Category
- Microscopy and Imaging » Confocal Microscopy
Booking Details
Facility Management Team and Location
Facility Features, Working Principle and Specifications
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 Specifications:
Microscope Details: Zeiss Observer Z1 is an inverted, motorized, and computer-controlled fluorescence microscope equipped with a high-speed microlens-enhanced Nipkow spinning disc (Yokogawa CSU-X1 automated model).
Cameras: Peltier-cooled monochrome EMCCD camera with 56 fps frame rate at full resolution (512x512) for confocal imaging.
1.4 MP monochrome camera with one-stage Peltier cooling for non-confocal (widefield) imaging.
Incubation Stage: Temperature (from 3°C to 45°C) and CO2-controlled incubation stage. Stage insert available for glass slides and 35mm Petri dishes.
Focus System: Proprietary Definite Focus from Zeiss with NIR LED Hardware Focus to eliminate drift during long-term imaging.
Software: Zeiss ZEN Blue image acquisition software.
Objectives:
- 10X/0.45 NA (air)
- 20X/0.8 NA (air)
- 40X/1.2 NA (air)
- 40X/1.3 NA (oil)
- 63X/1.4 NA (oil)
- 100X/1.4 NA (oil)
DIC and brightfield imaging is possible with all the objectives.
Lasers: 405 nm, 488 nm, and 561 nm solid state lasers as excitation sources.
Filters for Widefield Imaging:
Filters currently mounted for widefield (non-confocal) imaging: DAPI, Alexa Fluor 488, Rhodamine, Cy5.
Instructions for Registration, Sample Preparation, User Instructions, Precautionary Measures and Charges
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.
We shall accept online registration only through the IRCC webpage. 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.
USB drives are strictly prohibited for copying data to minimize virus-related issues. You are requested to bring a new blank CD to transfer your data.
Please mention what fluorophores you have used in your sample (excitation/emission spectra) when you make a request.
Users must be present during the entire slot.
Applications
Imaging Techniques:
Single Colour Confocal Imaging: Technique for capturing high-resolution images of a single fluorescent color using confocal microscopy.
Three-Colour (RGB) Confocal Imaging: Method to visualize and capture images in three different fluorescent colors (Red, Green, Blue) simultaneously using confocal microscopy.
Four Color Fluorescence Imaging: Utilizes fluorescence microscopy to image samples labeled with four different fluorophores, enabling multi-channel imaging.
Brightfield Imaging or DIC Imaging: Traditional microscopy techniques for imaging samples without fluorescence, either in brightfield for contrast based on light absorption or DIC (Differential Interference Contrast) for enhanced contrast of transparent specimens.
Fast Dynamic Processes: Techniques optimized for capturing rapid changes in biological processes, often using high-speed cameras or time-resolved imaging methods.
Z-Stacks: Acquisition of multiple images from different focal planes along the Z-axis, which are then computationally combined to produce a 3D representation of the sample.
Multi-position Imaging: Automation of imaging across multiple spatial positions on a sample, facilitating comprehensive analysis of different regions of interest.
Time Series Imaging: Sequential imaging of the same sample over time intervals, crucial for studying dynamic biological processes and changes over time.
Live Cell Imaging: Techniques allowing observation and imaging of living cells under controlled conditions, often requiring specialized equipment like environmental chambers.
Sample Details
Incubation Stage: CO2-controlled incubation stage