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Category
- Microscopy and Imaging » Confocal Microscopy
- Microscopy and Imaging » Confocal Microscopy
Booking Details
Facility Management Team and Location
Facility Features, Working Principle and Specifications
Facility Description
A spinning disk confocal microscope, when coupled with a sensitive electron-multiplying charge-coupled device (EMCCD) camera, can exceed the sensitivity of a laser scanning confocal microscope system. The fast acquisition time of a spinning disk confocal microscope also allows the collection of three- or four-dimensional data that may not be possible on other systems. For example, it takes seconds rather than minutes to acquire data of a 15-μm Z-stack with individual slices separated by 0.25 μm (68 μm by 68 μm field of view). The acquisition time is dictated by the intensity of the fluorescence signal, which depends on protein concentration, laser power, and the camera's sensitivity. Since the immobilization of live animals may not be complete, fast acquisition time also minimizes the probability of sample movement during image acquisition. This in turn allows high-quality reconstruction of all signals in a three-dimensional space. The spinning-disk confocal microscope facility is suitable for capturing fast-changing phenomena, such as imaging swimming bacteria, lipid droplets, etc. Most powerful tools for live cell imaging.
Working Principle:
There is a trade-off between image acquisition at high-resolution and at high speed with conventional scanning probe confocal microscopy. This is because each point in the sample plane is scanned in a sequential manner to obtain a 2D image. In a spinning-disk confocal microscope, multiple points within the sample are excited simultaneously. The image is recorded using an array detector like a high-speed sensitive EMCCD camera instead of a point detector (e.g. PMT). This system is suitable for capturing fast changing phenomena, such as, imaging swimming bacteria, etc.
The Yokogawa CSU-X1 spinning disc has a combination of upper and lower disks rotated by a motor. The laser light is first defocused to expand to a larger spot size. This larger laser beam spot is then translated into ~20000 small focused laser beam spots by the upper microlens array disk. These laser beam spots then pass through a dichroic mirror and are perfectly aligned to pass through corresponding pinholes on the lower pinhole array disk. The laser spots are then focused by the objective lens onto the sample. Fluorescent light from the specimen returns along the same path through the objective lens and pinholes, is reflected by a dichroic mirror, and is focused at a camera. Thus, the light beams can illuminate the entire observation area of the specimen and form a confocal optical slice at the camera.
Zeiss Observer Z1 Microscope Specifications:
Microscope and spinning disc: Zeiss observer Z1 inverted motorized and computer-controlled fluorescence microscope fitted with high-speed microlens-enhanced nipkow spinning disc.
- Objectives: 10X/0.45 NA (air), 20 X/0.8 NA (air), 40 X/1.2 NA (air), 40 X/1.3 NA (oil), 63 X/1.4 NA (oil) & 100 X 1.4 NA (oil). DIC imaging is possible with all the objectives.
- Lasers: 405nm (50mW), 488nm (50mW), and 561nm (20mW) solid-state lasers as excitation sources.
- HXP 120W metal-halide illuminator for wide-field fluorescence.
- Fluorescence filter sets (wide-field; non-confocal):
- Image acquisition at 50 fps at full resolution.
- Three colour (DAPI, Alexa fluor 488, and Rhodamine) confocal imaging is possible.
Instructions for Registration, Sample Preparation, User Instructions and Precautionary Measures
Spinning Disc Confocal Microscope Facility Guidelines:
- Only online registration through the IRCC Drona webpage will be accepted.
- The form should be completely filled out, and all sample details must be provided in the requisition form.
- Each slot is 2 hours long, and a maximum of 4 samples are allowed per slot; charges apply per slot.
- If an appointment is given but cannot be honored, notify sdconfocal@iitb.ac.in immediately to cancel the slot.
- USB drives are prohibited due to potential computer virus issues.
- After analysis, users should retrieve data from the analysis PC.
- All data must be transferred within 7 days of imaging, without exceptions.
- Subsequent slots are allocated only after the current slot is completed and payment is made.
Imaging Guidelines: Currently, we can image fixed samples sealed between a glass slide and a cover slip. Please do not bring samples without sealing them with a cover slip.
Petri Dishes: For imaging in 35 mm or 55 mm diameter petri dishes, please use specially available imaging petri dishes with cover slip bottoms if you wish to use oil immersion objectives.
- Users should know what kind of sample preparation is required for his/her samples.
- Please mention what fluorophores you have used in your sample (excitation/emission spectra) when you make a request.
