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where z is the distance from the output plane, λ is the wavelength, C is a constant, and Ri and Ro are the input and output beam half-widths, respectively. When developing a beam shaping application, the value of β is crucial since higher values are correlated with greater beam shaping performance. This formula also suggests that designing beam shapers for bigger beams, shorter wavelengths, and closer focus distances will be simpler.

In this video, we're going to talk about the components and the magnification of a compound light microscope. The compound light microscope actually has several different components as you can see from the image below that you likely need to be familiar with. It's called a compound light microscope because there are 2 lenses that are compounded together, or 2 lenses that are used together. Light is going to pass via 2 lenses, the objective lens and the ocular lens. Each of these lenses is going to provide additional magnification to help increase the apparent size of the image. If we take a look at this image that we have below, notice that we're showing you the components of a compound light microscope. Notice that the compound light microscope is going to be compounding 2 lenses together. There is the ocular lens where these eyepieces are, and then there's also the objective lenses, which are these lenses, right here in this position. These lenses are rotatable lenses. You can actually rotate this little nose piece right here to get different objective lenses, and each one provides different magnification. You can see the red one provides 4 × magnification. The yellow one provides 10 × magnification. The blue one provides 40 × magnification. And, usually, there's a 4th one that's in the background that you can't really see right here, but it would provide a 100× magnification. The objective lens and the ocular lens are compounded together, and that is why we call this a compound light microscope. Then we have here is the stage, where you place your glass slide containing your specimen. And you have a slide holder here, which is basically this little clip that's going to hold your slide in place so that it doesn't move around. You have a condenser right here at this position, which is going to be focusing and controlling the amount of light. Then you have the light source at the very bottom. This is where the light is actually going to be originating from. Over here you have a little lever, that you can use to control the intensity of the light. Then you have 2 knobs here that are referred to as the coarse focus and the fine focus. The larger one in the back, this larger piece here in the back, is called the coarse focus, and the smaller knob right here is called the fine focus. What these knobs do is they raise vertically and lower vertically the stage. The coarse focus is going to quickly move the stage, and the fine focus will slowly raise the stage. This is all about helping to focus the image to make sure that the image, the specimen that you're trying to view is actually in focus and is as clear as it can be. These are the main components of a compound light microscope.

The irradiance and phase profile of laser light is often redistributed in refractive beam-shaping assemblies. Diffractive optical elements and aspheric or freeform lenses, which are field-mapping phase elements, are primarily used in such assemblies. A refractive field mapper uses wavefront distortion and the energy conservation constraint to convert the Gaussian beam profile into a flat top profile. In a Keplerian or Galilean lens assembly, the incident beam's amplitude and phase are altered after passing through both components. Within the boundaries of the design, the resultant beam shaping is extremely effective and wavelength agnostic. Flat phase fronts and homogeneous irradiance distribution are made possible by refractive beam shapers.​​​​​​

Now when it comes to determining the total magnification of the compound light microscope, what we need to know is that the total magnification of the specimen is going to be the multiplication. It's the multiplication of the magnification provided by each lens. Usually, when we're calculating the total microscope magnification, we just multiply the ocular lens magnification by the objective lens magnification. The ocular lens magnification is usually always going to be 10 × magnification. And so the ocular lens magnification usually does not change, and that's fairly standard for the ocular lens magnification on a compound light microscope. The objective lens magnification can change, and so it depends on which objective lens is being used. The red, the yellow, the blue, or the black providing a 100 ×. And so, if the 4 × objective lens were being used, then to calculate the total magnification of the microscope, you do 10 × times 4×, and so 10 times 4 is 40, and the total magnification would be 40 if the 4× were being used. If the 10 × objective lens were being used, then you would do 10 ocular lens magnification times 10 objective lens magnification. And so 10 times 10 is a 100×, and the total magnification would be a 100×. If the 40 × objective lens were being used, then you would take the ocular lens magnification 10 × and multiply it by 40 ×, and so that would give you 400× magnification. Last but not least, if the 100 × objective lens is being used, then to get the total microscope magnification, you do the ocular lens magnification of 10 ×, times the 100× of the objective lens. And so 10 times a 100 is 1,000× magnification. Be careful not to confuse total magnification with the magnification of each individual lens. It's important to remember that you need to always multiply the objective lens magnification by the 10 × ocular lens magnification to get the total microscope magnification. We'll be able to get some practice applying this concept right here as we move forward in our course. But for now, this here concludes our brief introduction to the components and magnification of a compound light microscope. I'll see you all in our next video.

