eliminates out-of-focus light in specimens and enables 3-D imaging of thick specimens
Confocal imaging involves serially scanning the specimen to create computer-generated optical sections down to 250 nm thickness using visible light. These optical sections may be stacked to provide a 3-D digital reconstruction of the specimen.
There are different types of single-photon confocal system:
- Laser scanning - where a single focused point of laser illumination (spot or slit) is scanned across a sample in the X, Y and Z directions (alternatively, but less commonly, the beam is stationary while the microscope stage moves through the beam). Signals pass back to a detector (photomultiplier) through a pinhole aperture, which blocks light from other regions of the specimen.
Systems for fast confocal imaging:
i. Spinning disc - where a spinning disk (Nipkow disk) with multiple pinholes inserted between the light source and the specimen. Signals are collected through the same pinholes, which also block out-of-focus light. Data is collected by a CCD camera.
ii. Sweptfield confocal - Instead of a Nipkow disc system, a Sweptfield system scans the specimen using a slit of light. The illuminating photons can also be focused through a plate containing a linear array of 32 stationary pinholes so that each point in the specimen is illuminated up to 260 times per second. Emission photons are descanned and focused onto a high sensitivity CCD camera. Because the pinholes are stationary, the plate containing them can contain arrays of four different size pinholes, allowing selection of the optimum pinhole size to maximize both axial and lateral resolution for all of the most commonly used objective lenses. Two different slit sizes meet demands for faster acquisition, while maintaining confocality and reducing phototoxicity.
In confocal imaging, image data is confined to a defined plane through the specimen. It avoids interference from light emanating from above and below this plane. Signal-to-noise is dramatically improved compared with widefield techniques. Confocal imaging is most frequently associated with fluorescence techniques. It can be used with both fixed and live specimens.
Fluorescence confocal imaging is important in live cell imaging, where multiple probes can be identified at high spatial and temporal resolution. Dynamic events in cells can be imaged with the help of time-lapse imaging and can be viewed in 3-D. Spinning disk and Sweptfield confocal systems are ideal for the imaging of high-speed intracellular events such as calcium ion dynamics. Confocal imaging is fundamental to advanced imaging techniques such as FLIM, FRET, FRAP and FLIP and can be used in association with TIRF imaging.
There are several Nikon confocal systems from entry-level C2+ general laboratory systems, to the A1+si spectral imaging and state-of-the-art A1R+ laser scanning confocal systems.These can be mounted on any upright or inverted research-level microscope. The LiveScan Sweptfield system can be configured on an Eclipse Ti series microscope or FN-1 microscope system.
The C2+ laser scanning confocal system incorporates intuitive EZ-C1 software for the set up all operating parameters. Further data processing and deconvolution processing can be achieved with additional software. The LiveScan Sweptfield and A1 systems incorporate NIS-Elements for multi-dimensional imaging (X,Y,Z,Lambda (wavelength),T, multipoint) with support for capture, display, peripheral device control, and data management. It also offers sophisticated image processing features, such as an extremely powerful deconvolution module.
A number of objectives for fluorescence imaging are available (Plan Fluor, Super Fluor, Plan Apochromat, Plan Apochromat VC), Apo TIRF). TIRF objectives are ideal for live cell confocal imaging because they temperature correction. Plan Apochromat VC lenses are ideal for use with a 405 diode laser - enabling chromatically correct imaging from 400-700nm.
For routine live cell research, the C2+ laser scanning confocal system on a Ti series inverted microscope with CLEM, PFS and environmental control incubator. For advanced, high resolution, "state-of-the-art" live cell imaging, Nikon's LiveScan Sweptfield or A1+ confocal systems with Ti-PFS inverted microscope with multiple laser ports, environmental control incubator and Piezo stage.
Fundamental Concepts in Confocal Microscopy