Analyzing Fish Scales Through Microscopy to Determine Aquatic Health Teledyne Lumenera

24 Mar.,2023

 

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Posted on Dec 4, 2020 Posted in Did you Know Specs and Techs | By Teledyne Lumenera

The analysis of fish is an essential process in understanding the health of bodies of water such as lakes and rivers. This blog investigates the key components within a microscopy imaging system needed for this type of analysis. Additionally, some crucial information such as the Nyquist ratio will be broken down with examples to assist with choosing the right camera.

How to determine fish age through microscopy? 

The typical way to determine the age of a fish is by its size. However, this is not a precise method and is really just a way to get a vague idea of whether a fish is young or old. Through microscopy, analysis of bones and scales can reveal growth rings that help identify the age of the fish, as seen in Figure 1.



It is important to consider the different types of optics when analysing various samples. To get a more precise measurement of the number of growth rings on a scale, lens options such as the 20 times objective used in Figure 2 might be considered.



Additionally, the type of lighting used for the sample is very important to capture as much detail as possible. With proper lighting such as the oblique fibreoptic lamp in Figure 3, it is possible to achieve a sharp contrast between the layers and capture an image that clearly depicts the depth of the sample.



For samples that are opaque such as the otolith in Figure 4, having an additional lighting can help determine the age of a fish as well. The otoliths of a fish can be large (sagittae) and some are quite small (lapilli and the asteriscii). The structure of an otolith continues to grow as a fish ages and is much denser than the bones, which makes it more reliable for determining the age of a fish. The shape of an otolith is also specific to a species. This makes analysis of a sample that has been found eaten by a seal or other animal very useful for determining their diets.



The importance of the Nyquist ratio for determining the best camera for a microscopy solution 

One of the most important calculations to ensuring a properly designed microscopy system involves the Nyquist ratio. This ratio is used to determine if a camera will be able to capture an image with the full potential of the optics equipped to a microscope. There are a few factors that determine if a camera has the correct specifications to achieve the Nyquist ratio. These include but are not limited to, optical resolution, pixel size, and magnification.

Deciding what camera is best for a microscope can be challenging when there are always different specifications on new models. When resolution is one of those factors, the obvious choice is to choose the camera with the highest resolution. However, this does not directly translate to a higher quality image. The Nyquist ratio is the relationship between camera pixel size and the resolution of the lens for a microscope. This is a vital calculation for understanding if a camera will be able to fully capture the maximum resolution of the sample on the microscope slide. The theorem states, when sampling a signal (such as an analog signal to digital) there must be a sufficient sampling frequency larger than two times the input signal. By using a sample rate that meets this requirement the original signal can be reconstructed from the sampled signal.

Calculating the Nyquist ratio is a three-step process. First, calculate the optical resolution. Then calculate the camera pixel size at the sample plane. Finally, calculate the Nyquist ratio.

Optical resolution is calculated by using the formula:
(λ * 1.22) / (2 * NA)

In this formula, λ represents the wavelength of light being used and NA represents the numerical aperture of the microscope.

The second step is to calculate the camera sensor pixel size at the optical plane. The formula is detailed below:
(camera sensor pixel size) / (mount magnification) * (objective magnification)

Together, with both the optical resolution and the pixel size at the optical plane we can calculate the Nyquist of the optical system by dividing these two values. An example of these calculations can be seen below using the following information.

Wavelength: 550nm
10 times magnification objective
NA: 0.3
Teledyne Lumenera INFINITY 3-6UR camera with a 4.54 micrometer pixel size and a c-mount with a 1 times magnification

Optical resolution:
(550nm)*(1.22)/(2)(0.3) = 1.1 micrometers

Pixel size at sample plane:
(4.54 micrometer)/(1*c-mount)*(10x objective) = 0.454 micrometers

Therefore, the Nyquist ratio can be calculated as:
(1.1)/(0.454) micrometers = 2.423 ratio

With a Nyquist ratio above 2, the requirement to have the sampling frequency be at least double the source is achieved. Therefore, all the image data will be able to be captured. With this type of result, measuring fish scales can take full advantage of a high-resolution camera.


What is unique about Micro Optical Solutions? 

After many successful projects with the various fishery groups in the United States, the populations density of many lakes and areas in the ocean have been determined through a Micro Optical Solutions system. By developing an all in one system that includes a microscope, Teledyne Lumenera INFINITY camera, as shown in Figure 5, and computer with ready-to-use analysis software, Micro Optical Solutions provides reliable set-ups for microscopy.



The imaging systems developed by Micro Optical Solutions are unique because instead of being limited to the zoom of a particular lens, a system like the one shown in Figure 6 can adjust the stand and alter the position and FoV. By capturing an image of a fish scale or bone cross section through the use of analysis software, measurements can be taken on samples and recorded with their images. Additionally, providing the correct lighting accessories is a key part of enhancing the z-profile on samples to clearly find details such as growth rings. Contact Richard Schneider from Micro Optical Solutions at 978-255-2220 (office) or 978-697-2216 (cell) to get started on designing a custom microscopy system.



More Information 

To learn more about USB3 camera options for microscopy, visit the Teledyne Lumenera INFINITY camera page or reach out to our microscopy imaging experts at [email protected]

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