- Users must be available throughout the imaging.
- Only online registration through the IRCC webpage will be accepted. If you need to cancel your slot, send an email immediately with an explanation.
- Slots will be provided on a first-come first-served basis.
- The slots are from 9 am - 11 am, 11 am - 1 pm, 2 pm - 4 pm, 4 pm - 6 pm. You can request two consecutive slots only once a week. If your experiment needs more time (e.g. A long time live cell imaging, etc.), please drop an email to sdconfocal@iitb.ac.in or confocal002@gmail.com and CC Prof. Swati Patankar patankar@iitb.ac.in so that we can deal with your specific requirement.
- The non-office hours slots are of 3 hours and it starts from 6 pm to the next day 9 am. (6 pm - 9 pm, 9 pm - 12 am, 12 am - 3 am, 3 am - 6 am, and 6 am - 9 am)
- USB drives are strictly not allowed for copying data to minimize virus-related issues. The data can be shared in the cloud or you need to bring a new blank CD/DVD to transfer your data. All data must be transferred within 15 days of imaging. Without exception.
Charges for Analytical Services in Different Categories
Applications
- High-speed imaging and versatility.
- 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)
- Multi-point with or without time-lapse imaging
- Co-localization analysis, 3D volume rendering and 3D measurement
- Single-color and three-color imaging and brightfield/DIC imaging.
- Fast dynamic processes.
- Multi-position imaging (up to 100 hours).
Sample Details
Incubation Stage: CO2-controlled incubation stage
SOP, Lab Policies and Other Details
Publications
Research Publications:
Rao, V. K., Ashtam, A., Panda, D., and Guchhait, S. K. (2024). Natural-Product-Inspired Discovery of Trimethoxyphenyl-1,2,4-triazolosulfonamides as Potent Tubulin Polymerization Inhibitors. ChemMedChem. 19, e202300562.
Batra, P. J., Kumari, A., Liao, V. W. Y., Hibbs, D. E., Groundwater, P. W., and Panda, D. (2023). 3,5-bis(styryl)pyrazole inhibits mitosis and induces cell death independent of BubR1 and p53 levels by depolymerizing microtubules. The Journal of Biochemistry. 174, 143–164.
Giri, P., Batra, P. J., Kumari, A., Hura, N., Adhikary, R., Acharya, A., Guchhait, S. K., and Panda, D. (2023). Development of QTMP: A promising anticancer agent through NP-Privileged Motif-Driven structural modulation. Bioorganic & Medicinal Chemistry. 95, 117489.
Kurian, J., Ashtam, A., Kesavan, A., Chaluvalappil, S. V., Panda, D., and Manheri, M. K. (2023). Hybridization of the Pharmacophoric Features of Discoipyrrole C and Combretastatin A-4 toward New Anticancer Leads. ChemMedChem. 18, e202300081.
Venkatramani, A., Ashtam, A., and Panda, D. (2024). EB1 Increases the Dynamics of Tau Droplets and Inhibits Tau Aggregation: Implications in Tauopathies. ACS Chem. Neurosci.. 10.1021/acschemneuro.3c00815.
Sakunthala, A., Datta, D., Navalkar, A., Gadhe, L., Kadu, P., Patel, K., Mehra, S., Kumar, R., Chatterjee, D., Devi, J., Sengupta, K., Padinhateeri, R., & Maji, S. K. (2022). Direct Demonstration of Seed Size-Dependent α-Synuclein Amyloid Amplification. Journal of Physical Chemistry Letters. 13(28), 6427–6438.
Raza, M. R., George, J. E., Kumari, S., Mitra, M. K., & Paul, D. (2023). Anomalous diffusion of E. coli under microfluidic confinement and chemical gradient. Soft Matter. 19(34), 6446–6457.
Venkatramani, A., Mukherjee, S., Kumari, A., and Panda, D. (2022). Shikonin impedes phase separation and aggregation of tau and protects SH-SY5Y cells from the toxic effects of tau oligomers. Int J Biol Macromol. 204, 19-33.
Siddiquie, R.Y., Gaddam, A., Agrawal, A., Dimov, S.S., and Joshi, S.S. (2020). Anti-Biofouling Properties of Femtosecond Laser-Induced Submicron Topographies on Elastomeric Surfaces. Langmuir. 36(19), 5349-5358.
Pradhan, A., Mishra, S., Surolia, A., and Panda, D. (2021). C1 Inhibits Liquid-Liquid Phase Separation and Oligomerization of Tau and Protects Neuroblastoma Cells against Toxic Tau Oligomers. ACS Chem Neurosci. 12(11), 1989-2002.
