Laser scanning confocal facility @BSBE
Laser scanning confocal microscopy employs spatial filtering techniques to eliminate any out-of-focus light in specimens with finite thickness and leads to the formation of high-resolution images.
Make and Model
Carl Zeiss, LSM 780
Available mode for use
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Single colour and two-colour imaging and brightfield imaging
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Z-stacks
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Time series (with or without Z-stack)
Specifications/Features
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Travel range of 250 μm.
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Objectives: Plan-apochromat 10X/0.45 NA(air), 20X/0.8 NA (air), C-apochromat 40x/1.2 NA (water) for FCS measurements, Plan-Apochromat 40x/1.3 NA (oil) iPlan-apochromat 63x/1.4 NA (oil), iPlan-apochromat 100x/1.4 (oil). DIC imaging is possible with the last three objectives.
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Lasers: Ar+laser, DPSS laser and HeNe laser, Titanium sapphire multiphoton laser
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Zen 2012 acquisition software
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Temperature and CO2 -controlled incubation stage for long term live cell imaging.
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Lifetime imaging through a FLIM attachment
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Non de-scanned detector available for use with the multiphoton laser for deep tissue imaging.
Application
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- Multi-colour imaging
- Z- stacks and 3D image reconstruction
- Time series (with or without Z-stack)
- Tile scanning to image different parts of the sample automatically over long durations)
- FRET, FRAP, FCS/FCCS
- FLIM
Facility in-charge
Contact Email
lsmconfocal[at] iitb[dot] ac[dot] inContact no.
022-2159 6746Location
Central Instrumentation Room, Ground floor
Biosciences & Bioengineering Department.
I.I.T. Bombay, Powai,
Mumbai - 400 076.
Other contact person(s)
- Pradip Shinde
- Santosh Panigrahi
Facility Management Member(s)
(w.e.f. )
Prof. Santanu K. GhoshProf. Anirban Banerjee
Prof. Rajesh Parkar
Prof. Shobhna Kapoor
Prof. Abhijit Majumder
Registration Link:
Information-IIT Bombay users
Internal Users Registration Details and FormRegistration form-External users
External Users Registration Details and Form.pdf589.93 KB
- Microscope : Fully motorized and computer-controlled Zeiss Axio-Observer Z1 microscope (inverted) with motorized stage and proprietary Definite Focus technology. Piezo-driven stage for scanning stages with a maximum travel range of 250 ïm.
- Objectives : Plan-Apochromat 10X/0.45 NA (air), Plan-Apochromat 20X/0.8 NA (air), C-Apochromat 40x/1.2 NA (water) for FCS measurements, Plan-Apochromat 40x/1.3 NA (oil) iPlan-Apochromat 63x/1.4 NA (oil), iPlan-Apochromat 100x/1.4 (oil). DIC imaging is possible with the last three objectives.
- Scanning Module : Up to 8 frames/sec scanning speed at 512 x 512 pixels. 32 channels in a proprietary GaAsP spectral detector (8nm per channel) and two PMT fluorescence channels available. One of the PMTs is low-noise for imaging in the red/far red. One transmission detector for DIC imaging is available.
- Lasers : Ar+ laser (458nm, 488nm and 514 nm @25 mW), DPSS laser (561 nm @20 mW) and HeNe laser (633 nm @5 mW), Titanium Sapphire multiphoton laser (690 nm - 1040 nm with 2.5W @800nm)
- Filters : DAPI (only through multiphoton laser), Alexa Fluor 488, Rhodamine.
- Zen 2012 acquisition software from Zeiss with 3D, ROI, FRAP and stitching modules.
- Temperature and CO2-controlled incubation stage for long term live cell imaging.
- Lifetime imaging through a FLIM attachment (Becker and Hickle)
- FRET, FRAP, FCS/FCCS.
- NDD (Non de-scanned detector) available for use with the multiphoton laser.
Light coming from multiple out-of-focus planes leads to blurred images or loss of information during conventional (widefield) fluorescence microscopy. Laser scanning confocal microscopy employs spatial filtering techniques to eliminate any out-of-focus light in specimens with finite thickness and leads to the formation of high-resolution images. A focused laser beam scans the sample in a line-by-line manner in the X-Y plane to generate a 2D image. The focus of the laser is then changed to a different Z-plane and the X-Y scanning operation is repeated. A confocal microscope is able to generate a high-resolution 3D image of a sample with a finite thickness in this manner. It should be noted that the “confocal†operation is only possible with a laser light source (i.e. coherent source) and not with the normal fluorescent light sources.
