How to prevent blobs under the microscope?

How to prevent blobs under the microscope?

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My task is to take photographs of fungus spores under a microscope at x100 using oil immersion.

My problem is that I get all these little blobs. How do I prevent the blobs?

So, I have been instructed to first put the spores on the glass slide, then add a teensy drop of water to it (I use a syringe so I have exact control over the droplet) and then put the coverslip on top of that. Then I tap the coverslip down with the eraser-end of a pencil to force the excess water out from under the coverslip. The immersion oil goes on top of that and then I can view the spores and take the photos.

This is what I mean:

All those little blobs aren't supposed to be there. I can't tell what are spores and what aren't, or if these spores have ornaments or not. However, I happen know this spore does have ornaments (rhodotus palmatus).

At least some of the blobs that you have appear to be air. Seating the coverslip carefully will help to prevent air-bubbles from forming under the coverslip. Lay one edge against the fluid and slowly ease it down.

You mention using an oil immersion lens. It is possible that water has come into contact with your lens. Oil finding its way beneath the coverslip could also introduce refractory blobs as well.

Leaf Experiments

Leaf cells have a special feature: pigment-containing chloroplasts in certain cells that enable them to produce energy and their own food through photosynthesis.

What does that mean? Well, the chloroplasts within a cell contain different pigments, which are what gives a leaf its color.

Green chlorophyll is the most common type of pigment, but there are also xanthophylls (yellow), cartenoids (yellow, orange), and anthocyanins (red).

The chlorophylls usually hide the other pigments, except when autumn comes along and chlorophyll begins to break down. This is why leaves turn different colors in the fall.

So then, what is photosynthesis? Simply put, it’s the capture of light energy to produce food. Light energy from the sun is transmitted through a leaf’s cells to chloroplasts, where chlorophyll and other absorbing pigments serve as receptors to collect the energy. In the process of photosynthesis, carbon dioxide from the air is converted into energy-rich carbon compounds called carbohydrates. As this happens, oxygen is given off into the air, providing the oxygen that we breathe.

You can test the importance of light energy in plant growth by doing a simple experiment.

(Bean plants are a good choice, as they sprout quickly.)

You’ll need one to be the control, with normal growing conditions, either outside in sunlight or inside by a bright window. See how light effects growth by covering the other test plants with a paper bag or small box during part of the day.

Try covering one for four hours during the morning, and another for the whole day.

Observe changes to the plants over the course of a week. Which grows the best? What is the result of light-deprivation?

Observe Mold Up Close

To examine mold under the microscope, it is best to grow your own in a controlled environment. We recommend using soft bread that is preservative-free, but many fruits or vegetables such as potatoes or oranges will also work. A good sample of mold may take up to two weeks to form, so be sure to plan ahead for this project. Please note: we do not recommend this project for those with allergies to mold (including penicillin) or with severe asthma.

The easiest way to grow mold is on a piece of bread. Any preservative-free bread that is soft will work well. Bread with preservatives in it will take much longer to form mold. Leave the bread in the open for about an hour so it is exposed to contaminants in the air. Place the bread in a ziplock bag, and sprinkle water over it so it is damp. Seal the bag, leaving some air inside. Place the bag in a dark, warm place, away from other food items. A kitchen cupboard close to the stove may be one option. Or you could place it next to a window, with a bowl or plate covering it from the light. Mold will grow best in a moist environment. Mold should start forming in 2-3 days, but will take a week or more to get a good sample.

Check on the piece of bread every few days, and add more water if it is becoming dried out. Avoid opening the plastic bag as much as you can. If you touch the bread, be sure to thoroughly wash your hands afterwards. When sufficient mold has formed, you can prepare a slide and examine it under the microscope (student microscopes work well for this). You will need a sample that is about one inch across.

What You Need:

Safety Note: When handling mold, it is very important to cover as much skin as you can, and to wear gloves. Mold is an allergen and can be toxic. Working with mold as you are in the experiment typically only affects the very young or very old, or those with severe immune problems, but it is important to take precautionary measures (such as gloves) and to not do this project if you have allergies or asthma.

What You Do:

  1. Place a drop of water in the center of the slide, using an eyedropper if you have one, or the tip of a clean finger. You can use solution of methylene blue instead, which is a microscope stain, and makes the sample easier to see by coloring certain parts of the mold cells.
  2. Using a toothpick, scrape some of the mold off, and place it on the drop of water.
  3. Take the cover slip and set it at an angle to the slide so that one edge of it touches the water drop, then carefully lower it over the drop so that the cover slip covers the specimen without trapping air bubbles underneath.
  4. Use the corner of a paper towel to blot up any excess water at the edges of the cover slip.
  5. View the slide with a compound microscope, starting with a low objective.

