The Optical System. The optical system of a compound microscope consists of two lens systems: one found in the objective(s) lens(es) (Fig. 2, part 3); the other in the ocular (eyepiece) (Fig. 2 part 1). The objective lens system is found attached to a rotating nosepiece (Fig. 2, part 2). A microscope usually has three or four objectives that differ in their magnification and resolving power. Magnification is the apparent increase in size of an object. Resolving power is the term used to indicate the ability to distinguish two objects as separate. The most familiar example of resolving power is that of car headlights at night: at a long distance away, the headlights appear as one light; as the car approaches, the light becomes oblong, then barbell-shaped, and finally it becomes resolved into two separate lights. Both resolution and magnification are necessary in microscopy in order to give an apparently larger, finely detailed object to view.

5. Fine focusing knob: smaller of the two knobs, the fine adjustment knob brings the specimen into sharp focus under low power and is used for all focusing when using high power lenses such as the 100x oil immersion lens.

1. Eyepiece/Ocular lens: Lens in which the final magnification occurs. Often is at 10X magnification, but can be different.

Due to their different shooting styles, you want specific ND filters for shooting photography versus videography. When shooting video, you typically want a variable ND because your camera settings stay relatively stable, so you need to adjust the amount of light hitting your camera’s sensor. But if you’re shooting with a camera like a drone or GoPro, VNDs usually are not an option. PolarPro is the leading brand for high quality ND filters for drones and GoPros.

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What are neutral density (ND) filters and how do you use them? Today I’ll explain what ND filters are, how they differ from other filters such as polarizers, and when to use them when shooting photos or videos.

There is also a variable neutral density filter (VND). This filter is circular and changes power as the filter is rotated. It’s a convenient way to get multiple ND filters in one, but the only catch is that you have to be extra vigilant while shooting and change the ND filter according to the light conditions.

The first microscope was developed in 1590 by Dutch lens grinders Hans and Zacharias Jansen. In 1667, Robert Hooke described the microscopic appearance of cork and used the term cell to describe the compartments he observed. Anton van Leeuwenhoek was the first person to observe living cells under the microscope in 1675—he described many types of cells, including bacteria. Since then more sophisticated and powerful scopes have been developed that allow for higher magnification and clearer images.

Microscopy is used by scientists and health care professionals for many purposes, including diagnosis of infectious diseases, identification of microorganisms (microscopic organisms) in environmental samples (including food and water), and determination of the effect of pathogenic (disease-causing) microbes on human cells. This exercise will familiarize you with the microscopes we will be using to look at various types of microorganisms throughout the semester.

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microorganism, magnification, resolution, working distance, parfocal, parcentric, prokaryotic, eukaryotic, bacillus, coccus, spirillum, spirochete, morphology, bacterial arrangements, depth of field, field of view, taxonomic classification

The Illumination System. The objective and ocular lens systems can only perform well under optimal illumination conditions. To achieve these conditions, the light from the light source (bulb) must be centered on the specimen. (In most inexpensive microscopes, the manufacturer adjusts this centering. In more versatile microscopes, the centering becomes more critical and is a function performed by the operator.) The parallel light rays from the light source are focused on the specimen by the condenser lens system (see Fig. 2) The condenser can move up and down to affect this focus. Finally, the amount of light entering the condenser lens system is adjusted using the condenser diaphragm. It is critical that the amount of light be appropriate for the size of the objective lens receiving the light. This is important to give sufficient light, while minimizing glare from stray light, which could otherwise reduce image detail. The higher the magnification and resolving power of the lens, the more light is needed to view the specimen.

A compound light microscope has a maximum resolution of 0.2 µm, this means it can distinguish between two points ≥ 0.2 µm, any objects closer than 0.2um will be seen as 1 object.  Shorter wavelengths of light provide greater resolution. This is why we often have a blue filter over our light source in the microscope, it helps to increase resolution since its wavelength is the shortest in the visible light spectrum.  Without resolution, no matter how much the image is magnified, the amount of observable detail is fixed, and regardless of how much you increase the size of the image, no more detail can be seen. At this point, you will have reached the limit of resolution or the resolving power of the lens. This property of the lens is fixed by the design and construction of the lens. To change the resolution, a different lens is often the only answer.

Magnification refers to the process of making an object appear larger than it is; whereas resolution is the ability to see objects clearly enough to tell two distinct objects apart. Although it is possible to magnify above 1000X, a higher magnification would result in a blurry image. (Think about magnifying a digital photograph beyond the point where you can see the image clearly). This is due to the limitations of visible light (details that are smaller than the wavelength of light used cannot be resolved).