Fred M. Dickey, Louis S. Weichman, Richard N. Shagam, "Laser beam shaping techniques," Proc. SPIE 4065, High-Power Laser Ablation III, (16 August 2000); https://doi.org/10.1117/12.407361

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Next, we have this piece that's right below the stage here that we call the condenser, which is important for focusing and controlling the amount of light that passes through. We can go ahead and cross off the condenser.

Ilamaran Sivarajah is an experimental atomic/molecular/optical physicist by training who works at the interface of quantum technology and business development.

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Beam shaping affects the basic characteristics of light. The Heisenberg uncertainty principle related to position and momentum governs how effective it is.

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Diffractive beam shapers modify the laser beam into a certain irradiance distribution by using diffraction rather than refraction. Etching is a technique used by diffractive elements to produce a specific nanostructure in a substrate. Usually, the height and zone spacing have an impact on the element's function and design wavelengths. Therefore, it is crucial to use a diffractive optical component at the design wavelength to prevent performance issues. Diffractive elements are also more reliant on alignment, divergence, and the beam location in the plane to refractive beam shapers. However, since diffractive optical components often consist of a single element rather than a number of refractive lenses, they are far more favorable in laser systems with limited space.​​​​​

A lens or other focusing device is used to superimpose the beamlets at the target plane in a laser beam integrator, also known as a homogenizer. Multiple Ienslets are used to split the beam into an array of smaller beams, called beamlets. Both coherent laser beams and other light sources can be input into a laser beam integrator. The total of the diffraction patterns generated by the lenslet array typically represents the final output beam profile. A homogenized flat top profile is produced by most laser beam integrators using incident Gaussian beams.​​​

Bright field microscopy is a common technique that produces a bright background, allowing for the examination of stained and unstained specimens. The compound light microscope, a key type, utilizes two lenses—ocular and objective—to enhance magnification. Total magnification is calculated by multiplying the ocular lens (typically 10x) by the objective lens magnification (e.g., 4x, 10x, 40x, or 100x). This method is essential for observing cellular structures, though unstained transparent organisms may lack contrast, necessitating alternative microscopy techniques for better visualization.

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Bright field microscopy is a common technique in light microscopy that produces a bright background, allowing for the examination of both stained and unstained specimens. It works by passing light through the specimen, which is then magnified by two lenses: the ocular lens and the objective lens. The light that passes through the specimen forms an image that is darker than the bright background, making it easier to observe cellular structures. This method is particularly useful for stained specimens, as the contrast between the stained structures and the bright background is enhanced.

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Gaussian beams may be physically trimmed by an aperture to generate a pseudo-flat top profile in low-performance systems when cost is a deciding issue. Although this is ineffective and wastes energy on the Gaussian profile's periphery, it reduces system complexity and expense.

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Sivarajah, Ilamaran. 2023. A Guide to Laser Beam Shaping Techniques. AZoOptics, viewed 09 November 2024, https://www.azooptics.com/Article.aspx?ArticleID=2432.

Different beam profiles other than the conventional Gaussian profile of a laser are sometimes advantageous for specific applications. For instance, flat top profiles are preferable to Gaussian beams in applications like materials processing systems because the flat top profile provides more precise and predictable cuts and edges.

Sivarajah, Ilamaran. "A Guide to Laser Beam Shaping Techniques". AZoOptics. https://www.azooptics.com/Article.aspx?ArticleID=2432. (accessed November 09, 2024).