Did you know molds are actually fungi? Fungi are found in all sorts of environments, and some are helpful, while others can be harmful. Mushrooms are actually a type of edible fungi. Mold is certainly a nuisance on food, but some antibiotics have been made from mold similar to the kind you grew.

The colorful growth on the bread is made of connected thread structures called hyphae. These form a mold colony which was started by a single mold spore. The hyphae may look soft and fuzzy, or it could be very colorful. By looking at the hyphae under a microscope, you will be able to identify what kind of mold it is.

Rhizopus feeds on starch or sugar, making it a common mold on bread. This type of mold may start off as white hair-like structures and eventually will form solid black spots. Under the microscope, Rhizopus appears as short strands with oval-shaped heads, looking like a balloon on a string. The head is where the spores of this type of mold are contained.

Aspergillus is another mold commonly found on food items, especially grains. It is a typically a bluish green color, with a thin ring of white around each colony. Some species of Aspergillus are black in color. You can identify this type of mold by making a slide and viewing it under the microscope. It has a thin branch-like structure, with heads that look like blooming flowers, and release spherical spores.

Penicillium, which is where the powerful antibiotic Penicillin comes from, can be blue-green or gray, often with a fuzzy white edge. This type of mold is common, and often looks like Aspergillus with the naked eye. If you examine it under a microscope, you will see that the head has thinner structure than Aspergillus, with several strand segments branching out from the main strand. At the end of each segment of the head you should be able to see small spores.

Clean-up: When the experiment is finished, put the bread and anything that touched it (including the gloves and apron) in a heavy-duty plastic zip lock bag, and throw it away. The slide will not be permanent, and should be disposed of as well. Clean the area you were working in thoroughly with bleach wipes (such as Clorox) or soap and water.

Ordinary Microscope Sees in Super-Resolution With Specially Engineered Light-Shrinking Material

Electrical engineers at the University of California San Diego developed a technology that improves the resolution of an ordinary light microscope so that it can be used to directly observe finer structures and details in living cells.

The technology turns a conventional light microscope into what’s called a super-resolution microscope. It involves a specially engineered material that shortens the wavelength of light as it illuminates the sample — this shrunken light is what essentially enables the microscope to image in higher resolution.

“This material converts low-resolution light to high-resolution light,” said Zhaowei Liu, a professor of electrical and computer engineering at UC San Diego. “It’s very simple and easy to use. Just place a sample on the material, then put the whole thing under a normal microscope — no fancy modification needed.”

The material mounted on the stage of an inverted microscope. Credit: Junxiang Zhao

The work, which was published in Nature Communications, overcomes a big limitation of conventional light microscopes: low resolution. Light microscopes are useful for imaging live cells, but they cannot be used to see anything smaller. Conventional light microscopes have a resolution limit of 200 nanometers, meaning that any objects closer than this distance will not be observed as separate objects. And while there are more powerful tools out there such as electron microscopes, which have the resolution to see subcellular structures, they cannot be used to image living cells because the samples need to be placed inside a vacuum chamber.

“The major challenge is finding one technology that has very high resolution and is also safe for live cells,” said Liu.

The technology that Liu’s team developed combines both features. With it, a conventional light microscope can be used to image live subcellular structures with a resolution of up to 40 nanometers.

Comparison of images taken by a light microscope without the hyperbolic metamaterial (left column) and with the hyperbolic metamaterial (right column): two close fluorescent beads (top row), quantum dots (middle row), and actin filaments in Cos-7 cells (bottom row). Credit: Nature Communications

The technology consists of a microscope slide that’s coated with a type of light-shrinking material called a hyperbolic metamaterial. It is made up of nanometers-thin alternating layers of silver and silica glass. As light passes through, its wavelengths shorten and scatter to generate a series of random high-resolution speckled patterns. When a sample is mounted on the slide, it gets illuminated in different ways by this series of speckled light patterns. This creates a series of low-resolution images, which are all captured and then pieced together by a reconstruction algorithm to produce a high-resolution image.

The researchers tested their technology with a commercial inverted microscope. They were able to image fine features, such as actin filaments, in fluorescently labeled Cos-7 cells — features that are not clearly discernible using just the microscope itself. The technology also enabled the researchers to clearly distinguish tiny fluorescent beads and quantum dots that were spaced 40 to 80 nanometers apart.

The super-resolution technology has great potential for high-speed operation, the researchers said. Their goal is to incorporate high speed, super-resolution, and low phototoxicity in one system for live-cell imaging.

Liu’s team is now expanding the technology to do high-resolution imaging in three-dimensional space. This current paper shows that the technology can produce high-resolution images in a two-dimensional plane. Liu’s team previously published a paper showing that this technology is also capable of imaging with ultra-high axial resolution (about 2 nanometers). They are now working on combining the two together.

