NIGHTSEA® for Keyence

Fluorescence solutions for most Keyence lenses 

Our adaptations to the Keyence system are concerned with the lens, not the microscope model (VHX-5000, -6000, -7000 etc.). We offer variants of our fluorescence adapter system that work with the most commonly used Keyence lenses – the VH-Z00, -Z20, -Z50, -Z100, -Z500, -ZST, and with two of the lenses incorporated in the VHX-7100 Fully Integrated (FI) head 
(VHX-E20 and -E100). Our system is not recommended for use with the higher magnification lenses in the FI head. 

The key elements of any fluorescence system are: 

• A light source that produces sufficient energy in the appropriate 
  wavelength range to excite fluorescence in the sample of interest 
• A barrier filter in the viewing path that blocks reflected excitation light 
  while transmitting the fluorescence emitted by the sample 

NIGHTSEA implements these for the Keyence system with: 

• high intensity LED light sources available in five excitation wavelength 
  ranges (see list on next page) 
• emission barrier filters that can be added to the Keyence lenses easily 
  and non-invasively 

The components of a NIGHTSEA system that adds a single excitation/emission wavelength combination on a Keyence microscope are: 

• Flexible gooseneck lamp base with LED drive circuit and intensity control;
• Universal power supply (120/240VAC, 50/60Hz) with international plug set
• Excitation light source 
• Emission barrier filter 
• Barrier filter glasses for fluorescence viewing and eye safety 
• Padded carrying case 

Additional wavelength sets are implemented by adding a matched set (light plus filter set) consisting of a light source, barrier filter, and filter glasses.

Comprehensive solution kit

We have configured Keyence fluorescence adapter kits that include the three most useful excitation wavelengths for varied industry applications, and that are suited to various combinations of the supported Keyence lenses. The kit price is significantly less than purchasing the items separately. 

Each Industry Kit includes the following items, or a subset that is suited to the particular Keyence lenses you want to outfit: 

• Three excitation light heads – Ultraviolet (UV), Violet (VI), and Royal Blue (RB) This recommendation is based on our experience with varied applications. Read the explanation below.
• Three barrier filters, of one or more types depending on the lens(es)
  to be supported
• Matching barrier filter glasses for each excitation wavelength
• Accessory hardware for mounting the NIGHTSEA light head over the lens' fiber optic input port (only with VH-Z50, -Z100, -500 and -ZST)
• Gooseneck lamp base with dimming control
• Universal power supply – 120/240VAC, 50/60Hz, with international plug set
• Packing/carrying case with custom-cut foam

Lens-specific solutions

The Keyence lens you are using (not the microscope model) determines how the NIGHTSEA light source and barrier filter will be added to the system. The light source will either placed to the side of the microscope stage or mounted over the Keyence light source input port. The barrier filter will either be slipped over the bottom of the lens or inserted in the Analyzer slot. The options are summarized below.

• VH-Z00, -Z20
  • Light source – placed to the side of the microscope stage
  • Barrier filter – slips over the bottom of the lens and attaches with thumbscrews
• VH-Z50, -Z100
  • Light source – placed to the side of the microscope stage or mounted over the Keyence light source input port 
  • Barrier filter – inserts in the Analyzer slot at the top of the lens
• VH-Z500
  • Light source – mounts over the Keyence light source input port 
  • Barrier filter – inserts in the Analyzer slot at the top of the lens 
• VH-ZST
  • 20-200 lens
    • Light source – placed to the side of the microscope stage
    • Barrier filter – inserts in the Analyzer slot at the top of the lens
  • 200-2000 lens
    • Light source – mounts over the Keyence fiber optic input port (except for UV) providing enhanced performance at high magnification
    • Barrier filter – inserts in the Analyzer slot at the top of the lens
      NOTE – The Ultraviolet (UV) light source does NOT work well when mounted over the Keyence fiber input at either high or low magnification. Consider our Violet (VI) light source as an alternative for many applications.
• VHX-7100 Fully Integrated (FI) Head
  • VHX-E20 and -E100
    • Light source – placed to the side of the microscope stage
    • Barrier filter – inserts in the Analyzer slot at the right side of the FI head
  • VHX-E500 and -E2500
    • Use with these lenses is likely not practical due to short working distance

Why we recommend a set of three excitation wavelengths for industry needs

We have found that the combination of the Ultraviolet, Violet, and Royal Blue excitation/emission sets addresses a wide variety of industrial imaging challenges. The most common misconception we encounter is that fluorescence is uniquely associated with ultraviolet (UV) light and many customers immediately ask for that wavelength. However, we have found in several cases – even some in which UV was specifically recommended by a dye manufacturer – that either Violet or Royal Blue provided superior performance. For example: 

A prospective customer wanted to image concrete thin sections impregnated with fluorescent dye. The dye supplier recommended UV, and it is true that UV will make the dye fluoresce nicely. We found that our Royal Blue excitation produced significantly brighter fluorescence. 

A prospective customer sent us a sample of material that was known to fluoresce under UV. The goal was to see the distribution of non-fluorescent particles in a fluorescent matrix. While both UV and Royal Blue excited fluorescence in the sample, the best imaging contrast was achieved with Violet excitation. 

We achieve enhanced performance with the ZST lens by mounting the light head on the lens's fiber optic port, but that path does not transmit UV well. In many cases, for example to inspect conformal coating, Violet is an excellent alternative to UV. 

Acquiring the three-color system will provide the highest chance of success and will equip you with a versatile toolkit to address new fluorescence imaging challenges as they arise. Contact us if you would like to discuss your application further. 

Wavelength sets

There are five excitation/emission wavelength combinations available for the system.

Performance expectations

The microscopes in the Keyence VHX series were not designed for fluorescence. That said, the NIGHTSEA adapter system adds a fluorescence capability that is proving to be valuable for a growing number of users. Several companies have purchased multiple adapters so that they could replicate the performance across multiple microscopes. In at least one case the customer only purchased their Keyence system because of the added NIGHTSEA fluorescence capability that enabled a critical analysis function for them. There is a direct correlation between the fluorescence intensity of the sample and the imaging magnification that can be achieved. With some of our solutions – for the -Z00, -Z20, and the FI head – the excitation light from our freestanding source is 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. With other lenses – the -Z50, -Z100, -Z500, and -ZST – the excitation light source can be mounted over the Keyence light source fiber optic input port, resulting in a brighter spot on the subject.

We have a growing body of experience with a number of fluorescence applications with the Keyence VHX series digital microscopes and associated lenses, as shown in the image gallery. This includes a biological application, looking at mosquito midguts immunostained with a fluorescent antibody.

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