Confocal / Imaging Facility

The Bio-imaging Facility, located in the C-Wing, Ground Floor of the Division of Biological Sciences provides "open access" confocal laser scanning microscopy (CLSM) services to the research community of the Indian Institute of Science. It is actively involved in image acquisition in various areas of bio-medical research catering to many departments of the Indian Institute of Science, not only for the Division of Biological Sciences, but also for departments like Materials Research Centre (MRC), Materials Engineering (ME), Mechanical Engineering, Inorganic Physical Chemistry (IPC), SSCU, MBU, Organic Chemistry, and CNS.

The contributions of this facility is reflected by a striking increase in the number of activities and projects that it has been supporting over the past years and the facility now runs with even more dedicated, trained personnel to operate the high end microscopes.

Scientific Staff:

Convenor: Prof. Dipshikha Chakravortty (Microbiology and Cell Biology)

Research Scientist:

  • Dr. Uttara Chakraborty (Sep 2014- till date, SRS)
  • Dr. Santosh Podder (June 2013- June 2015, JRS; July 2015- till date, SRS)
  • Project Assistants: Deepti Bapat (Feb 2012- Oct 2014)
  • Leepika Baid (Oct 2014- May 2015)
  • Saima Kauser Nasir (May 2015- till date)
  • Divya S. (May 2015- till date)

Scientific Personnel

Dr. Dipshikha Chakravortty (Associate Professor, MCB)

Dr. Uttara Chakraborty (Senior Research Scientist, Bio-imaging and Flow Cytometry

Dr. Santosh Podder (Senior Research Scientist, CIDR and Bio-imaging Facility)

Divya S. (Project Assistant)

Saima Kauser Nasir (Project assistant)

The research scientists and project assistants are supported by the IISC-DBT partnership program.

Students and researchers from almost 36 labs of 11 different departments of the Institute extensively use confocal microscopy in order to visualize a variety of samples, from monolayers and small organisms, such as developing fly and fish embryos, to very thick sections from brain and other organ tissues. Our facility has experience imaging in bacteria, mammalian cells, yeast cells, Drosophila and Zebra fish embryos and ovaries, drosophila sarcomeres, sections of brain and other tissues, carbon nanodots and nanoparticles, in both fixed and live specimens. This is accomplished using a variety of optical imaging modalities in the form of 5 state-of-the-art imaging platforms in the facility, to get a clearer picture of macro, cellular, and sub-cellular structures and function. We can image single or multiple labeled specimens, using magnifications from 10X to 100X with a temporal resolution ranging from 1/13 to 1/430 sec.

Laser scanning confocal microscopy (LSCM) represents one of the most significant advances in optical microscopy ever developed, primarily because the technique enables visualization deep within both living and fixed cells and tissues and affords the ability to collect sharply defined optical sections from which 3D renderings can be created.

Equipments:

  • Zeiss 510 meta confocal microscope

  • Olympus IX81 spinning disk microscope.

  • Leica TCS SP5 confocal microscope

  • Leica DMI 6000B inverted fluorescence live imaging microscope

  • Zeiss LSM 880 with Airyscan

  • Fully dedicated 5 workstations are available for the researchers to analyze and process their images.

The system of booking slots for the different microscopes is online, links for each microscopy system are available and bookings are given based on request mails sent to the facility mail ID, slot availability can be visualized on web calendars.

Recommended guidelines to use the Bio-imaging facility

  1. Number of slots allotted: 2 slots/person/week, 16 slots/lab/month. Further requests for slots should come from the concerned PI. If there are free slots available with no user demand, it will be allotted to those in que as per requests.

  2. Booking for all the microscopy systems in the facility will be through online calendars from August, 01, 2015, 15 days in advance.

  1. Zeiss LSM510 Meta- http://www.brownbearsw.com/cal/confocalschedule (pw-schedule1)

Mail ID: confocaliisc@gmail.com

  1. Confocal Leica SP5- http://www.brownbearsw.com/cal/leica (pw-confocal)

Mail ID- leicaconfocal2015@gmail.com

  1. Live Imaging Leica DMI6000- http://www.brownbearsw.com/cal/Liveimaging (liveimaging)

Mail ID- leicaconfocal2015@gmail.com

  1. Links for Airyscan

http://www.brownbearsw.com/cal/Airyscan (password: resolution)

  1. Links for Olympus spinning disk microscopy

http://www.brownbearsw.com/cal/olympus (password: Olympus)