Reference: “Metamaterial assisted illumination nanoscopy via random super-resolution speckles” by Yeon Ui Lee, Junxiang Zhao, Qian Ma, Larousse Khosravi Khorashad, Clara Posner, Guangru Li, G. Bimananda M. Wisna, Zachary Burns, Jin Zhang and Zhaowei Liu, 10 March 2021, Nature Communications.
DOI: 10.1038/s41467-021-21835-8

This work was supported by the Gordon and Betty Moore Foundation and the National Institutes of Health (R35 CA197622). This work was performed in part at the San Diego Nanotechnology Infrastructure (SDNI) at UC San Diego, a member of the National Nanotechnology Coordinated Infrastructure, which is supported by the National Science Foundation (grant ECCS-1542148).

How to prevent blobs under the microscope? - Biology

The focus in this chapter lies in creating an overview of the more basic and general types of microscopy, for example optical microscopy, however, we DO NOT go into detail of more specialized and advanced techniques at this point (like confocal, phase contrast, interference microscopy, etc.). Hopefully, we will be able to fill in such gaps in the future. A fantastic resource on microscopes, how they work, advantages/dissadvantages, etc., can be found here.

Stereo microscopes are optical devices, designed for lower magnifications (usually up to 100x) and the observation of small to medium sized samples. Illumination is reflected from the sample and can come from an external source. The light travels through 2 optical paths, which improves the 3-dimensional visualization. Due to the available space between the sample and the optical parts, the set-up also allows for sample manipulation and further preparation for other methods.

Keywords: stereo, binocular, microscope

Commercial stereo microscopes cost from 150€ upwards, very fancy ones up to 1000€. The most expensive part of the device is probably the optics, which is hardly compensated by DIY devices, since quality lenses are extremely hard to produce DIY. In addition to the classical devices, there are also digital microscopes on the market, which are monocular, very cheap (25 - 60€) and have a similar constitution to the classical stereo microscopes and could serve the same function. They also offer very high magnification, although the resolution and focusing are questionable.

3. Available DIY resources

As mentioned, it is irrational to make lenses and other optics from scratch, however, if there are resources for these, which are relatively cheap, it could make sense. There is just one DIY design available at the moment, which uses a combination of different cheap binoculars. Binoculars are already available at amazon for a little more than 10€, so the construction seems worth-while.

4. Is DIY good enough and reasonable?

It is difficult to comment on the DIY soultions at this point, since not many are available. The requirements for a stero microscope are, however, relatively low, so a low cost device, as mentioned above, seems reasonable.

The magnification and the focus, need to be gradually adjustable by hand. A minimum satisfactory maginifcation is probably about 50x, however, the device needs to have enough space between smaple and the lenses, to allow manipulation.

Classical optical microscope

Microscopes were developed for observation of very small samples. Traditionally, the first encounter with microscopy comes via a classical optical microscope. In what we call "bright field" microscopy, light from a source is transmitted through a very thin sample, the image is then focused and magnified, through a system of lenses. To improve resolution and observability of the sample, this can be prepared in advance, by fixating, very thin sectioning, staining. Commonly, optical microscopes enable magnifications of up to 1000x. Due to the limitations of visible light, highest magnifications mostly require immersion oils, for focus improvement.

Keywords: Optical, bright, field, microscope

Hobby microscopes can often be acquired for 150€ or less, these, however, typically have very poor optics, with low resolutions, depth of field, focus, etc. and consequently aren't really suitable for research. The simplest lab-grade microscopes are availabe from 300€ upwards, with a limit in the thousands of €. High prices are due to the optical requirements, however, new microscopes also have expensive features, which aren't always neccessary.

3. Available DIY resources

There are a few DIY microscopes available at the moment, which use the optics of available devices, such as web-cams (example 1, example 2), smartphones, etc. These can come very cheap (if you already have a smartphone) and are suitable for simple microscopy, but it depends, whether, they will suit your needs. A very important aspect of microscopy is also the precise control of the sample, relative to the objective. The OpenFlexure design, allows the precise manipulation, presumably below 100nm, which is a powerful tool to integrate with other solutions.

4. Is DIY good enough and reasonable?

The important issues in microscopy are, as mentioned, sufficient lighting, focus and magnification. Lighting can be controlled with a light source, and the condenser (which also determines the angle and focus, at which light hits the sample), focus and magnification are typically determined by the objective lenses and the ocular. There are usually other issues as well. Microscopes usually have a very near depth of field, it is therefore crucial, that the samples are very thin. Also, since the light normally passes through different media before it hits the objective, it can diffract and distort the image. Therefore, at higher magnifications immersion oils are used to place between the objective and a cover slip, on top of the sample. With the available DIY resources, all of these factors are difficult to control, it is therefore questionable if they are good enough.

A good microscope should have an adjustable light source (adjustable intensity, however, providing a very even ilumination), and the capabilities to focus light with a condenser. The microscopy optics should also enable good magnification, while retaining resolution. An optical microscope can be purely digital and based, for example, on a web-cam, however, we feel that improvements can be done on the existing models.

