This facility hosts following state-of-the-art instruments: Super Resolution Microscope; Laser Scanning Confocal Microscope and High Content Imaging System.

1. Super Resolution Microscope: Elyra PS1, Carl Zeiss

Structured illumination microscope is a widefield technique in which a grid pattern is generated through interference of diffraction orders and superimposed on the specimen while capturing images. The grid pattern is shifted or rotated in steps between the capture of each image set. Following processing with a specialized algorithm, high-frequency information can be extracted from the raw data to produce a reconstructed image having a lateral resolution approximately twice that of diffraction-limited instruments and an axial resolution upto 120 nanometers.

About Microscope: The Microscope is framed in an inverted fluorescence research microscope (Axio observer 7) with motorized stage. There are other super resolution modules available with the system which are as follows:

• Total Internal Reflection Fluorescence Microscopy (TIRF)
• Photo Activation Localization Microscopy (PALM with 3D)
• Stochastic Optical Reconstruction Imaging (STORM)

It has a metal halide illuminator HXP120 for sample observation and 4 diode lasers to make imaging comprehensive. The lasers are:

• 405 nm Laser 50 mW
• 488 nm laser 100 mW
• 561 nm laser 100 mW
• 640 nm laser 150 mW

It is equipped with varied range of microscope objectives:

• 20X Plan-Apochromat (NA0.80)
• 63X C- Apochromat Water immersion (NA 1.2)
• 63X Plan-Apochromat Oil immersion (NA 1.40)
• 100X alpha Plan-Apochromat Oil immersion (TIRF Objective) (NA 1.46)

The system uses two different cameras for imaging:

• Andor EM CCD camera iXon DU897 (For TIRF, PALM and STORM imaging)
• Camera pco.edge sCMOS Version 4.2 (for SIM imaging)

Applications: System is used to find super resolved images (resolution up to 120 nm) of cells and their network filaments/fibers, organelles.

2 Laser Scanning Confocal Microscope: LSM 880, Carl Zeiss

The coherent light (excitation laser) passes through a pinhole aperture that is situated in a conjugate plane (confocal) with a scanning point on the specimen and a second pinhole aperture positioned in front of the detector (a photomultiplier tube). As the laser is reflected by a dichromatic mirror and scanned across the specimen in a defined focal plane, the emission from points on the specimen (in the same focal plane) pass back through the dichromatic mirror and are focused as a confocal point at the detector pinhole aperture. Due to this aperture, the extraneous light (out of focus light) is not detected by the detector thereby resulting in sharp images.

About Microscope: The Microscope consists of an inverted fluorescence research microscope (Axio observer 7) with following objectives:

• 10X Plan Apochromat (NA 0.45)
• 40X Plan-Apochromat Oil immersion DIC (NA 1.30)
• 63X Plan-Apochromat Oil immersion DIC (NA 1.40)

Observation and imaging units: A metal halide illuminator (HXP120) and HAL100 illuminator for sample observation and 5 lasers including Multi Argon lines for confocal imaging.

1. Diode laser 405 nm
2. Laser Argon Multiline 458/488/514 nm
3. Laser HeNe 543 nm
4. Laser HeNe 594 nm
5. Laser 633nm

Detection unit:

1. Transmitted light detector T-PMT
2. LSM 880 confocal scan head with 2MA-PMT and 1 GaAsP (for spectral detection)
3. High resolution digital microscopy camera AxioCam 503 mono

Applications: Fluorescence imaging of tissues, cells, live imaging and spectral imaging and dynamics experiments such as FRAP and FRET.

3. Micro Confocal High Content Imaging System: ImageXpress, Molecular Devices

The ImageXpress system is an integrated cellular imaging and analysis system that is designed for rapid, automated screening of fluorescently labelled fixed and live cells in microplates. It also provides environmental control for live cell imaging, moves fluids to and from cells for compound addition. The ImageXpress Confocal system is a high-content imaging system that can switch between widefield imaging and confocal imaging. The rapid autofocus and precision sample movement features of the microscope allow large numbers of high-resolution images to be acquired in the shortest possible time. All key optical and mechanical elements are motorized, with asynchronous command execution, allowing complete real-time control of the instrument configuration through the MetaXpress high-content image acquisition and analysis software. When used in combination with the powerful image analysis capabilities of the MetaXpress software, the instrument becomes an extremely flexible and programmable device, ideally suited for user-defined, high-speed automated assays.

Instrument Specifications

• ImageXpress Micro Confocal System is a fully automated confocal microscope with pinhole sizes of 50 µm and 60 µm.
• It is capable of taking images from a microplate (96 well/ 384 well / 1536 well) or from a microscopic slide.
• It is capable of detecting and imaging multiple fluorescent signals simultaneously.
• Transmitted light imaging can be done for non-fluorescent cells.
• Z-stack images can be acquired and analysed.
• Environmental control- multi-day, live cell time-lapse imaging under appropriate temperature, humidity and atmospheric conditions can be performed.
• This system also contains single-channel, fluidics robot. This robot picks up tips from a tip box and draws fluid from and delivers fluid to a given well on the compound and sample plates. The robot is capable of dispensing volumes between 3 μL and 200 μL.
• This system also has a hotel shelf with capacity for 88 standard microplates, where plates can be stacked for imaging. With the help of Sample Handling Robotic Arm, the plates can be removed from hotel shelf and can be loaded onto ImageXpress Micro Confocal System where images are acquired, and then returned to a home location.
• This entire system has been integrated with Cytomat 2C automated incubator through a Sample Handling Robotic Arm so that multiple plates can be scanned for time-lapse live cell imaging. This automated incubator has a capacity for 42 standard microplates.
• Automated barcode reader is also a part of this system.

Light Source

The external light source for the ImageXpress Micro Confocal System is a solid-state light source that has a rated lifetime of more than 20,000 hours. It has light source for excitation of DAPI, FITC, TEXAS RED, Cy3, Cy5 wavelengths.

Objectives

• 4X Plan Apo λ NA 0.20
• 10X Plan Apo NA 0.45
• 20X S Plan Fluor ELWD NA 0.45
• 40X S Plan Fluor ELWD NA 0.60
• 60X S Plan Fluor ELWD NA 0.70
• 100X CFI L Plan EPI NA 0.85

Applications

Application modules are the foundation of the MetaXpress Software analysis workflow. These modules provide automated image analysis for biology-specific applications, interactive image segmentation, and data results, and options for data recording and management. Currently, Our MetaXpress software has the following modules:

• Angiogenesis Tube Formation
• Cell Cycle
• Cell Health
• Cell Proliferation
• Cell Scoring
• Live Dead
• Micronuclei
• Multi Wavelength Scoring
• Translocation
• Neurite Outgrowth