4. Coarse focusing knob: larger of the two knobs, the coarse adjustment knob moves the stage up or down to bring the specimen into focus. It is very sensitive, even small partial rotation of this knob can bring about a big change in the vertical movement of the stage. ONLY use coarse focusing at the beginning with the 4X, 10X low powered objectives in place.  If you use it with the higher powered objectives, it can damage the objective if you crash the lens through your glass specimen slide.

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The limit of resolution of the human eye is about 0.1 mm, or 100 microns (see Table 1 for metric review). Objects that are smaller than this cannot be seen clearly without magnification. Since most cells are much smaller than 100 microns, we need to use microscopes to see them.

At a wavelength of 550 nm (0.55µm), the 100X objective lens with a N.A. of 1.25 has a resolving power of 0.22 µm. Visible light has of wavelength from about 400-750 nanometers (nm). Since the limit of resolution decreases at the shorter wavelengths, microscopes are usually fitted with a blue filter. The resolving power of the lens separates the details of the specimen, and the magnification increases the apparent size of these details so that they are visible to the human eye. Without both resolution and magnification, you would either see nothing (good resolution, no magnification) or a big blur (poor resolution, good magnification).

7. Illuminator: contains the light source, a lamp made either of an incandescent tungsten-halogen bulb or an LED. There is normally a switch to turn on/off or a rheostat located on the side that you can use to adjust the brightness of the light.

For example, with a 10X objective lens and a 10X ocular, the total magnification of the microscope is 100X. If the objective lens is changed to a 20X objective, then the total magnification is now 200X, whereas if a 10X objective is used with a 12.5X ocular lens, the total magnification is now 125X. The use of objective and ocular lenses with different magnifications allows greater flexibility when using the compound microscope.  Due to the size of most bacteria (ranges widely from ~1um to over 100um), generally we require the use of the 100x oil immersion lens with a 10x ocular lense to view bacteria in a standard brightfield light microscope.

Some cameras out there such as drones and GoPros offer ND filters, but the cameras are designed in a way that makes it tough to adjust settings or ND filters while filming. Specifically, the GoPro never shows you your exact settings (shutter speed, ISO, aperture), or tools like a histogram or zebra patterns while shooting. This makes it challenging to appropriately film cinematic footage. However, if you leave the camera on auto settings, ND filters can still be helpful if you are shooting in ultra-bright conditions, such as a sunny day in the tropics. It works like this: the ND filter darkens your image to a certain degree, and this will lower your auto shutter speed. It probably won’t get exactly down to cinematic settings, but this can help your image from getting blown out.

The objective lens system produces an image of the specimen, which is then further magnified by the ocular lens (eyepiece). The magnification of this lens is engraved on the ocular. The total magnification of the microscope is determined by the combination of the magnification of the objective lens and ocular lens that is in use, that is:

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The microscope is one of the microbiologist's greatest tools. It allows for the visualization of small particles, including microbes, which individually are too small to be seen with the human eye. With the help of proper illumination, a microscope can magnify a specimen and optically resolve fine detail. This introduction to microscopy will include an explanation of features and adjustments of a compound brightfield light microscope, which magnifies images using a two lens system.

To avoid this unnatural movement, video camera settings should have a shutter speed that is double your frame rate. Typically, videos are shot in either 24 frames per second or 30 frames per second. That would require your shutter speed to be at either 1/48 sec or 1/60/sec. Doing so will usually cause your image to seem blown out, especially if shooting in bright lighting conditions. This is when ND filters help to block out light so that you can maintain a low shutter speed while shooting video.

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The limit of resolution of a standard brightfield light microscope, also called the resolving power, is ~0.2 µm, or 200 nm.  Biologists typically use microscopes to view all types of cells, including plant cells, animal cells, protozoa, algae, fungi, and bacteria. The nucleus and chloroplasts of eukaryotic cells can also be seen—however smaller organelles and viruses are beyond the limit of resolution of the light microscope (see Figure 1).

Look at the engravings on the objective lenses and note both the magnification (for example:  10X, 40X, 100X) and the resolution given as N.A. = numerical aperture, from which the limit of resolution can be calculated:

2. Revolving nose piece: Holds multiple objective lenses in place. The base of the nose piece can rotate, allowing each of the lens to be rotated into alignment with the ocular lens.

What does it mean to be microscopic? Objects are said to be microscopic when they are too small to be seen with the unaided eye—they need to be magnified (enlarged) for the human eye to be able to see them. This includes human cells and many other types of cells that you will be studying in this class. The microscope you will be using uses visible light and two sets of lenses to produce a magnified image. The total magnification will depend on which objective lens you are using—the highest magnification possible on these microscopes is typically 1000X—meaning that objects appear 1000X larger than they actually are.