Figure 1. The main optical elements within a confocal microscope are shown.
[https://www.uni-due.de/biofilm-centre/mikro_service_en.shtml]
| Presented Date | Presentation File | Presentation by (Prof.) | Department | |
|---|---|---|---|---|
| 04-12-2017 | View Presentation5.61 MB | Prof. Santanu K. Ghosh | Biosciences and Bioengineering | |
| 28-02-2019 | View Presentation991.17 KB | Prof. Santanu K. Ghosh | Biosciences and Bioengineering |
This FAQ deals with the operational aspects of the facility. If you would like to suggest a question, do feel free to drop an email to <debjani [dot] paul [at] iitb [dot] ac [dot] in>.
1. I need to do simple slide imaging. Which confocal microscope should I use?
You could use either as long as you image up to two fluorophores (red and green). If you want to image DAPI, you have to use the scanning probe confocal microscope. There is no laser for DAPI excitation in the spinning disc system. If you want to image more than two fluorophores, you need to use the scanning probe confocal microscope.
2. What consumable items should I bring with me? What items will be provided at the facility?
The facility will only provide the immersion oil for the objectives and the lens cleaning tissues. Everything else that you may need during imaging (e.g. gloves, pipettes, tips, regular tissue rolls, aluminium foil to cover NDD, etc.) you will have to bring yourself. If you are in doubt, please speak to the operators or one of the conveners in advance.
3. I need to use the confocal microscope. Do I need to train as a TA?
If your usage is infrequent (less than twice per month), one of the operators or the existing TAs can do the imaging for you. If your research project heavily depends on the use of the confocal facility, it would be better if you trained as a TA. Do remember, training as a TA comes with certain duties, such as, imaging other people’s samples.
4. What does being a TA involve?
The job of a TA is to help us run the facility smoothly and image other people’s samples. You will have a do a minimum of 6 hours of TA duty per week just like the TAs allotted to other central facility equipment. This is non-negotiable. If you are a first year PhD student with loads of coursework, we suggest that you come back after a year. The upside is that you will get really proficient in using a stateof-the-art confocal microscope. You will also be able to book slots during ‘off’ hours (between 6pm and 9am) to run samples for yourself or your research group. On the whole it should be a very useful learning experience for you.
5. I think I need to train as a TA. What should I do?
The first thing you should do is to check with your advisor on whether both of you agree with the time commitment. If you are a non-BSBE student, you need to contact the TA coordinator of your department to see if you could be assigned as a TA in the central facility. If the answer to both questions is ‘yes’, send an email to the convener of the microscope where you want to train. We will take over from there.
6. I booked a slot, but my sample is not ready. What should I do?
This can happen once in a while, so don’t worry. Send an email to and call the operator on his mobile phone as soon as you realize that you cannot make it to your slot. This is a matter of courtesy to ensure that other people can use your slot. If this happens too many times, clearly you are not planning your experiments very well and we will take a strict view of it.
7. I need to do live cell imaging. Which microscope should I go for?
If you are imaging swimming bacteria, sperm cells, etc. you should definitely choose the spinning disc system. If your live cell imaging involves three or more fluorophores (unlikely though!), you have to choose the scanning probe confocal system. If your sample is tagged with a single fluorophore (green or red) and you want to image for an hour or so, you can choose either. If you want to do longer experiments (e.g. exploring the motility of a mammalian cell), we will assign you the spinning disc system unless there is a compelling reason to use the other microscope. Such experiments should be scheduled at night (after 8pm). If they need to run longer than 12 hours, you should plan to do these experiments over the weekend. In case of initial overnight operation, you (or the TA) will need to check on your sample every couple of hours.
8. I need to book more than one consecutive slot. Can I get it?
Sure, if you can justify why. Having ~20 odd samples to image at one go is not a good enough reason. We need to be fair to every user while assigning slots. We will give you as many slots as you need to image all your samples, but they will be distributed over several days.
9. I don’t have any fluorophore in my sample. Can I still use the confocal system?
That depends. If you are doing live cell imaging (with CO2 and temperature) in brightfield/DIC mode for a long enough time, you certainly can use the system, as there is currently no other microscope in IITB that has this facility. Just remember that it won’t be confocal imaging, i.e. you won’t be blocking the out-ofplane light. In such a case, the Definite Focus feature will be very useful to you to ensure that at least one of the Z-stack images remains in focus throughout.
10. Can I request a particular TA to image my sample?
No. All TAs have done the same training and it should not matter who images your sample. The conveners have framed this policy to ensure that no single lab/TA monopolizes the use of the facility. If you have any apprehensions about any TA, feel free bring it to the notice of the convener immediately.