Pradhan, A., Mishra, S., Basu, S.M., Surolia, A., Giri, J., Srivastava, R., and Panda, D. (2021). Targeted nanoformulation of C1 inhibits the growth of KB spheroids and cancer stem cell-enriched MCF-7 mammospheres. Colloids Surf B Biointerfaces. 202, 111702.
Mukherjee, S. and Panda, D. (2021). Contrasting Effects of Ferric and Ferrous Ions on Oligomerization and Droplet Formation of Tau: Implications in Tauopathies and Neurodegeneration. ACS Chem Neurosci. 12(23), 4393-4405.
Kumari, A., Shriwas, O., Sisodiya, S., Santra, M.K., Guchhait, S.K., Dash, R., and Panda, D. (2021). Microtubule-targeting agents impair kinesin-2-dependent nuclear transport of beta-catenin: Evidence of inhibition of Wnt/beta-catenin signaling as an important antitumor mechanism of microtubule-targeting agents. FASEB J. 35(4), e21539.
Sane, A., Sridhar, S., Sanyal, K., and Ghosh, S.K. (2021). Shugoshin ensures maintenance of the spindle assembly checkpoint response and efficient spindle disassembly. Mol Microbiol. 116(4), 1079-1098.
Saha, R., Patkar, S., Maniar, D., Pillai, M.M., and Tayalia, P. (2021). A bilayered skin substitute developed using an eggshell membrane crosslinked gelatin-chitosan cryogel. Biomater Sci. 9(23), 7921-7933.
Patwardhan, S., Mahadik, P., Shetty, O., and Sen, S. (2021). ECM stiffness-tuned exosomes drive breast cancer motility through thrombospondin-1. Biomaterials. 279, 121185.
Malankar, G.S., Sakunthala, A., Navalkar, A., Maji, S.K., Raju, S., and Manjare, S.T. (2021). Organoselenium-based BOPHY as a sensor for detection of hypochlorous acid in mammalian cells. Anal Chim Acta. 1150, 338205.
Mehendale, N., Sharma, O., Pandey, S., and Paul, D. (2018). Clogging-free continuous operation with whole blood in a radial pillar device (RAPID). Biomedical Microdevices: BioMEMS and Biomedical Nanotechnology. 20, 1-1.
Surve, M.V., Bhutda, S., Datey, A., Anil, A., Rawat, S., Pushpakaran, A., Singh, D., Kim, K.S., Chakravortty, D., and Banerjee, A. (2018). Heterogeneity in pneumolysin expression governs the fate of Streptococcus pneumoniae during blood-brain barrier trafficking. PLoS Pathogens. 14.
Venugopal, B., Mogha, P., Dhawan, J., and Majumder, A. (2018). Cell density overrides the effect of substrate stiffness on human mesenchymal stem cells' morphology and proliferation. Biomaterials Science. 6, 1109-1119.
Sharma, H., John, K., Gaddam, A., Navalkar, A., Maji, S.K., and Agrawal, A. (2018). A magnet-actuated biomimetic device for isolating biological entities in microwells. Scientific Reports. 8.
Srivastava, S., and Panda, D. (2018). A centrosomal protein STARD9 promotes microtubule stability and regulates spindle microtubule dynamics. Cell Cycle. 17(20), 5349-5358.
Das, S., Kumar, R., Jha, N. N., and Maji, S. K. (2017). Controlled Exposure of Bioactive Growth Factor in 3D Amyloid Hydrogel for Stem Cells Differentiation. Adv. Healthcare Materials.
Srivastava, S., and Panda, D. (2018). A centrosomal protein FOR20 regulates microtubule assembly dynamics and plays a role in cell migration. Biochemical Journal.
Vinchurkar, M., Ashwin, M., Joshi, A., Singh, A., Tayalia, P., and Rao, V. R. (2017). MEME Aptasensor for Label-free Detection of cancer cells. Department of Electrical Engineering, Indian Institute of Technology Bombay, Mumbai, India, School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India, Indian Institute of Technology Delhi, Delhi, India.
Sthanam, L. K., Barai, A., Rastogi, A., Mistaria, V. K., Ana Maria, R., Kauthale, R., Gatne, M., and Sen, S. (2016). Biophysical regulation of mouse embryonic stem cell fate and genomic integrity by feeder derived matrices. 2016.