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To use an oil immersion lens, place a drop of oil on top of the dried specimen on the slide and carefully focus the microscope so that the objective lens is immersed in the oil. Any lens, which requires oil, is marked "oil" or "oil immersion." Conversely, any lens not marked "oil" should NOT be used with oil and is generally not sealed against oil seeping into and ruining the objective.

In photography, ND filters can help you slow down motion in bright conditions for shooting long exposure photography. Think a smooth flowing waterfall in broad daylight, or cars driving on a road that gets blurred out. Typically, you can only achieve this type of photography in dark conditions after the sun goes down. However, you can use an ND filter to trick your camera into thinking broad daylight is darker than it really is. This helps you shoot at low shutter speeds in bright conditions. There’s also another type of ND filter out there called the graduated ND filter. This filter is half regular (see-through), and half darkened. The best use of this filter is when shooting a landscape where the sky is brighter than the foreground. A graduated ND helps balance out the light in the scene.

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3.1: Introduction to the Microscope is shared under a not declared license and was authored, remixed, and/or curated by LibreTexts.

Objective lenses used for observing very small objects such as bacteria are almost always oil immersion lenses. With an oil immersion lens, a drop of oil is placed between the specimen and the objective lens so that the image light passes through the oil. Without the oil, light passing through the glass microscope slide and specimen would be refracted (bent) when it entered the air between the slide and the objective lens. This refracted light might still be able to contribute to the image of the specimen if the objective lens is large. However, at the higher magnification, the objective lens is small, so is unable to capture this light. The loss of this light leads to loss of image detail. Therefore, at higher magnifications, the area between the slide and the lens is modified to have the same (or nearly the same) refracting qualities (refractive index) as the glass and specimen by the addition of immersion oil.  Watch this NC BioNetwork video (https://youtu.be/-0EvnroWpVc) on oil immersion. For more information, read this article (https://www.microscopeworld.com/t-us...rsion_oil.aspx).

Before reading the following discussion of the theory of the microscope, please familiarize yourself with the names of the microscope parts shown in Figure 2 and their function.

6/9. Stage & Mechanical stage: The horizontal surface where you place the slide specimen is called the stage. The slide is held in place by spring loaded clips and moved around the stage by turning the geared knobs on the mechanical stage. The mechanical stage has two perpendicular scales that can be used to record the position of an object on a slide, useful to quickly relocate an object.

Polarizers and ND filters are similar in that they are filters that go in front of camera lenses. They also block light, but the difference is the type of light they block, and how that impacts your resulting shot. ND filters block light in general and only affect the brightness of your image. On the other hand, polarizers block light that is reflected off of surfaces, such as water or glass. The resulting impact is on your image color, and polarizers can help saturate colors in your image.

However, photography typically does not call for super-specific camera settings, so static ND filters are best. If you’re serious about landscape photography, consider getting an entire kit made by brands such as Lee, Cokin, and Formatt HiTech. They can be pricey, especially if you go for glass filters (the best quality). So if you’re looking to save some money, go for resin filters instead. They cost less and also are harder to scratch or damage if you drop them. I went the resin route via Formatt HiTech and have been very happy with their filters. Just note that these kits involve buying a filter holder, a size-specific ring to attach to your camera lens, and filters. It might sound like a lot, but these kits are usually more flexible than having to buy filters for each lens that you own.

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3. Objective lenses: Initial magnification of your specimen occurs here. Most brightfield light microscopes have 3 objective lenses seated into the resolving nose piece base.

For videography, ND filters also help block out light to blur your video footage. You might be wondering, “why would I want blurry video footage?” Well, it depends on what kind of video you are trying to film. If you want a cinematic, movie-like film, your video should have a slight blur when filming a fast-moving subject, such as a waterfall, or an athlete running fast. If you don’t have this motion blur and are shooting at a high shutter speed, like 1/8000 sec, your subject might appear like it is moving unnaturally fast. It’s a subtle thing, but the trained video eye will notice this irregularity.

An ND filter goes on the front of your camera lens and it blocks light from entering your camera frame. If you are using an interchangeable lens camera such as a DSLR or mirrorless camera, the ND filter is circular and size-specific depending on the filter size of your lens. ND filters also exist for other cameras such as drones and GoPros, but they are very size-specific, so you’ll have to buy them from vendors that sell filters made exactly for the product that you are using. ND filters come in a variety of strengths, such as ND 8, ND 16, and ND 32. The higher the number, the stronger the ND filter and the more light gets blocked out.

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8. Diaphragm and Condenser: the diaphragm controls the amount of light passing from the illuminator through the bottom of the slide, there is a small lever used to achieve the optimal lighting.  The condenser is a lens system that focuses the light coming up from the illuminator onto objects on the slide.