NIGHTSEA Model SFA Fluorescence Adapter for Keyence VHX Series Digital Microscopes (continued) Imaging Tips for Keyence Microscopes


Recommendations for imaging fluorescence with the Keyence systems are primarily driven by the fact that the fluorescence emitted by your sample tends to be weaker than the reflected white light that the system is designed to image. This means that you will want to adjust things to either get more light to the camera, or do more with the light you have.


Exposure Adjustment Aperture


If your lens has an aperture control set this to Open. You want to let as much light in as you can.


Brightness Control


In the Lighting section go to Brightness and take control of your integration time (shutter speed). Click Manual and move the slider to the right, or for even more exposure click Super-charge and adjust there. Depending on the brightness of the subject we have found it not unusual to got 100’s of milliseconds of exposure time. You can also go to the Gain section, select Manual, and increase the gain. Some of these adjustments can also be made from the console controls.


HDR and Texture


In some cases we have seen it helpful to use HDR and Texture modes, but in other cases this has not worked well. Experiment as needed.


Subject Selection


Fluorescence is often not bright and, depending on the subject, may be restricted to a small area. If your exposure time (shutter speed / integration time) is in the hundreds of milliseconds there will be a noticeable image lag when you move the sample. Combine these factors and it can at times be difficult and annoying to try to make large sample movements in fluorescence mode. If you know generally the area you want to examine it can be more convenient to first use white light (with its short integration times) to home in on the area of interest and then switch over to fluorescence mode.


Focusing


When you are imaging fluorescence you need a barrier filter that blocks reflected excitation light and transmits the fluorescence of interest. This introduces an extra optical element and some accompanying refraction of the light, causing a shift in the lens-to-subject distance for optimal focus. If you use white light to find an area of interest and focus on it, then switch to fluorescence and add the barrier filter you will need to refocus by a small amount. An alternative is to insert the barrier filter before doing your white- light sample area selection and focusing, and the image will still be in focus when you switch to the fluorescence excitation source.


Artifact Detection


Another byproduct of the long integration time needed to image fluorescence is the potential to have non-fluorescent artifacts in the image. These arise from ambient room light reflected from the sample. A lot of this can be avoided by turning off room lights or providing something to shade the sample area from light sources. You can easily determine if a feature in an image is true fluorescence or not by turning off the excitation light source. Any true fluorescence will disappear immediately, while artifacts will be unaffected.


Note that all techniques may not function equally well with all of the Keyence lens options. Experiment to find the best solution for your particular equipment configuration. If you are having problems imaging, contact your local Keyence sales rep or NIGHTSEA.


Cracks in Sonaspection test plate, fluorescent penetrant


TAM panel starburst with fluorescent penetrant


Next we show some cross sections of electronic components that were embedded in epoxy, cross-sectioned, and polished, after which indications were highlighted with an epoxy-like dye with Morton Fluorescent Yellow G as the fluorescent ingredient.


Crack and Failure Analysis with Fluorescence Fluorescence is a valuable tool for failure analysis, helping investigators see what they might otherwise miss. Fluorescence indicators of various types – including fluorescent penetrants, magnetic particles, and other fluorescent dyes – are commonly used to highlight cracks or defects that would otherwise be difficult or impossible to see. The fluorescence makes them stand out in high contrast. The NIGHTSEA fluorescence adapter systems can be used with microscopes at a variety of scales for detailed examination of these features. Here we show images made with a stereo microscope and with a Keyence digital microscope.


Note that all of the images below were made using the Royal Blue excitation/emission option. While this kind of analysis is normally associated with Ultraviolet, we find that the Royal Blue option is an excellent choice for working with a wide variety of fluorescent indicators.


First, some small cracks highlighted with fluorescent penetrants.


Circuit Board Conformal Coating Inspection


The conformal coating used on electronic circuit boards fluoresces under excitation by either Ultraviolet or Violet light. The fluorescence is a powerful tool for checking coating integrity.


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