List%20of%20Publications%20and%20Conference%20Presentations.pdf
1. Yadav, P., S. Chaturvedi, S.K. Biswas, R. Srivastava, K. Kailasam, A.K. Mishra, and A. Shanavas, Biodegradable Protein-Stabilized Inorganic Nanoassemblies for Photothermal Radiotherapy of Hepatoma Cells. ACS Omega, 2022. 7(10): p.8928-8937.
2. Sthanam, L.K., T. Roy, S. Patwardhan, A. Shukla, S. Sharma, P.V. Shinde, H.T. Kale, P. Chandra Shekar, K. Kondabagil, and S. Sen, MMP modulated differentiation of mouse embryonic stem cells on engineered cell derived matrices. Biomaterials, 2022. 280: p.121268.
3. Singh, D., P. Singh, A. Pradhan, R. Srivastava, and S.K. Sahoo, Reprogramming Cancer Stem-like Cells with Nanoforskolin Enhances the Efficacy of Paclitaxel in Targeting Breast Cancer. ACS Appl Bio Mater, 2021. 4(4): p. 3670-3685.
4. Singh, B., R. Bahadur, M. Rangara, M.N. Gandhi, and R. Srivastava, Influence of Surface States on the Optical and Cellular Property of Thermally Stable Red Emissive Graphitic Carbon Dots. ACS Appl Bio Mater, 2021. 4(5): p. 4641-4651.
5. Shirke, P.U., H. Goswami, V. Kumar, D. Shah, S. Beri, S. Das, J. Bellare, S. Mayor, K.V. Venkatesh, J.R. Seth, and A. Majumder, "Viscotaxis"- directed migration of mesenchymal stem cells in response to loss modulus gradient. Acta Biomater, 2021. 135: p. 356-367.
6. Shenoi, P.R., V.B. Kokane, H.V. Thawale, R.R. Kubde, M.K. Gunwal, and S.P. Shahu, Comparing marginal microleakage in Class V cavities restored with flowable composite and Cention-N using confocal microscope-an in-vitro study. Indian J Dent Res, 2021. 32(3): p. 348-353.
7. Sane, A., S. Sridhar, K. Sanyal, and S.K. Ghosh, Shugoshin ensures maintenance of the spindle assembly checkpoint response and efficient spindle disassembly. Mol Microbiol, 2021. 116(4): p. 1079-1098.
8. Saha, R., S. Patkar, D. Maniar, M.M. Pillai, and P. Tayalia, A bilayered skin substitute developed using an eggshell membrane crosslinked gelatin-chitosan cryogel. Biomater Sci, 2021. 9(23): p. 7921-7933.
9. Patwardhan, S., P. Mahadik, O. Shetty, and S. Sen, ECM stiffness-tuned exosomes drive breast cancer motility through thrombospondin-1. Biomaterials, 2021. 279: p. 121185.
10. Mundhara, N., A. Majumder, and D. Panda, Hyperthermia induced disruption of mechanical balance leads to G1 arrest and senescence in cells. Biochem J, 2021. 478(1): p. 179-196.
11. Malankar, G.S., A. Sakunthala, A. Navalkar, S.K. Maji, S. Raju, and S.T. Manjare, Organoselenium-based BOPHY as a sensor for detection of hypochlorous acid in mammalian cells. Anal Chim Acta, 2021. 1150: p. 338205.
12. Kar, N., D. Gupta, and J. Bellare, Ethanol affects fibroblast behavior differentially at low and high doses: A comprehensive, dose-response evaluation. Toxicol Rep, 2021. 8: p. 1054-1066.
13. Joshi, R., P.D. Murlidharan, P. Yadav, V. Dharnidharka, and A. Majumder, Histone deacetylase inhibitor overrides the effect of soft hydrogel on the mechanoresponse of human mesenchymal stem cells. bioRxiv, 2022: p. 2022.01.04.474891.
14. Jahan, I., J. Pandya, R. Munshi, and S. Sen, Glycocalyx disruption enhances motility, proliferation and collagen synthesis in diabetic fibroblasts. Biochim Biophys Acta Mol Cell Res, 2021. 1868(4): p. 118955.
15. Garlapati, C., S. Joshi, R.C. Turaga, M. Mishra, M.D. Reid, S. Kapoor, L. Artinian, V. Rehder, and R. Aneja, Monoethanolamine-induced glucose deprivation promotes apoptosis through metabolic rewiring in prostate cancer. Theranostics, 2021. 11(18): p. 9089-9106.