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One major limitation of bright field microscopy is its poor contrast when observing unstained, transparent specimens. These specimens do not absorb much light, making them difficult to see against the bright background. Additionally, staining procedures, which can enhance contrast, may kill the organisms, which is not always desirable. To overcome these limitations, other microscopy techniques like phase-contrast or differential interference contrast (DIC) microscopy are used, as they provide better contrast without the need for staining.

The total magnification of a compound light microscope is calculated by multiplying the magnification of the ocular lens by the magnification of the objective lens. The ocular lens typically has a magnification of 10x. For example, if you are using a 40x objective lens, the total magnification would be calculated as follows:

Next, we have the actual stage itself. This large square here is the stage where the glass slide containing the specimen is going to be placed. We can label this as the stage. Then, next we have this little clip right here, this clip that is going to hook and hold the glass slide in place. This is what we call a slide holder because it holds the glass slide in place.

Both of these knobs can be used by an experienced microscopist to help focus an image and focus the specimen under the microscope. This here concludes this example problem. Now that we've labeled the fine focus, the coarse focus, and the slide holder here, we can cross all of those off. We'll be able to get some more practice applying the concepts that we learned as we move forward. I'll see you all in our next video.

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Laser beam shapers that are either diffractive or refractive are frequently used in higher-performance applications that need more efficiency.​​​​​

There is a drawback to bright field microscopy, as unstained transparent organisms can sometimes create very poor contrast with a bright background. If you have unstained transparent organisms, then they may not create much contrast with their background. Sometimes, staining is not a solution because some staining procedures may kill the organism, which might not be what the scientist wants. There are other types of light microscopes that help to resolve this drawback by helping to increase contrast. We will talk about light microscopes that increase contrast later in our course.

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Next, we have the actual light source, and so we can go ahead and label this as the light itself. This is where the light will originate from. Then, right here, we have a knob that is going to be controlling the light intensity. We can label this as the light intensity control and then go ahead and cross off light intensity control from our list.

Before we end this video, there is a very important type of bright field microscope called the compound light microscope. The compound light microscope is one of the most commonly used bright field microscopes and uses two lenses, an ocular lens and an objective lens, to increase magnification. We'll talk more about the compound light microscope in our next video. The compound light microscope is one that you're most likely going to use in your introductory biology courses, labs, and microbiology labs. It is important to be familiar with the compound light microscope, which we will discuss more in our next lesson video. But for now, this concludes our brief introduction to bright field microscopes. We will apply these concepts as we move forward. So, I'll see you all in our next video.

The design of beam shapers is constrained by the uncertainty principle. For instance, the spatial frequencies become less defined for a design with a highly well-defined location. A characteristic parameter is determined by applying the uncertainty principle to diffraction theory, specifically the Fresnel integral of the Fourier transform relation.

A compound light microscope has several key components: the ocular lens (eyepiece), objective lenses, stage, slide holder, condenser, light source, coarse focus knob, and fine focus knob. The ocular lens is where you look through, and it typically provides 10x magnification. The objective lenses are rotatable and provide varying magnifications (e.g., 4x, 10x, 40x, 100x). The stage holds the specimen slide, and the slide holder keeps it in place. The condenser focuses and controls the light, which originates from the light source at the base. The coarse and fine focus knobs adjust the stage's height to bring the specimen into focus.

Edmund Optics. . Laser Beam Shaping Overview. [Online] Edmundoptics.com. Available at: https://www.edmundoptics.com/knowledge-center/application-notes/optics/laser-beam-shaping-overview/

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Apart from the beam shaping techniques described above, various other methods are also broadly employed, dependent on the demands of particular applications.  Most methods excel at eliminating spherical aberration and reducing other optical aberrations while increasing throughput. A thorough overview of the practicalities of beam shaping in many areas for prospective applications, supported by significant efforts to uncover the real nature of structured light and its characteristics, is provided in a number of literary publications.​​​​​

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Circularizing a beam is a new kind of laser beam shaping that entails changing an oval or other non-circular profile to a circular one. Cylinder lenses employ curved surfaces to converge or diverge light, much like conventional spherical lenses do; however, they only have optical power in one direction. The perpendicular dimension of light is unaffected by cylindrical lenses. Standard spherical lenses are unable to do this because they force light to equally concentrate or diverge in a rotationally symmetric way. Because of this quality, cylinder lenses are effective in circularizing elliptical beams and forming laser light sheets.

