450nm, 488nm, 561nm, 594nm, 647nm, 785nm.

  • Dr. Ashwini N. Atre Technical Officer C
  • Mrs. Trupti P. Kulkarni Technical Officer A

INFORMATION FOR EXTERNAL USERS:

  • The Charges for External Users are Rs. 2000 per hour + 18 % GST for academic users and Rs. 3000 per hour + 18 % GST for non-academic users.

User will be charged according to the microscope usage time. User can bring DVD or pen drive for collecting data/ images. Outsiders will be allowed to use NCCS Microscopy facility maximum of two hours per week per microscope. To book the slots contact us @ bioimaging@nccs.res.in  020-25708265 / 8260 before 11 AM on previous Friday.

SERVICES PROVIDED:

  • Expert consultation is provided through the Facility In-Charge and our technical specialists.
  • Confocal microscopes are selected for complementary functions.
  • Equipment use is accessible through dedicated technicians / operators.
  • All services are provided on chargeable basis.
  • Assistance with data analysis can be customized to the needs of individual investigators and research projects.
  • Image manipulation and enhancement using Photoshop, ImageJ, Fiji, Matlab and Cell Profiler, IrfanView.
  • Assistance in image and data analysis related to morphometrics, particle counting, particle tracking, numeric analysis, statistical analysis, etc.
  • Data backup and archiving.

Requisition form

 Method for Immunofluorescence staining 

 

Material required:

  1. 4% Paraformaldehyde (PFA).
  2. 1x PBS solution.
  3. 0.01% Triton x solution.
  4. Blocking solution (5% FBS solution in PBS).
  5. DAPI/Hoechst solution: dissolve 1mg in 1ml of 1X PBS. Objectives: 2x, 10x, 20x, 40x to 60x.
  6. Antibody against specific protein.

 

Procedure:

  1. Culture the cells on coated glass coverslips/chamber slides.
  2. Wash the monolayer thrice with 1X PBS.
  3. Fix in 4 % PFA for 10 min in RT.
  4. Wash with 1X PBS three times.
  5. Permeabilize with 0.01% triton x 100, 10mins at RT.
  6. Wash with 1X PBS three times.
  7. Add blocking solution and incubate at 37° for 1 hr.
  8. Add primary antibody (1:200 dilution in blocking solution), overnight at 4C.
  9. Wash three times with PBS.
  10. Add secondary antibody (1:1000) for 1 hr in dark at 37°C.
  11. Wash 3 times in PBS.
  12. Counter stain with DAPI/ Hoechst solution in PBS for 10 min.
  13. Wash 3 times in PBS.
  14. Air dry the slide at and mount on a glass slide.(Note Before mount use mounting agent and close the cover slips with nail polish)

 

Useful links:

 

Microscopy resources:

Dr. Jomon Joseph, In-charge, Bio-Imaging Facility

Contact Information

 


020-25708265/8260

 


bioimaging@nccs.res.in

 


Bio-Imaging Facility, 
National Centre for Cell Science, 
NCCS Complex, P.B. No. 40, Ganeshkhind Post, 
S. P. Pune University Campus, 
PUNE – 411 007, 
Maharashtra State, India. 

Directions to Bio-Imaging Facility: Situated in NCCS New Building Ground Floor (Next to Reception)

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