At 3i we incorporate the Yokogawa CSU line of spinning disk confocals in our turnkey imaging systems. Along with electron multiplication CCDs (EMCCD), LaserStack laser launch, TTL Synchronization electronics and SlideBook software, the systems capture data rapidly with minimal photodamage.
Rat basophilic leukemia (RBL) cells.
CSU-X1 | Speed
• Highest speed spinning disk confocal fast 3D imaging
• 50µm pinholes optimized for high NA objectives
• Far lower photodamage than point scanning
• Available for 1 or 2 cameras
CSU-W1 | Deep and clear
• Increased pinhole spacing for decreased cross-talk and deep tissue imaging
• 50µm and 25µm pinhole disks for high and low NA objectives
• 17mm field of view for large format cameras
• Available simultaneous 2-camera imaging or 2-channel split-view imaging
• Double-disk design includes bypass position for widefield imaging
• Available near IR excitation to 785nm
The CSU SDCs use a proprietary disk configuration with two disks, one with pinholes for sharp confocal imaging and the other with microlens-covered pinholes to capture illuminating light which otherwise would be blocked by the disk. The result is illumination from the microlens disk through the pinhole disk for strong excitation of fluorophores, causing a fluorescence emission which in turn passes through the pinhole disk with high confocality. Detection of the signal via an electron multiplication CCD camera allows image capture at speeds to hundreds of frames per second as demanded today by live cell imaging. Furthermore 3i proprietary TTL Synchronization electronics and SlideBook software allow blanking of the illuminating laser light in the time between camera exposures resulting in the minimum possible unwanted photobleaching of cells.
Confocal imaging via spinning disk involves scanning a field with laser light from a number of pinholes arranged in a pattern on a modified Nipkow disk. Unlike laser scanning confocal microscopes (LSM) which scan one point of laser light across an entire field, a spinning disk confocal scans approximately 1,000 points of laser light across the field simultaneously resulting in much faster image production. In a traditional LSM, the detector is a photomultiplier tube which can register the signal from only one point of light (pixel) at a time and with a typical quantum efficiency of 40-50%. In an SDC the detector is a CCD camera which can register the signal from a quarter million or million pixels simultaneously with a quantum efficiency upwards of 95%. The result is that while LSMs can typically image on the order of one full frame per second, SDCs can image at over 1,000 frames per second. This significant speed difference combined with the superior sensitivity of high-end CCDs has made spinning disk confocal a must-have technology for advanced live cell imaging labs.
| CSU-X1 | CSU-W1 | |
|---|---|---|
| Pinhole Diameter | 50µm disk | 25µm disk and 50µm disk |
| Number of Disks | One | One or two with motorized switching |
| Disk Bypass for Widefield Imaging | Available | Standard |
| Acquisition of Speed | 2000 FPS | 200 FPS |
| Field of View | 10mm x 7mm | 17mm x 16mm |
| Near IR Excitation | Up to 640 nm | Up to 785 nm |
Z Projection from 50µm thick mouse brain section imaged with a 63x 1.4NA objective with a CSU-W1 and sCMOS camera. Genetically express YFP (Green), GAD65 stained with Alexa 555 (Blue), and NeuN stained with Alexa 647 (Red).
CSU-W1 SoRa
Super-Resolution via Optical Re-Assignment
Super-resolution microscopy techniques that overcome the spatial resolution limit of conventional light microscopy are increasingly used for advanced research in cell biology. However temporal resolution is low so it is difficult to observe dynamic events in living cells. Furthermore, existing techniques have restrictions on fluorophores that can be used and specimens that can be observed. There is an increasing need for a versatile super-resolution microscopy technique that can visualize fine structures of living cells with high temporal resolution. To meet this need, Yokogawa has developed a high-speed super-resolution confocal scanner based on its confocal scanner unit (CSU), a technology that has proven to be ideal for observing living cells.
CSU-W1 SoRa is an easy-to-use super-resolution microscopy solution utilizing a dual Nipkow disk pair with microlenses on both the illuminating and pinhole disks. The resulting raw images have a 1.4x resolution improvement and with deconvolution one can achieve twice the resolution of raw spinning disk data. The high-speed benefits of spinning disk confocal are well established, with a maximum speed of 200fps, low photodamage compared to other super-resolution methods, and no limitation on dyes or fluors for sample labeling. SoRa is available for new systems as well as an upgrade for existing CSU-W1 systems.
Widefield
CSU-SR Raw
CSU-SR+ Deconvolution
Microtubules (Thermo Fisher Scientific®, FluoCells® prepared slide #2 F14781), Scale bar:1μm OLYMPUS IX71 UPLSAPO100XO NA1.4
Golgi captured with CSU-W1
Golgi captured with CSU-W1 SoRa
| Specifications | |
|---|---|
| XY Resolution | PSF FWHM (XY)= Appx. 160nm * |
| Z Resolution | PSF FWHM (Z)=Appx. 400nm * |
| Deconvolution | Expected value： FWHM (XY)=Less than 120nm, FWHM (Z)=Less than 350nm * |
| Objective lens | 100x, 63x |
| Magnification changer for SoRa | 2.8x for 100x objective, 4.0x for 63x objective |
| Effective field of view | 61×57μm for 100x objective, 71×67μm for 63x objective |
| Switch to confocal | Available (motorized) |
| Scanning speed | Up to 200 Hz |
| Excitation laser | 405 – 640nm |
| Upgrade | Available for all shipped CSU-W1 |