• 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 m.
• 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]

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.paul@iitb.ac.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.

________________________________

1. Saha, R. & Tayalia, P. Clove Oil-Incorporated Antibacterial Gelatin-Chitosan Cryogels for Tissue Engineering: An in Vitro Study. ACS Biomater. Sci. Eng. (2022) doi:10.1021/acsbiomaterials.2c00546.

2. Navalkar, A. et al. Oncogenic gain of function due to p53 amyloids occurs through aberrant alteration of cell cycle and proliferation. J. Cell Sci. (2022) doi:10.1242/jcs.259500.

3. Chatterjee, D. et al. Co-aggregation and secondary nucleation in the life cycle of human prolactin/galanin functional amyloids. Elife (2022) doi:10.7554/eLife.73835.

4. Mehra, S. et al. α-Synuclein Aggregation Intermediates form Fibril Polymorphs with Distinct Prion-like Properties. J. Mol. Biol. (2022) doi:10.1016/j.jmb.2022.167761.

5. Sakunthala, A. et al. Direct Demonstration of Seed Size-Dependent α-Synuclein Amyloid Amplification. J. Phys. Chem. Lett. (2022) doi:10.1021/acs.jpclett.2c01650.

6. Singh, N. et al. Amyloid fibril-based thixotropic hydrogels for modeling of tumor spheroids in vitro. Biomaterials (2023) doi:10.1016/j.biomaterials.2023.122032.

7. Ray, S., Singh, N., Patel, K., Krishnamoorthy, G. & Maji, S. K. FRAP and FRET Investigation of α- Synuclein Fibrillization via Liquid-Liquid Phase Separation In Vitro and in HeLa Cells. in Methods in Molecular Biology (2023). doi:10.1007/978-1-0716-2597-2_26.

8. Rani, S. et al. 4D nanoprinted sensor for facile organo-arsenic detection: A two-photon lithographybased approach. Chem. Eng. J. (2023) doi:10.1016/j.cej.2022.140130.

9. Srivastava, A. K., Desai, U. & Singh, A. Effect of graphene coating on modified and pristine carbon fibers on the tribological response of carbon fiber epoxy composites. Compos. Part B Eng. (2023) doi:10.1016/j.compositesb.2022.110412.

10. Sharma, S. & Sharma, K. P. Light-responsive self-assembled microstructures of branched polyethyleneimine at low pH. Chem. Commun. (2022) doi:10.1039/d2cc04996a.

11. Menon, A. P. et al. Mutually Exclusive Interactions of Rifabutin with Spatially Distinct Mycobacterial Cell Envelope Membrane Layers Offer Insights into Membrane-Centric Therapy of Infectious Diseases. ACS Bio Med Chem Au (2022) doi:10.1021/acsbiomedchemau.2c00010.

12. Adhyapak, P. et al. Lipid Clustering in Mycobacterial Cell Envelope Layers Governs Spatially Resolved Solvation Dynamics. Chem. - An Asian J. (2022) doi:10.1002/asia.202200146.

13. Lin, H. Y. J. et al. Discovery of 2′,6-Bis(4-hydroxybenzyl)-2-acetylcyclohexanone, a Novel FtsZ Inhibitor. Molecules (2022) doi:10.3390/molecules27206993.

14. Mishra, M. & Kapoor, S. Multifaceted roles of mycobacterium cell envelope glycolipids during host cell membrane interactions. in Biology of Mycobacterial Lipids (2022). doi:10.1016/B978-0-323-91948-7.00004-X.

15. Poudyal, M. et al. Liquid condensate is a common state of proteins and polypeptides at the regime of high intermolecular interactions. bioRxiv (2022).

16. Pushpakaran, A., Battaje, R. R. & Panda, D. Vitamin K3 inhibits FtsZ assembly, disrupts the Z-ring in Streptococcus pneumoniae and displays anti-pneumococcal activity. Biochem. J. (2022) doi:10.1042/BCJ20220077.

17. Kureel, S. K., Sinha, S., Purkayastha, P., Barretto, S. & Majumder, A. Substrate Stiffness Controls the Cell Cycle of Human Mesenchymal Stem Cells Via Cellular Traction. JOM (2022) doi:10.1007/s11837-022-05392-z.

18. Bachal, K., Yadav, S., Gandhi, P. & Majumder, A. Design and validation of a flowless gradient generating microfluidic device for high-throughput drug testing. Lab Chip (2022) doi:10.1039/d2lc00879c.

19. Yadav, S. & Majumder, A. Biomimicked large-area anisotropic grooves from Dracaena sanderiana leaf enhances cellular alignment and subsequent differentiation. Bioinspiration and Biomimetics (2022) doi:10.1088/1748-3190/ac7afe.

Conference Presentations:
Ketaki Bachal , Deboshmita Sarkar, Arpita Ghosh, Shilpee Dutt , Abhijit Majumder., ‘Selfassembled tumoroid formation on soft substrate mimics in vivo like features of tumorigenesis’, 4th International Symposium on Mechanobiology, Sydney, Australia, 2022.

Bachal K*, Yadav S., Gandhi P., Majumder A., ‘Lithograhy-less, Scalable and Frugal Static Gradient Generating Microfluidic Device for High-Throughput Drug Testing’, poster presentation at WRCB on ‘Low cost diagnostic for healthcare affordable, 3rd June 2022.

Bachal K*, Sarkar D., Ghosh A., Majumder A. and Dutt S., ‘Self-assembled tumoroid formation on soft substrate mimics in vivo like fearures of tumorogenisis’, poster presentation at EMBO lecture series Centre for Predictive Human Model Systems (CPHMS), Hyderabad, 2022

Yadav S*, Joshi R. and Majumder A., ‘Bio-mimicked anistropic groove curvature alters cellular morphology’, Centre for Predictive Human Model Systems (CPHMS), Hyderabad, 2022

Yadav S*, Tawade P., Bachal K., Rakashe M., Pundlik Y. and Majumder A., ‘Scalable Large Area Microf luidic Concentration Gradient Generator for Drug Dilution Application’, poster presentation at WRCB Summit on ‘Next Generation Therapeutics’, 18 November 2022, IIT Bombay.

Gautam Sharma, Swati Pund, Rajkumar Govindan, Mehar Un Nissa, Deeptarup Biswas, Sanniya Midha, Koustav Ganguly, Mahesh Padukudru Anand, Rinti Banerjee, *Sanjeeva Srivastava, ‘Evaluation of cysteamine nanoemulsions and the cellular pathways associated with cigarette smoke-induced Chronic Obstructive Pulmonary Disease’, Human Proteome Organization (HUPO) 2022 World Congress, Cancun, Mexico, in December 2022 (4th -8th December 2022)

• 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@iitb.ac.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.