16. Dwivedi, N., S. Das, J. Bellare, and A. Majumder, Viscoelastic substrate decouples cellular traction force from other related phenotypes. Biochem Biophys Res Commun, 2021. 543: p. 38-44.
17. Deshmukh, P.P., G.S. Malankar, A. Sakunthala, A. Navalkar, S.K. Maji, D.P. Murale, R. Saravanan, and S.T. Manjare, An efficient chemodosimeter for the detection of Hg(II) via diselenide oxidation. Dalton Trans, 2022. 51(6): p. 2269-2277.
18. Cotta, K.B., S. Ghosh, and S. Mehra, Potentiating the Anti-Tuberculosis Efficacy of Peptide Nucleic Acids through Combinations with Permeabilizing Drugs. Microbiol Spectr, 2022. 10(1): p. e0126221.
19. Biswas, A., S.B. Singh, C.S. Todankar, S. Sudhakar, S.P.P. Pany, and P.I. Pradeepkumar, Stabilization and fluorescence light-up of G-quadruplex nucleic acids using indolyl- quinolinium based probes. Phys Chem Chem Phys, 2022. 24(10): p. 6238-6255.
20. Bhutda, S., S. Ghosh, A.R. Sinha, S. Santra, A. Hiray, and A. Banerjee, Differential Ubiquitination as an Effective Strategy Employed by the Blood-Brain Barrier for Prevention of Bacterial Transcytosis. J Bacteriol, 2022. 204(1): p. e0045621.
21. Barai, A., A. Mukherjee, A. Das, N. Saxena, and S. Sen, alpha-Actinin-4 drives invasiveness by regulating myosin IIB expression and myosin IIA localization. J Cell Sci, 2021. 134(23).
22. Asadullah, S. Kumar, N. Saxena, M. Sarkar, A. Barai, and S. Sen, Combined heterogeneity in cell size and deformability promotes cancer invasiveness. J Cell Sci, 2021. 134(7).
23. Anil, A., S. Apte, J. Joseph, A. Parthasarathy, S. Madhavan, and A. Banerjee, Pyruvate Oxidase as a Key Determinant of Pneumococcal Viability during Transcytosis across Brain Endothelium. J Bacteriol, 2021. 203(24): p. e0043921.
Minutes of the user meeting held on 28th Sept, 2016
Location: BSBE seminar room A
A joint meeting of the users (students, project staff and faculty members) of the laser scanning probe and spinning disc confocal systems was arranged. The meeting was attended by 23 student/project staff users, 5 faculty members (including the faculty members in-charge Santanu Ghosh and Debjani Paul) and the two operators.
A. Technical issues:
It was decided to request the Zeiss technical support team to come down and sort out the following technical problems.
1. A 405 nm laser source is urgently needed for DAPI imaging in the spinning disc (SD) confocal microscope so as to overcome the underuse of the system during working hours. We will send a request to IRCC with usage statistics and quotations.
2. Many users require DIC images along with fluorescence. Currently it is not clear how both can be done together in the spinning disc system. The engineers need to be contacted to check (i) whether this is possible, and (ii) what additional accessories are required to do this.
3. Several users reported a strong Z-drifting in the SD system, such that the entire sample goes out of focus within an hour. It happens during Z-stack acquisition at a single location, as well as while imaging at the multiple locations. This problem has not been seen in the LSM system. We need a clarification from Zeiss whether the definite focus systems are working differently in LSM and SD microscopes. We also need the Zeiss engineers to solve this at the earliest.
4. The ZEN Blue software in the SD system continues to crash repeatedly. This problem was reported at the last user/TA meeting as well.
Action items:
a) Faculty members in-charge and operators to follow up with Zeiss for technical support.
b) Operators to generate the usage statistics for users who request DAPI imaging. Faculty members in-charge to submit the request for DAPI laser to IRCC with quotes and usage statistics.
B. Slot booking options on Drona after working hours:
5. The slot booking maintained on the Google calendar and Drona need to match. Currently there is no way to book slots after 6pm on the Drona calendar. We need to send a request to IRCC software team to allow booking of slots between 6pm and 9am.
Action item: Faculty-in-charges to send request to IRCC software team.
C. Policy for long duration imaging in the SD system:
6. Many users book the SD system only for live cell imaging for long durations. It is convenient to do these experiments overnight. Since the last TA duty slot gets over by 6pm, it was decided that these slots can start from evening 6:00pm itself and the sample can be taken out at 9am the next day.