  1. User who needs the slot must only book and not anybody else on his/her behalf.

  2. Timings for the facility to run in 2 shifts are: 10 AM-1 PM and 2 PM- 5 PM. We request all the users to come on time and finish within the stipulated time allotted. In case of delays, kindly call the facility and inform beforehand. We can extend the wait for another half an hour on request but not more. For those who do not inform, the slot will be given to the next request in que. *

  3. We are aware that both the confocal systems, Zeiss LSM 510 Meta and Leica TCS SP5 are aged systems and have been giving us service for the last 8-10 years. Owing to the heavy workload in the facility kindly bring samples judiciously for imaging. We are unable to provide you images in triplicates for all your samples in certain cases, as instrument fails to respond. Samples which have not worked should not be imaged unnecessarily.

  4. 2 users from the same lab cannot share a single slot without any prior notice.

  5. Users should provide their E-mail IDs and contact number so that we can circulate facility related information to all.

  6. After acquisition all data must be collected by user in CDs. We will not be keeping any backup for more than 3 months.

  7. Imaging done in our facility should always be acknowledged in your publications, as that acts as a boost for the facility in terms of quality and performance.

  8. One slot every month will be blocked for our journal club presentations, with prior notice to all users.

* Delay from the facility end will also be informed in advance for genuine reasons.


Confocal Microscopes

Zeiss 510 Meta LSCM, inverted (installed in March 2004)

 

Detailed specifications: Laser scanning confocal microscope (LSCM) with META detector module which can scan across a range of wavelengths to allow digital separation of overlapping spectra (such as GFP and YFP). The achievable scan speed is from a range of 5 fps to 70 fps for 512x 512 pixel resolution.

Lasers:

  • Argon laser (458, 477, 488, 514 nm, 30 mW)

  • HeNe- laser 543 nm, 1mW (Integrated)

  • HeNe- laser 633nm, 5mW (Integrated)

Objectives:

  • 10x/0.45, Objective Plan-Apochromat WD= 2.1 mm

  • 20x/0.75 Objective Plan-Apochromat WD= 0.61 mm

  • 40x/0.95 Objective Plan-Apochromat Corr WF= 0.16 mm

  • 40x/1.20 water: Objective C-Apochromat Corr WD= 0.22 mm

  • 63x/1.3 oil: Objective Plan-Apochromat oil DIC

  • 100x/1.3-oil: Objective Plan-Apochromat Corr

For further technical details visit our webpage (under construction)

Leica TCS SP5 Confocal microscope, upright (installed in Sep, 2007)

Detailed specifications: This microscope is equipped with a high speed scanner (a resonant scanner) which works at more than 25 fps, ideally meant for live cell imaging. It has three conventional detectors for fluorescence imaging and one for transmitted light, therefore the system can be used well for routine multi-dye imaging.

Lasers:

  • Diode laser 405 nm

  • Multi Argon laser (458, 476, 488, 496, 514 nm)

  • DPSS 561 nm

  • HeNe- laser 633nm

Objectives:

  • 10x/0.30 water, HCX APO L U-V-I

  • 40x/0.80 water , HCX APO L U-V-I

  • 63x/0.9 water, HCX APO L U-V-I

  • 63x/1.4 oil: HCX PL APO lambda blue

For further technical details visit our webpage (under construction)

Zeiss LSM 880 with Airyscan Superresolution microscope (installed in June, 2015)

Principle of Airyscanning

The Airyscan detector consists of 32 elements based on GaAsP PMT technology with each element detecting the emission signal of a focal spot much smaller than the typical 1 Airy volume. This detection scheme together with adapted deconvolution algorithms allows resolving fluorescently labelled structures to a much higher degree. The resolution reaches 140 nm in x and y and 400 nm along the optical axis. The emission signal is filtered using standard emission filters. The detected signal is processed online for immediate judgement of the resulting 2D image. As the detector works along the confocal principle an increase in resolution is achieved in 3D and using standard specimens prepared for confocal imaging. Another detection mode provides the ability to virtually open or close the pinhole after the image has been acquired. In addition the detector can be used as an additional confocal detector and in combination with other detectors of the system. The achievable scan speed is from a range of 13 fps to 430 fps for 512 x 512 pixel resolution. Fully spectral confocal laser scanning microscope with superresolution and FCS/FCCS for single molecule localization

 

Microscope features and functionalities:

1. Fully motorized and computer controlled inverted fluorescence research microscope

2. Definite focus attachment (LED based automated focus drift compensation device)

3. Motorized XY-scanning stage with Z-Piezo insert for tile, mosaic, faster imaging in Z in confocal and SR mode. Scanning stage 130 x 100 STEP for Axio Observer and Axiovert 100/135/200

