The images above have been cropped from the original and have not been cleaned up (they still show all the imperfections from the camera sensor – microscopy will show everything on the sensor, even when you think you have cleaned it). Below is a cleaned up image of the Synedra superba diatom slide using 405nm oblique illumination with the Olympus 40x Dplan Apo UV objective.

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As you experiment with the demonstration program, please make sure you observe the following characteristics of diffuse reflection.

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Oblique lightingexample

It can be shown that the dot product of two vectors is equal to the cosine of the angle between the two vectors divided by the length of the two vectors. In code format, this means that:

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Diffuse reflection is the reflection of light or other waves or particles from a surface such that a ray incident on the surface is scattered at many angles ...

So what is oblique illumination? Basically, it is offsetting the direction of the incoming light to the subject compared with normal bright field microscopy. Changing the angle of light emphasizes some features within a subject, and the direction of the incoming light can be altered to highlight specific features depending on their orientation. As an example let’s take a look at three images of a Synedra superba diatom, captured using an Olympus 40x Dplan Apo UV objective, using 405nm light, on my Olympus BHB. Firstly, normal bright field, with the light coming from directly below.

Examine the plot of a cosine curve to the right. Notice that when the angle is zero, the cosine of zero is 1.0. As the angle increases, the cosine of the angle curves to zero. When the angle is 90 degrees, the cosine of 90 is 0.0. This is lambert’s cosine law. The cosine values are treated as percentages of color. When the angle is zero, cos(0) is 1.0, and you get 100% color. When the angle is 90 degrees, cos(90) is zero and you get 0% color. When the angle becomes greater than 90 or less than -90 the cosine goes negative. This is an indication that the front side of the triangle is pointing away from the light source. You can’t have a negative percentage of light, so we clamp the cosine of the angle to values between 0.0 and 1.0.

Oblique lightingphotography

To perform diffuse lighting calculations each vertex that defines a triangle must have an associated normal vector. The normal vector defines a direction that is projecting out from the front side of the triangle. The normal vector defines how light will reflect off of a surface defined at the vertex. The normal vector may be 90 degrees to the surface of the triangle, or it might be at some other angle to simulate a curved surface.

Oblique lightingeye exam

I hope that has been interesting for you to see. Microscopy continues to amaze me, as do the details I am able to see with my little custom made Olympus BHB UV microscope. Thanks for reading, and if you’d like to know more about this or any other aspect of my work, you can reach me here.

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I’d also like to give a bit of a ‘shout out’ to a company – J.B Microscopes Ltd. When I bought the 40x Dplan Apo UV objective on eBay, the coverslip thickness correction collar was stuck and wouldn’t move (as far as I know the objective was bought new and then kept in a cupboard for 40 years, so presumably the grease had set). J.B Microscopes were able to fix this for a very good price and send it back to me within a few days of sending it to them, so I would definitely consider using them again in the future.

Oblique lightingmeaning

Light that directly strikes an object and then reflects in all directions is called “diffuse” light. The amount of light reflection is determined by the angle between the light ray and the surface normal vector. In Physics, Lambert’s cosine law provides an equation for calculating diffuse color.

Transmittedlighting

I’m a bit of an imaging geek, so at this stage I’ll show the objectives and condenser that were used for this work, and the slide itself (which cost me about £30 on eBay).

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Oblique lightingforensic science

The diagram to the right labels the pieces needed to calculate diffuse reflection. We need to calculate the angle between the vertex’s normal vector and a vector pointing at the light source from the vertex. This angle is labeled “theta” in the diagram.

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Oblique lightingmeans that light is directed at an angle to the document

There is a big step up in resolution with this objective, as expected given its much higher NA. However showing the images at this size online doesn’t really show that, as they have to be reduced in resolution for the website. Below is a crop from the image above, which has also had to be shrunk in resolution).

If both v0 and v1 are normal vectors that have a length of 1, the dot product gives the cosine of the angle without any division.

Obliqueillumination microscopy

Using shorter wavelength UV light for microscopy can (and does) improve resolution, allowing smaller features to be resolved than with longer wavelength light. There are however times when using UV is impractical, or impossible, and in those circumstances, other approaches to improve the image are required. Today I’ll cover some basics about one of those approaches – the use of oblique lighting to illuminate the subject. With oblique lighting, we get to emphasize certain features on a subject, and this approach was used to capture the image shown below using my custom modified Olympus BHB microscope.

As can be seen from the images, using oblique illumination highlights certain features, and by changing the direction the light comes in from different features can be emphasized.

It is interesting to note in this image the ‘imperfections’ in the ‘perfect’ structure. However as mentioned, even this was reduced in resolution for sharing. Below we go in even tighter, and keeping the original image resolution.

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The oblique illumination gives an almost 3D appearance to the image which I really like, and there is certainly plenty of resolution given the NA of the objective (NA 0.85). Note, I have flipped this horizontally, as I like the composition better this way round. However I still wanted to go further, so I broke out the 60x Olympus SPlan Apo (NA 1.4) oil immersion objective. The objective and condenser were oiled to the slide and this is what an image from that looks like using 405nm oblique illumination.

The dot product of two vectors is defined as the sum of the products of their associated terms. 3D vectors are normally stored as arrays, where (v[0], v[1], v[2]) is the values of the vector. Therefore, the dot product of vectors v0 and v1 is:

The example WebGL program above was based on a “point light source”. If you had a different type of light source, such as a sun light source, the shader programs would have to be changed because the definition of your light source would change, but the fundamental math would be the same.

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