A fluorescence microscope can be used for samples with luminescent (self-light-emitting) properties, like fluorescence, or photophorescence, where instead of transmitted or reflected light, the emitting light from the samples themselves is observed. In principle, its build-up is similar to a classical optical microscope, but in addition, it has a second light source with appropriate wavelengths, to excite the sample and cause it to emit its own light (with slightly longer wavelengths). Typically the excitatory light is reflected of a dichronic mirror (transmits some wavelenghts and reflects others), then travells through the objective, where it focuses on the visible part of the sample. The fluorescent light then travels back through the objective, but also through the dichronic mirror, towards the ocular.

Keywords: fluorescence, photophorescence, luminescence, optical, microscope

While it seems that the modification is a small one, fluorescent microscopes are substantially more expensive, compared to the classical optical ones, costing several thousand €, the cheaper ones around 3000€. The dichronic filters (mirrors) alone can cost 500-1000€, depending on their wavelength specificity. Also, the second light source will contribute to the expensiveness.

3. Available DIY resources

DIY fluorescence microscopes typically use existing bright field microscopes as a base and integrate additional lighting and filters. Great examples are example 1 suitable for GFP and DsRED, and the more sophisticated example 2, also suitble for GFPs. The most advanced is probably the portable, bright field and fluorescence microscope - The Global Focus microscope, which is built from scratch for 240$, uses a simple flashlight for the light source and reaches a resolution of 0.8μm at a 1000x magnification.

4. Is DIY good enough and reasonable?

Maybe a bit surprising, though more difficult to make, DIY fluorescence microscopes seem to be more advanced than their classical bright-field counterparts. The Global Focus microscope project, seems to fulfill the expectations of a laboratory microscope. It could be even improved upon by integrating a steady, adjustable light source and a condensor lense for an improved signal. Also an additional digital camera would allow the capturing of images directly on a computer, which is important for recordings.

A fluorescent microscope should in principle have the same optical capabilities as a bright-field microscope, with the addition of creating and detecting fluorescence. The latter is strongly depends on the light source and filters, and needs to be adjusted to the fluorescent material. When performing immunohistochemical analysis, the fluorophores/antibodies need to be acquired in accordance with the capabilities of the microscope.

Microtomes are the main tool of sample preparation for optical microscopy. They mainly consist of a sample holder, containing the sample (which is fixed in somekind of solid medium like parafin, epoxi resins. ), a knife and a cyclic mechanism to cut the sample into thin slices. Typical "semi-thin" sections, suitble for optical microscopy are around 1μm thin, but for different purposes also sections of up to 100μm are used. There are a few methods of microtomy, like cryosection (rapid freezing of samples, allows the sectioning and analysis of a sample, while the rest of the tissue is kept intact), or ultramicrosection (creating very thin,

70nm thick sections, suitable for electron microscopy), all using the same basic principle.

Keywords: microtome, sectioning, semi, thin

There are different forms of microtomes on the market, most common are rotary microtomes, which still have many variants. Manual to automatic, using steel, glass, or even diamond knives, appropriate for parafin, epoxy, or frozen samples. The most common semi-automated microtomes are fairly expensive, costing from 2000 - 10000€. It is crucial, that the sections are as thin as adjusted and cut evenly (up to 20nm thin), which requires very precise mechanics, thus the high price.

3. Available DIY resources

DIY projects are a rarity at the moment, the reason for which is unknown. It is possible, that this is due to a high discrepancy between the research standards, compared to hobby requirements. Most DIY methods (example 1, example 2, example 3) use a manual system, where the sample is placed on top of a bolt and the section thickness is set with a nut. Sections are prepared manually, by sliding over the nut with a blade. The most sophisticated DIY microtome at the moment is actually built from LEGOs and can reach an estimated section thinness of 250μm.

4. Is DIY good enough and reasonable?

The current DIY status of microtomes is far from sufficient for serious microscopy. In any case, it should be possible build a DIY, maybe 3D prinatlbe microtome, producing even slices, the greater challenge would be achieving the required 1μm section range, or even go beyond, to ultrathin sections. For this endeavor, possibly a combination of mechanics and alternative processes can be used (for example temperature dependent expansion).

As it is currently lacking, we will set out to develop a DIY microtome, which can be made from 3D printable parts, assebled easily, but will meet the requirements (or come very close at least) of commercial devices.

Transmission electron microscope

####1. Background The resolution of an optical system has its physical limitations. Due to diffractional properties of electromagnetic radiation, an important one is the wavelength. As a consequence, we cannot look far into the sub-micrometer scale with only visible light. However, this is not always neccessary. Electrons can have a significantly shorter wavelength than visible light, which allows the observation of even smaller objects, sophisticated devices can reach a sub Ångström resolution. A transmission electron microscope (TEM) uses a focused electron beam, which is projected through a very thin (

70nm), contrasted sample (some structures absorb the electrons, others don't), on a CCD, which is then used to produce a visible grey-scale image.