7. Until the Z-drift problem is sorted and the DAPI laser is bought, the SD system is not used heavily during the day. Therefore, it was decided that multiple slots to a single user or slots for long duration imaging can be allotted during the day. Tuesdays and Thursdays are to be kept exclusively for long duration imaging, in addition to after 6pm and on weekends.
Action item: Disseminate the information to the confocal user group by email.
D. Additional TA duty:
8. Since there is a high usage of the spinning disc system after hours and also during the weekends, it was decided to allot TA duty for an additional slot after 6pm (most likely 6- 8pm) and also during the day on Saturday and Sunday (2 hour each). This is required for the users who are not TAs to be able to check on their samples during these long imaging slots.
9. There is also a requirement to train more TAs to manage the after-hour and weekend slots. A request will be sent around the institute in November/December for training of another batch of TAs. So far, 8 TAs have been trained on each microscope and the two operators also help in imaging.
10. Cleaning of the cell culture incubator and laminar flow hood, and emptying of the dehumidifier at least once a day on Saturdays, Sundays and holidays are to be included in TA duty. These duties will be carried out by all the TAs in rotation once a week.
11. Vaccuuming of the confocal rooms is to be done to minimize the dust related problems. A request to procure a vaccuum cleaner exclusively for use in the BSBE ground floor central facility will be made.
Action items:
a) Disseminate the information to the TA group by email
b) Operators to draw up new duty chart for TAs starting next week.
E. Slot cancellation policy:
12. The users are requested to inform of any cancellation in advance by any one of the three ways: (i) sending an email to the confocal users Google group, or (ii) sending a text message on the Whatsapp group of the users, or (iii) sending a text message to either the TA or operators in-charge. The TA/operator will then immediately send out an email to the user group informing of the available slot. Only verbal intimation for slot cancellation will not be accepted.
13. The first person to respond to the slot cancellation message by any of the above ways gets to use the cancelled slot. Verbal requests for slot booking will not be accepted.
14. If a user does not show up for 15 minutes into the slot without informing the TA or the operators, then that user will not be given a confocal slot for the next 1 month as a disciplinary action.
Action item:
a) Disseminate the information to the confocal user group by email.
b) Operators to inform new users about the cancellation policy when assigning slots.
F. User groups:
15. The confocal user Google group has recently been formed. It includes all users and their advisors. Other than reporting operational issues, this group will also be used by users to report any technical problem at the earliest.
16. A Whatsapp group of the users will be made by the operators for reporting slot cancellation only.
Action items:
a. Disseminate the information to the confocal user group by email.
b. Operators to make a Whatsapp group of the user base as a back-up contact strategy.
G. Information given to the users:
17. Both internal and external users will eventually be charged for using the two confocal systems. The modality for charging is being worked out by the facility management group in consultation with IRCC. The users will be informed as soon as a decision is reached.
18. A workshop on the usage of the two confocals will be organized to increase the user base within the institute.
19. We will organize a similar lecture and demonstration workshop to increase external (outside IIT) usage.
20. FLIM, FCS and FCCS training will be scheduled in November.
21. It was decided to showcase as artwork some of the good images obtained using the facility. A competition will be organized to select the images.
22. Users are requested to acknowledge the IRCC Central Facility whenever they use these images in any publication, conferences, etc.
Action item: Disseminate the information to the confocal user group by email.
- Currently we can image fixed samples sealed between a glass slide and a cover slip. Do not bring samples without sealing them with a cover slip.
- 35 mm diameter petri dishes. Please use specially available imaging petridishes with cover slip bottoms if you wish to use oil immersion objectives.
- Only online registration through the IRCC webpage will be accepted. If you need to cancel your slot, send an email immediately to with an explanation.
- Slots will be provided on a first-come first-served basis.
- The slots are from 9am - 11am, 11am - 1pm, 2pm - 4pm, 4pm - 6pm. You can request two consecutive slots only once in a week. If your experiment needs more time (e.g. long time live cell imaging, etc.), please drop an email to and CC Prof. Santanu Ghosh santanughosh [at] iitb [dot] ac [dot] in so that we can deal with your specific requirement.
- USB drives are strictly not allowed for copying data to minimize virus-related issues. You need to bring a new blank CD to transfer your data. All data must be transferred within 7 days of imaging. Without exception.
- Users must be available throughout the imaging.
- Please mention what fluorophores you have used in your sample (excitation/emission spectra) when you make a request.
- Register online through the IRCC webpage.
- After the slotbooking request is accepted, please contact the operator (Pradip Shinde or Santosh Panigrahi at 4770) to discuss the details of your experiment.