The coarse focus and fine focus knobs on a compound light microscope both adjust the stage's height to bring the specimen into focus, but they do so at different speeds. The coarse focus knob, which is larger, moves the stage quickly and is used for initial focusing. The fine focus knob, which is smaller, moves the stage slowly and is used for precise, fine-tuning adjustments. Using both knobs allows for clear and sharp imaging of the specimen.

So here we have an example problem that wants us to complete the following diagram down below by labeling each part of the compound light microscope. Notice over here on the left-hand side we have a word bank with all of the components of the compound light microscope. We have to use this word bank to fill in all of these blanks labeling each piece appropriately. I'm going to start at the very top. What we have here are the two eyepieces where the ocular lens is found. We can label this piece here as the ocular lens and then cross off "ocular lens" from our list.

Other forms of optics for circularizing elliptical beams include pairs of anamorphic prisms. Two prisms are combined to distort a laser beam in anamorphic prism pairs. They can create additional elliptical beam profiles in a range of sizes, although they are primarily employed to convert elliptical beam profiles into circular distributions. The reshaping is accomplished using the same optical technique as cylinder lenses: refraction. While one axis, or direction, of light is twisted, the other axis stays unchanged. This accounts for the various divergence angles of the beam.​​​

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In this video, we're going to continue to talk about light microscopy by focusing specifically on the bright field microscopes. Bright field microscopes are the most common type of light microscope. As its name implies, it generates a bright background, hence why it's called a bright field microscope. These microscopes are routinely used to examine both stained and unstained specimens, helping to form darker images or objects on a lighter and brighter background. If we take a look at our image below, notice we're focusing on bright field microscopy. On the left, notice that we have some cells undergoing mitosis that are stained. Recall that mitosis is the division of the nucleus in eukaryotic cells. These cells appear darker compared to their lighter and brighter background. This is what we call bright field microscopy. On the right-hand side, we're showing you some chloroplasts within some moss cells. The chloroplasts are not stained here; they naturally have this green appearance. We'll talk more about these chloroplasts later in our course. Notice that the cells and the structures are darker with respect to their lighter and brighter background. These are some examples of bright field microscopy.

Then, over here, we have two knobs. We have a larger knob in the back, and then we have a smaller knob here in the front. The larger knob in red is called the coarse focus, and the smaller knob is called the fine focus. Both of these knobs essentially do the same thing. They both will either raise or lower the stage vertically. The difference between them is the speed at which they raise and lower the stage. The coarse focus will raise and lower the stage much more quickly because it's a larger knob. The fine focus is going to raise and lower the stage much slower.

The irradiance distribution and phase of a laser beam are often used to describe its form. The latter is crucial for assessing a beam profile's homogeneity along its propagation distance. Typical irradiance distributions include the Gaussian, which shows a decline in irradiance with increasing radial distance, and the flat-top beams, which show a constant irradiance across a certain region.

Next, what we have here is labeling these objective lenses, which recall are rotatable lenses that have different magnifications. We can go ahead and label these as the objective lenses and then cross off "objective lenses" from our list.

Sivarajah, Ilamaran. (2023, May 15). A Guide to Laser Beam Shaping Techniques. AZoOptics. Retrieved on November 09, 2024 from https://www.azooptics.com/Article.aspx?ArticleID=2432.

The characteristic properties of a laser beam can be modified by beam-shaping techniques to meet the demands of specific applications. In order to achieve a desirable beam profile that is maintained over the intended propagation distance, beam shapers redistribute the irradiance of an optical beam with appropriate adjustments of its phase.