It’s not difficult to imagine that a high solid angle light can become an increasingly lower solid angle and less intense light (inverse square rule) as the working distance is increased, effectively turning it into a more directional point source!

Surfaces prone to uneven reflectivity are often challenging to inspect – particularly with bright-field point sources commonly used in machine vision systems.  To achieve more uniform contrast over such surfaces, one solution is to apply diffuse lighting techniques.

We also see that this short working distance contributes to an effective lighting geometry.  However, this is not an efficient sampling strategy, as the 75 x 125 mm tray would require multiple X & Y traverses to inspect the entire tray.

To maintain sufficient incident light intensity, uniformity, and angular distribution, Diffuse Lights are best placed close to the intended inspection surface.  This is particularly crucial for the Diffuse Dome Light for two reasons:

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We see that the Diffuse Dome Light and Flat Diffuse Light work equally well for relatively small areas of the PCB (Figures 3A & 3B).  In comparison, the large Diffuse Dome is less effective than the Flat Diffuse Light on larger fields-of-view, primarily because the dome has to be oversized by at least 50% of the intended field-of-view size (Figures 3C & 3D).

Mosaic and MicroPoint are two products that are designed to suit such photostimulation experiments, each having some benefits that suit particular usage scenarios.

MicroPoint provides a flexible and field-proven tool for photo-stimulation. Supplied with a patented compact, pulsed nitrogen pumped tunable dye laser it is capable of ablation,…

For illustration purposes, we can see the 2-D matrix code in close-up views (See Figures 5A–5D).  A 2” (50 mm) working distance is necessary for the code to be read and verified with a standard resolution CCD camera, while also being typical of the working distances required for effective diffuse and coaxial source lighting.

The FX0808 light is part of the Ai “Expandable Series” of lights.  This enables our team to build a light of this type from 1” x 1” (25 x 25 mm) to 24” x 24” (610 x 610 mm) in 1” (25 mm) increments, all available in 2 weeks.

Photoactivatable fluorescent proteins (PA-FPs) can undergo dramatic changes in their properties in response to illumination of a specific wavelength. In basic terms they can be converted from a dark inactive state to a fluorescing, active state. As with uncaging, there are a multitude of applications that this can be used for, most notably studying a specific dynamic cell process within the wider cell. Some of the main forms of PA-FPs are summarized as follows:

As discussed in our previous Lighting Education Blog Post, diffuse lighting in machine vision applications may be classified as full bright field illumination as opposed to partial, or directional bright field lighting.  The underlying concept behind full bright field illumination, defined as illumination with a larger “solid angle”, is that light is sourced from a large area and is incident on the object surface from multiple angles (Figure 1A).

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Advanced illumination offers a diffuse source to address the application shortcomings of other diffuse, bright field lights: the FX0808 Flat Diffuse Light and FD0808 Back-lit Flat Diffuse Light.  These Flat Diffuse Lights are a highly diffuse source with a viewing port in the center, allowing it to be used as front, or projection light (Figure 2).

What Mosaic allows is the total flexibility in illumination of the specimen. This means different shapes and sizes of illumination areas can be defined- even in complex, or multiples of different regions of different shapes and/or patterns of illumination. Since exact timing is kept across the full field of view using this technology, along with a range of triggering options, highly complex experiments are possible, as well as boosting productivity in each experiment. This makes Mosaic a highly effective way to perform photoactivation based experiments on your microscope.

PCBs can be challenging to illuminate correctly, particularly those with large or odd-shaped components, like capacitors or heat sinks. Figures 3A-3D illustrate the differences among the three diffuse lighting options: dome, flat, and coaxial.

Mosaic is a digital mirror-based array (DMD) that can be combined with light sources - from LED, arc lamps to higher power lasers including MicroPoint.

Figure 7A illustrates the entire tray lighted using the FX0808 Flat Diffuse Light at a 12” (305 mm) working distance.  In conjunction with a high-resolution CCD or CMOS camera, the FX0808 provides for a very robust and efficient inspection solution for larger fields of view.

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In order to perform these light-mediated experiments we need to control the delivery of illumination to the specimen in a tightly controlled manner. This includes control of illumination power and wavelength and also the timing of this illumination to region(s) of interest.

2) to maintain a high solid angle for even light distribution over curved or topographic surfaces, the low angle component from the dome interior must also be incident on the surface.

Let’s view images from samples that illustrate the advantages of applying the Flat Diffuse Light versus other diffuse lights, including a Diffuse Dome Light and a Square Coaxial Light.

The highly targeted effects that these techniques provide mean that there are a multitude of possibilities for optical labelling and tracking cells and any organelles of interest. Important intracellular molecules can also be observed and studied within cells where their general abundance can prohibit the understanding of a process in a localised region of the cell.

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MicroPoint is an illumination source for photostimulation applications. It uses a pulsed nitrogen pumped tunable dye laser system.

If we instead apply the same lighting schemes to a longer working distance view (Figures 6A-6E & 7A), one that may only require 1-2 image frames per tray, we see that only the Flat Diffuse Light and the DL097 Diffuse Dome Light are effective (Figures 6D & 6E, respectively).  Of course, a much higher resolution camera would be necessary to resolve the 2-D laser etched codes.

The image depicted in Figure 6E demonstrates that the DL097 Diffuse Dome Light effectively illuminates the tray at longer working distances.  But for larger sampling areas, we still require larger domes, which can be bulky, expensive, and may require longer lead times.

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Another typically challenging part to illuminate effectively is the biomedical culture or sample titer tray. These parts are often presented on a tray with a matrix of regularly spaced wells of varying size and depth. In this particular example, the wells are relatively small (5 mm wide) and shallow (3 mm deep), and spaced at approximately 8 mm in X and Y. Additionally, each well has a laser-etched 2-D matrix code that must be read and verified by a vision system as part of an FDA-requirement for sample correlation and tracking history.

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Figure 4 illustrates the limited size application of the 2”x2” Square Coaxial Light, where large size and set working distance are critical.

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Specifically, as illustrated in Fig 1A, a Diffuse Dome Light illuminates from nearly an entire hemisphere; hence it is described as having a relatively larger “solid angle”  than the directional Spot Light depicted in Figure 1B.

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While the Diffuse Dome Light is very effective on specular, curved, and topographic surfaces, and the Coaxial Light is effective for specular, flat, and angled surfaces of varying heights, both techniques have very specific application criteria that can present a challenge under some lighting circumstances.

MicroPoint is widely used to provide higher power densities for use in DNA or cell damage and repair studies as well as uncaging. However, it can be used at lower levels of illumination such as for photoactivation, making it a very flexible tool. Illumination from the MicroPoint can be directed to points of interest in the cell to activate PA-FPs with the fine degree of time control these experiments can demand.​

It’s also important to pair the lens focal length correctly in order to prevent vignetting, or “port-holing” – which is particularly true of the Diffuse Dome Light.