Keywords: transmission, electron, microscope, tem, wavelenght, resolution.

####2. Commercial variants Commercial TEMs are very expensive, costing several 10k€, up to a few 100k€ or even 1M€, depending on their capabilities. These are determined by the quality of the lens system (magnetic lenses), the range of electron acceleration. To prevent the electrons to interact with gases, the procedure needs to take place in a vacuum, which is also required for preventing an arc between the high voltage cathode and the ground. The system also requires cooling, a stable power supply and a vibration free ground, to create good quality, sharp images. In addition, TEM imaging requires the adequade sample preparation, using an ultramicrotome for sectioning, usually in combination with glass and diamond knives.

####3. Available DIY resources There seems to be only one resource of someone building a home-made transmission electron microscope, sadly, no documentation is available.

####4. Is DIY good enough and reasonable? The images produces with the mentioned system are of reasonalbe quality, possibly allowing the inspection of cell ultrastructure (with appropriate samples of course), which is good enough for general use!

####5. Requirements and plans As there are no resources on DIY TEMs available, they will have to be made from scratch. Luckily, there is one more documented project on scanning electron microscopy, which could maybe be used as a basis.

###Scanning electron microscope (SEM)

####1. Background In contrast to a TEM, a scanning electron microscope (SEM) does not project electrons through a sample. Rather, the beam is focused on the surface, which absorbs the electrons and emits secondary ones, which are then projected to CCD. For this to work, the sample surface needs to be conductive, which is why nonmetalic samples are coated with a metallic thin film (gold, silver), usually by sputtering, or phisical vapor deposition.

In addition to emitting secondary electrons after primary electron absorption, matter usually also emitts other electromagnetic radiation, in the x-ray spectrum. these are element specific and can be used to determine the chemical composition of a sample, using energy-dispersive x-ray spectroscopy (EDX). The sensors are mostly very sensitive and require to be cooled with liquid nitrogen.

Keywords: scanning, electron, microscope, sputter, coating, edx, ex-ray

####2. Commercial variants As TEMs, SEMs are very expensive devices, they do, however use lower voltages and typically have shorter cathode tubes. Used devices cost 10k to several 10k€, new SEMs go into 100k€ or into the million € range, for devices with additional EDX or STEM (a limited form of TEM using a SEM) capabilities.

####3. Available DIY resources There is one "fairly" documented DIY SEM project available, made by Ben Krasnow, who also made a few videos showing the capabilities of his, very imperssive device.

####4. Is DIY good enough and reasonable? The images provided by Krasnow show good-enough quality of his DIY-SEM. The magnification is the major limiting factor and whether it suffices is dependent on the samples. For the average DIY enthusiast it is surely good enough.

####5. Requirements and plans A good-enough SEM should be capable of providing a sharp image at magnifications of a few thousand-folds.

####1. Background Sputter coaters are used for depositing of metals on nonconductive samples for scanning electron microscopy. Usually, an inert (so it does not react with the wafer material) gas at low pressures is accelerated and ionized in a strong electric field, to very high speeds, at which it can remove molecules from a "wafer" it hits. Those molecules are then dispersed and can condense nearby surfaces. If the wafer is made from a metal, like Gold or Palladium (often used for SEM sputtering), the coated surfaces become conductive and thus suitable for SEM.

Keywords: sputter, coating, metal, deposition

####2. Commercial variants Commercial sputter coaters cost a few thousand €, even second-hand ones. The cheapest used variants with broken parts come for around 500€.

####3. Available DIY resources Interestingly, there seem to be more DIY sputter coater projects circulating the internet, than DIY SEMs. Example 1, example 2, example 3. Most use a simple vacuum chamber and many don't even use a noble gas, a simple magnet composition and a high voltage generator. Sputter coating is probably the most widely used method for SEM sample preparation, however, there are other alternatives as well, like physical vapor deposition by evaporation at very high temperatures, or even chemical methods like silver coating with silver nitrate. Whether a method is suitable, depends on the sample and the wished results.

####4. Is DIY good enough and reasonable? Existing DIY projects do seem good enough, however, most of them are poorly documented.

####5. Requirements and plans The pressure in the chamber needs to be low enough, to sustain a small amount of ionized gas particles, thus allowing high voltages without short-circuiting. An inert gas, like Argon is required, which does not react with the sputtering metal and controlled magnetic and electric fields are required to accelerate the gas towards the metal.

####6. Working around/without the device If you have access to a SEM or STM, but not a sputter coater (which is probably not that likely), there are alternative methods of coating a surface with a conductive material which may, however, not be as precise. One possibility is chemical deposition of silver, by soaking a sample into a silver nitrate mirror making mix. Another posibility is metal vapor deposition, by evaporating metals, such as silver by heating and placing the samples adjacently.