- max speed: 50 mm/s

- resolution: 140 nm

- absolute accuracy: +/- 5 µm

4. Stage insert Z-Piezo universal- to insert into stage top frame Z-Piezo of inverted microscope stands, applicable for petri dishes with dia. 25to 60 mm and specimen slides 76 x 26 mm, frame size 222 x 139 mm

LSM 880 Spectral confocal scan head with 2 MA-PMT + 32 GaAsP including lasers BLD 405 nm, Ar 458/488/514 nm, HeNe 543 nm, HeNe 594 nm & HeNe 633 nm with ZEN software

Laser modules: Diode 405 nm Laser Argon Multiline 458/488/514 Laser HeNe 543 nm Laser HeNe 594 nm Laser

Objectives

  • 10X/0.45 M27 Plan Apochromat (WD= 2.1 mm)

  • 20X/0.4 Corr M27 LD Plan Neofluar (D= 0- 1.5 mm)

  • 20X/0.8 M27 Plan Apochromat (WD= 0.55 mm)

  • 40X/1.3 oil DIC M27 Plan Apochromat (WD= 0.2 mm) (UV)VIS-IR

  • 40X/1.2 W Corr M27 C- Apochromat (D= 0.14- 0.19 mm) (UV)VIS-IR Selected for FCS

  • 63X/1.4 oil DIC M27 Plan Apochromat (WD= 0.19 mm)

  • 100X/1.4 oil DIC M27 Plan Apochromat (WD= 0.17 mm)

For further technical details visit our webpage (under construction)

Fluorescence microscopes

Olympus IX81 inverted spinning disk fluorescence microscope (installed in Feb, 2010)

Detailed specifications:

Microscope: This is a high speed motorized inverted spinning disk fluorescence microscope. In vivo microscopy of dynamic processes in cells and organisms requires very fast and sensitive acquisition methods. LSCM is inherently speed-limited by the requirement of beam scanning movements. In contrast to single beam scanning systems, the parallelized approach of multi-beam scanning is much faster. Spinning disk microscopes are therefore very well suited for fast in vivo imaging. It can be used both as an inverted fluorescent microscope (when the disc is not in the light path) as well as a semi-confocal system when the Olympus slit disc is used for imaging. In addition, an inbuilt robust 3 deconvolution algorithm helps to achieve near confocal images of samples in motion. The unique features of this microscope are

  1. High speed (semi) confocal imaging with Disc Scanning unit.

  2. High sensitive imaging with the help of Deep cooled (-80°) EMCCD camera

  3. Low bleaching with intense broadband (150W, Hg & Xenon arc lamp covering 350-700nm) for imaging in UV-NIR dyes.

  4. High speed & Low bleaching multi-dimensional (XYZʎ and T) imaging using fast switching devices like fast switching filter wheel, attenuator, shutter and real time control board for parallel switching of multiple devices during multi-dimensional imaging acquisition)

  5. A dedicated IR corrected 60X Super Apo Water immersion objective for live cell imaging and 100X oil immersion objective for higher magnification.

Dual Excitation light source: 100 W Mercury and 150W Mercury Xenon (MT20) with built in excitation filter wheel, attenuator and shutter for fast imaging (fluorescence)

Objectives:

  • 10x/0.30, 10X PLAN Fluorite, NA: 0.30, air

  • 20x/0.75: Plan Super Apo, NA: 0.75, air

  • 40x/0.95 Plan Super Apo NA: 0.95, air with correction collar

  • 63x/1.2: Plan super Apo NA: 1.2, water with correction collar

  • 100x/1.3: Plan fluorite, NA: 1.3, Oil

For further technical details visit our webpage (under construction)

Leica DMI 6000B Live Imaging Microscope (installed in Sep, 2007)

Detailed specifications:

Microscope: This is an inverted fluorescence microscope equipped for live imaging of samples. The Leica DMI6000 B inverted microscope offers differential interference contrast (DIC) for real life imaging of specimens with varying indices of refraction. This fully automated microscope is ideal for fluorescence, live cell, time-lapse imaging and available with temperature and CO2 /humidity control chamber.

Excitation light source:

Mercury arc lamp

Objectives:

  • 10x/0.30 dry, HC PL Fluotar

  • 40x/0.75 dry , HCX PL Fluotar

  • 63x/1.3 glycerine, HCX PL APO CS Glycerine 21°C