X-Light v2: Spinning Disk Confocal
X-Light V2 – Spinning Disk Confocal System
Confocal imaging system from Crest Optics
The X-Light V2 is a unique system for spinning disk confocal imaging. It features unparalleled versatility:
- Compatibility with all major inverted and upright microscopes
- Plug‐in spinning disk box, allowing a change of pinhole pattern in seconds
- Use of custom relay lenses for mitigation of aberrations of a large field of view
- Utilization of full field of view in sCMOS cameras
- Full compatibility with Crest Optics Video Confocal Super‐resolution module (“VCS”) for 3-D resolution enhancement
- Movable disk for widefield imaging
The X-Light V2 Confocal Imager is a full-spectrum spinning disk confocal imager that attaches to most major models of inverted or upright fluorescence microscope. It is designed to utilize the latest imaging technologies and is ideal for live cell imaging in biological applications.
The X-Light V2 Confocal Imager allows imaging at the extremes of performance of a current‐generation scientific camera, and it provides for objectives used when imaging with a camera with a smaller detector. It takes advantage of laser launches and LED light sources.
The result is a flexible confocal add‐on for existing microscopes that brings the latest commercialized technologies to microscopy. The X-Light V2 Confocal Imager is an extraordinary combination of capability and price.
Learn more about the Principles of Operation of the Confocal Spinning Disk.
A rapid motorized Disk In/DiskOut control provides access to widefield and confocal imaging modes. Switching between modes is automated by imaging software.
Nipkow Spinning Disk
A proprietary disk pattern design provides high confocal resolution, improved out‐of‐focus rejection and higher ratio of signal‐to‐noise. The result is < 800 nm confocal resolution with 60x 1.4 N.A. oil immersion objective.
Disks are available with pinholes sized for best results from different objectives:
- 40–μm holes, for objectives with N.A. < 1;
- 70–μm holes, for objectives with N.A. > 1;
- Custom pinhole diameters are available.
Disks are available to best suit particular camera setups.
- Single Hole Pattern: 22×22mm field of view for large chips of scientific CMOS cameras;
- Dual Hole Patterns: 12×12mm field of view for each pattern, for flexibility for smaller chips of CCD cameras.
The disk rotates at 15,000 revolutions per minute, providing excitation and imaging at speeds of current cameras.
- The excitation mount accomodates LED light sources or multimode fiber for lasers; its SMA connector provides high‐efficiency coupling;
- The gimbal mount for excitation eases alignment to produce the highest ratio of signal to noise;
- The motorized five‐position dichroic filter wheel and eight‐position emission filter wheel provide for automation and ease of interactive control.
Custom relay lenses mitigate aberrations of of a large field of view.
Ease of installation and maintenance
The X-LIGHT V2 addresses difficulties of both initial set-up and continuing maintenance of a confocal imager.
- The gimbal mount for excitation eases alignment that is required for maximal ratio of signal to noise;
- Adjustment of focus is accomplished without moving the camera;
- Fitler extraction tools ease insertion and removal of dichroic filters and emission filters.
X-Light V2 with VCS (VideoConfocal super-resolution)
VCS: Video Confocal Super-resolution
The VCS (“Video Confocal Super‐resolution”) module is an add‐on for the X-Light V2.
Patented Approach with High‐Speed Processing
CrestOptics VCS enhances the 3‐D resolution of the standard light microscope. It performs a two‐dimensional scan of the sample with a pinhole pattern and applies multiple algorithms to process the the emission, producing the image. The VCS technology is patented.
To speed the extensive calculation required, the VCS module uses CUDA technology from NVidia. The result is very fast processing.
Multiple Modes of Operation
The addition of the VCS module to the X-Light V2 provides a confocal imager with three modes of operation: Widefield Mode, Confocal Mode, and Super-resolution Mode.
Principles of Operation with VCS
VCS (“Video Confocal Super‐resolution”) is based on narrow-field illumination using scanning patterns and wide-field collection of raw images.
Detection algorithms already developed are able to super-resolve 3‐D structures in both compact and sparse specimens.
Although other techniques proposed and industrially developed mainly dedicate their efforts in extracting information from a relatively low spatial frequency range, VCM detection methods harness non-linear calculations exploiting the tops more than the belly of the raw signal intensity, collected as a function of illumination and detection positional parameters.
The figure below shows the schematic diagram for a basic VCS microscopy system. In this set‐up, the light source is focused onto a specified pattern that is optically conjugated to the microscope conjugate focal plane through a relay system. The mask is moved in the optical path with an X‐Y piezo motor system at each camera acquisition. The emission is collected widefield, and extensive processing performed to produce the final image.
The figure below shows a typical illumination pattern projected onto a sample.
Typical VCS Illumination Pattern Projected onto Sample
Below is a example using the X-Light Confocal System
Mouse Kidney axial sectioning
Three channels (Dapi, Alexa Fluor 488 WGA, Alexa Fluor 568 Phalloidin,) axial stack of Mouse Kidney sample. The video shows a comparison (not the same field of view) between Wide field, Confocal Spinning Disk and VCS super-resolution acquisitions of 8 microns kidney section. The total thickness of the sample is approximately 16 microns. The VCS reconstruction shows a much more clear axial localization of the nuclei (DAPI) within other cell structures with respect to the other techniques.