###Scanning tunneling microscope

####1. Background A scanning tunneling microscope (STM) is a device for imaging surfaces at scales of the atomic level. It uses a concept known as quantum tunneling, where a fine, conducting tip (under a small voltge) is placed near a sample. When the distance is short enough, electrons can "tunnel" through the empty space between them, whereas the measured current depends on the tip position relative to the sample, the voltage and the density of space of the sample. Using this technique, extremely precise surface profiles of a sample can be measured, even single atoms can be made visible.

Keywords: scanning, tunneling, microscope, atomic, scale, surface

####2. Commercial variants "Cheap" commercial STMs can be acquired for about 10 000€, however more high-end devices can cost from 30k to 150k€.

####3. Available DIY resources A few DIY STMs with good documentation are currently available, which can be assembled for less than a 1000€, but can still reach the atomic scale. Best described are example 1, example 2 and example 3.

####4. Is DIY good enough and reasonable? The current DIY STMs seem to be a serious competition to the commercial devices, at least for measuring conductive materials. The control and precision may not be quite as high, but seems to be more than sufficient to determine the crystal structure of metals for example.

####5. Requirements and plans Due to the small scale, possibly the most important issue to tackle is vibration isolation. There are different methods, how to achieve this, in general, the measurement device (containing the sample) requires a large inertia (needs to be relatively heavy), while resting on some sort of spring or pillow. Another important characteristic is tip size, which needs to be appropriately small and narrow, to achieve a good spatial resolution and finally, motion control of the sensor needs to be sufficient, which can be achieved for example, by controling the thermal expansion of the mount, or using a piezoelectric crystal, etc.

####1. Background Similar to the STM, an atomic force microscope (AFM) uses a very fine tip (on a cantilever), which can be (but doesn't have to) brought in contact with the sample surface and is then used to scan across it, while the cantilever displacement is measured. In addition to measuring the height profile, an AFM can also be used to measure other characteristics, such as mechanical or surface interaction properties of a sample. The cantilever deflection is often measured by a laserbeam, which is reflected of the cantilever and redirected by a displacement.

Keywords: atomic, force, microscope, surface, properties

####2. Commercial variants Commercial AFMs usually cost several 10k€ and up to a few 100k€. They come in many different forms, for a wide range of applicability (different sizes, mounted on a microscope, adjusted for wet samples, automated to different degrees, etc.).

####3. Available DIY resources In 2015, a DIY AFM project was published in Nature Nanotechnology, which can be built by school children in a few hours and can successfully measure particles with a size of 2.5μm. This was part of the OpenAFM project, supported by the LEGO foundation. One of the more advanced spin-offs for example, is the Strømlingo nano, which is also available commercially for 2999$.

####4. Is DIY good enough and reasonable? The available DIY resources were actually created for educational purposes, however, they do achieve respectable specifications for a very low price. Whether they suffice for high end research is questionable. However, it should be noted, that open source devices can generally be easily modified for specific purposes, which is especially useful for AFMs, with their wide range of (typically very costly) forms and adaptations.

####5. Requirements and plans Due to the mechanical interaction of the AFM tip with the samples, it is easily damaged and needs to be replaced over time. For this reason it is imporant for an AFMs construction to allow simple and fast cantilever exchange. It is also beneficial, if it allows the installation of several types of cantilevers, which bring additional functionality to the device (different geometries, materials, tip coatings, etc.). The scannig process requires fine control over cantilever movement, and a precise displacement detection! Similar to the STM, an AFM also needs some form of vibration reduction.

New Era in Coral Biology Research: Scientists Have Cultured the First Stable Coral Cell Lines

Researchers in Japan have established sustainable cell lines in a coral, according to a study published today (April 25, 2021) in Marine Biotechnology.

Seven out of eight cell cultures, seeded from the stony coral, Acropora tenuis, have continuously proliferated for over 10 months, the scientists reported.

“Establishing stable cells lines for marine organisms, especially coral, has proven very difficult in the past,” said Professor Satoh, senior author of the study and head of the Marine Genomics Unit at the Okinawa Institute of Science and Technology Graduate University (OIST). “This success could prove to be a pivotal moment for gaining a deeper understanding of the biology of these vitally important animals.”

Acropora tenuis belongs to the Acroporidae family, the most common type of coral found within tropical and subtropical reefs. These stony corals are fast growers and therefore play a crucial role in the structural formation of coral reefs.

However, Acroporidae corals are particularly susceptible to changes in ocean conditions, often undergoing bleaching events when temperatures soar or when oceans acidify. Establishing knowledge about the basic biology of these corals through cell lines could one day help protect them against climate change, explained Professor Satoh.

