Keyence and NIGHTSEA® Fluorescence – Performance Expectations

The digital microscopes in the Keyence VHX series were not designed for fluorescence, and the addition of the NIGHTSEA adapters will not provide the performance that one would find with a purpose-built system. That said, the NIGHTSEA adapter system adds a fluorescence capability that is proving to be valuable for a growing number of users, addressing real-world imaging challenges. We know of several who have purchased multiple adapters so that they could replicate the performance on multiple microscopes. In at least one case the customer only purchased the Keyence system because of the added NIGHTSEA fluorescence capability that enabled a critical analysis function for them.

Fluorescent mineral sample imaged with the Keyence + NIGHTSEA systems

There is a direct correlation between the fluorescence intensity of the sample and the imaging magnification that can be achieved. With all of the lenses except the VH-ZST the excitation light is applied from the freestanding lamp, directed in from the side. It is not focused onto the target area by the Keyence optics, with the result that the greater the magnification (the smaller the area you are looking at), the less of the light emitted by the fluorescence there is to collect. Fortunately many industrial applications make use of highly fluorescent dyes, and imaging at 500-800x has been achieved.

The Keyence VH-ZST is a dual objective zoom lens, with selectable low and high magnification objectives (20-200x and 200-2000x) at the bottom of the optic tube. With the lower magnification objective and relatively flat subjects we recommend applying the excitation light from the side with the freestanding lamp, as with the other lenses. With the higher magnification objective, or with samples with high relief that would produce unacceptable shadowing, a significant performance boost is achieved by mounting the NIGHTSEA fluorescence excitation light head directly above the Keyence fiber optic input port on the lens. Our system for the ZST lens includes a custom adapter that mounts over this port and holds the NIGHTSEA light head in the proper position. The excitation light is then being directed through the Keyence lens optics, producing a much more tightly focused spot on the sample. With this configuration we have made fluorescence images at up to 2000x, the high end magnification limit of the VH-ZST lens.

Note – Ultraviolet light and the VH-ZST lens: The fiber optic input port built into the lens transmits some ultraviolet, but it also seems to block quite a bit. The result is that the focused spot is not particularly strong. For our Ultraviolet light source we recommend that you apply the light from the side for all magnifications. This will impose some limit on achievable magnification that will be a function of the fluorescence intensity. Do not expect to achieve the highest magnifications with ultraviolet as a source. You can also consider alternatives to ultraviolet – many of the fluorescent materials used in industrial applications, such as the dye penetrants and epoxies used for highlighting cracks and separations or for concrete thin sections, fluoresce very well under Royal Blue excitation, while conformal coatings on circuit boards fluoresce well under Violet excitation.

The NIGHTSEA fluorescence adapter for the Keyence microscopes is not well suited for imaging biological subjects at high magnification. The method of delivering the excitation light, the fact that the Keyence camera is designed for higher light levels, and the fact that biological fluorophores tend to be weaker than man-made industrial dyes all combine to limit the expected performance.

For all applications you will need to use higher gain settings than you would normally use for white light imaging.

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NIGHTSEA® for Keyence
Keyence VH-ZST Lens – Adding NIGHTSEA® Fluorescence