Lambert Instruments

Confocal microscopy is an imaging technique used to increase micrograph contrast and/or to reconstruct three-dimensional images by using a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. The thickness of the focal plane is defined mostly by the objective lens, and also by the optical properties of the specimen and the ambient conditions.Only the light within the focal plane can be detected, so the image quality is much better than that of widefield images. Typical applications include life sciences, e.g. cell biology. [read more at Wikipedia]

Up to now, the LIFA (typically used for widefield fluorescence microscopy) is compatible with three different confocal microscopy techniques:

1]
The confocal scanning method of the CSU spinning disk system, based on the Nipkow disk scanner. The Nipkow disk has a spiral pattern of pinholes arranged to raster scan the specimen, with the light illuminating through the pinholes. Multibeam scanning is established by spinning the disk producing a widefield confocal image. By placing a microlens array in front of the Nipkow disk, the optical efficiency is improved about two orders. Details of the CSU can be found here or download the .pdf file with more images and movies of the widefield and confocal set-up here. More information on this confocal FLIM set-up is described at the product page, with a photo of the set-up. Moreover, at the application page more images are shown.

2]
The Olympus Disk Scan Unit (DSU). In this disk, the fluorescence excitation light from a ‘white’ light source (the Lambert Instruments modulated LED in this case) is first filtered for the required wavelength and then reflected via a dichromatic mirror. This reflected light passes through a unique, spinning slit confocal disk, (which is located in a conjugate position to the objective’s focal plane), through the objective and focused onto the specimen. Emitted fluorescence light from the specimen is then collected by the objective and sent back through the confocal disk. The passing of focused light back through the disk produces the required confocal effects. Fluorescence emission light (still modulated) is then selected for wavelength by a filter and focused on the modulated image intensifier, lens-coupled to a CCD camera to form visible images. The software of the LIFA system prepares the optical sectioned FLIM images as shown in the download "Optical Sectioning FLIM with LIFA and DSU Olympus Spinning Disk".

3]
The VT-Infinity 2D array scanner of Visitech. This is a high-speed multi-point confocal system. This system has been combined to the LIFA system and the results proved that confocal fluorescence lifetime images can be obtained
Its basic optical path consists of a stationary micro-lens array illuminated with a 405nm-laserdiode that was directly modulated by the LIFA system. A galvanometer mirror scanned the array over the sample and de-scanned the returning fluorescence light. This light was separated from the illuminating beam by a dichroic mirror, and passed through a stationary pinhole array to create the confocal image. This image was re-scanned in perfect synchronisation by the reverse side of the galvanometer mirror onto the Lambert Instruments modulated image intensifier, lens-coupled to a CCD camera. The galvanometer scanner was readily synchronised to the selected camera capture parameters, both exposure time and frame capture rate. The results are shown in the download "Multi-point confocal FLIM with LIFA and VT-Infinity of Visitech". More information about the VT-Infinity can be found here.