Creating the cultures

In the study, Professor Satoh worked closely with Professor Kaz Kawamura from Kochi University — an expert in developing and maintaining cell cultures of marine organisms.

Since adult coral host a wide variety of microscopic marine organisms, the group chose to try creating the cell lines from coral larvae to reduce the chances of cross-contamination. Another benefit of using larval cells was that they divide more easily than adult cells, potentially making them easier to culture.

The researchers used coral specimens in the lab to isolate both eggs and sperm and fertilize the eggs. Once the coral larvae developed, they separated the larvae into individual cells and grew them in petri dishes.

The microscope image shows three of the cell lines established in the study, ranging in color and form. Credit: OIST

Initially, the culture attempts ended in failure. “Small bubble bodies appeared and then occupied most of the petri dish,” said Professor Kaz Kawamura. “We later found that these were the fragments of dying stony coral cells.”

In the second year, the group discovered that by adding a protease called plasmin to the cell culture medium, right at the beginning of the culture, they could stop the stony coral cells from dying and keep them growing.

Two to three weeks later, the larval cells developed into eight different cell types, which varied in color, form and gene activity. Seven out of the eight continued to divide indefinitely to form new coral cells.

Exploring the symbiosis integral to coral survival

One of the most exciting advancements of this study was that some of the cell lines were similar in form and gene activity to endodermal cells. The endoderm is the inner layer of cells formed about a day after the coral eggs are fertilized.

Importantly, it is the cells in the endoderm that incorporate the symbiotic algae, which photosynthesize and provide nutrients to sustain the coral.

Corals are the one of the simplest animals, with only two layers of cells (called germ layers) forming in early embryonic development — an inner layer, the endoderm, and an outer layer, the ectoderm. Each germ cell layer ultimately develops into different types of cells, including digestive cells, muscle-like cells, nerve-like cells and stinging cells (cnidocytes) but how each cell type forms during development still requires investigation. Credit: OIST

“At this point in time, the most urgent need in coral biology is to understand the interaction between the coral animal and its photosynthetic symbiont at the cellular level, and how this relationship collapses under stress, leading to coral bleaching and death,” said Professor David Miller, a leading coral biologist from James Cook University, Australia, who was not involved in the study.

He continued: “Subject to confirmation that these cells in culture represent coral endoderm, detailed molecular analyses of the coral/photosymbiont interaction would then be possible — and from this, real advances in understanding and perhaps preventing coral bleaching could be expected to flow.”

For Professor Satoh, his interest is in how the photosymbiotic algae cells, which are almost as big as the larval cells, initially enter the coral.

“The algae are incorporated into the coral cells around a week after the larvae first develop,” said Prof. Satoh. “But no one has yet observed this endosymbiotic event on a single-cell level before.”

A new era for coral cell research

The scientists also found that the coral cell lines were still viable after being frozen with liquid nitrogen and then thawed. “This is crucial for being able to successfully supply the coral cell lines to research laboratories across the globe,” said Professor Satoh.

The implications for future research using these cell lines are far-reaching, ranging from research on how single coral cells respond to pollution or higher temperatures, to studying how corals produce the calcium carbonate that builds their skeleton.

Research could also provide further insight into how corals develop, which could improve our ability to farm coral.

In future research, the team hopes to establish cells lines that are clonal, meaning every cell in the culture is genetically identical.

“This will give us a much clearer idea of exactly which coral cell types we are growing, for example gut-like cells or nerve-like cells, by looking at which genes are switched on and off in the cells,” said Professor Satoh.

Reference: “Establishing Sustainable Cell Lines of a Coral, Acropora tenuis” by Kaz Kawamura, Koki Nishitsuji, Eiichi Shoguchi, Shigeki Fujiwara and Noriyuki Satoh, 26 April 2021, Marine Biotechnology.
DOI: 10.1007/s10126-021-10031-w

Tiny Blobs of Brain Cells Could Reveal How Your Mind Differs From a Neanderthal’s

Researchers grew clusters of brain cells in the lab with a gene carried by our ancient ancestors.

In recent years, scientists have figured out how to grow blobs of hundreds of thousands of live human neurons that look — and act — something like a brain.

These so-called brain organoids have been used to study how brains develop into layers, how they begin to spontaneously make electrical waves and even how that development might change in zero gravity. Now researchers are using these pea-size clusters to explore our evolutionary past.

In a study published on Thursday, a team of scientists describe how a gene likely carried by Neanderthals and our other ancient cousins triggered striking changes in the anatomy and function of brain organoids.

As dramatic as the changes are, the scientists say it’s too soon to know what these changes mean for the evolution of the modern human brain. “It’s more of a proof of concept,” said Katerina Semendeferi, a co-author of the new study and an evolutionary anthropologist at the University of California San Diego.

To build on the findings, she and her co-author, Alysson Muotri, have established the UC San Diego Archealization Center, a group of researchers focused on studying organoids and making new ones with other ancient genes. “Now we have a beginning, and we can start exploring,” Dr. Semendeferi said.

Dr. Muotri began working with brain organoids more than a decade ago. To understand how Zika produces birth defects, for example, he and his colleagues infected brain organoids with the virus, which prevented the organoids from developing their cortex-like layers.

In other studies, the researchers studied how genetic mutations help give rise to disorders like autism. They transformed skin samples from volunteers with developmental disorders and transformed the tissue into stem cells. They then grew those stem cells into brain organoids. Organoids from people with Rett Syndrome, a genetic disorder that results in intellectual disability and repetitive hand movements, grew few connections between neurons.

Dr. Semendeferi has been using organoids to better understand the evolution of human brains. In previous work, she and her colleagues have found that in apes, neurons developing in the cerebral cortex stay close to each other, whereas in humans, cells can crawl away across long distances. “It’s a completely different organization,” she said.

But these comparisons stretch across a vast gulf in evolutionary time. Our ancestors split off from chimpanzees roughly seven million years ago. For millions of years after that, our ancestors were bipedal apes, gradually attaining larger heights and brains, and evolving into Neanderthals, Denisovans and other hominins.

It’s been difficult to track the evolutionary changes of the brain along the way. Our own lineage split from that of Neanderthals and Denisovans about 600,000 years ago. After that split, fossils show, our brains eventually grew more rounded. But what that means for the 80 billion neurons inside has been hard to know.

Dr. Muotri and Dr. Semendeferi teamed up with evolutionary biologists who study fossilized DNA. Those researchers have been able to reconstruct the entire genome of Neanderthals by piecing together genetic fragments from their bones. Other fossils have yielded genomes of the Denisovans, who split off from Neanderthals 400,000 years ago and lived for thousands of generations in Asia.

The evolutionary biologists identified 61 genes that may have played a crucial role in the evolution of modern humans. Each of those genes has a mutation that’s unique to our species, arising some time in the last 600,000 years, and likely had a major impact on the proteins encoded by these genes.

Dr. Muotri and his colleagues wondered what would happen to a brain organoid if they took out one of those mutations, changing a gene back to the way it was in our distant ancestors’ genomes. The difference between an ancestral organoid and an ordinary one might offer clues to how the mutation influenced our evolution.

It took years for the scientists to get the experiment off the ground, however. They struggled to find a way to precisely alter genes in stem cells before coaxing them to turn into organoids.

Once they had figured out a successful method, they had to choose a gene. The scientists worried that they might pick a gene for their first experiment that would do nothing to the organoid. They mulled how to increase their odds of success.

“Our analysis made us say, ‘Let’s get a gene that changes a lot of other genes,’” said Dr. Muotri.

One gene on the list looked particularly promising in that regard: NOVA1, which makes a protein that then guides the production of proteins from a number of other genes. The fact that it is mainly active only in the developing brain made it more attractive. And humans have a mutation in NOVA1 not found in other vertebrates, living or extinct.

How to Prevent Parallax Error

Parallax error occurs when the measurement of an object's length is more or less than the true length because of your eye being positioned at an angle to the measurement markings. For example, a person viewing a car's speedometer from the driver's seat will get an accurate reading because she has a direct line of sight. A person viewing the speedometer from the passenger seat will overestimate the reading because of the angle between his eye, the meter and the arrow.

Orient your line of sight directly above the measurement marking on a ruler or similar device so that an imaginary vertical line connects your eye, the marking and the object. Parallax error is primarily caused by viewing the object at an oblique angle with respect to the scale, which makes the object appear to be at a different position on the scale.

Place the measurement device on its edge so it is level with the object being measured. If the measurement marking is above or below the object, it will magnify any parallax error caused by your line of sight being at an angle with respect to the marking.

Seek out the finest possible edge of the measurement device, or use a device with finer edges. A wider edge allows for a larger parallax error because the object could be higher or lower with respect to the true measurement marking.

Place your eye at the level of the appropriate measurement marking when measuring the level of a liquid in a graduated cylinder. Read the lower part of the curved surface of the liquid -- the meniscus -- to gain an accurate measurement and avoid parallax errors.

Ask other people to take measurements. Because parallax error is a type of random error, you can average multiple readings taken by different people to cancel out most of the parallax angle. It is likely that some readings will have positive parallax error and others will have negative error. The average of these readings will be closer to the true measurement.

Department of Biology

To learn biology is more than just learning facts! It’s learning how to answer questions, how to develop strategies to obtain answers and how to recognize the answers as they emerge. The department is dedicated to encouraging students to learn science in both an intuitive and logical way.

It encourages students to independently question, probe, experiment and experience the natural world around us as well as life under a microscope.

The curriculum is organized to provide students with a sound introduction to the major concepts of biology and to foster an appreciation for the diversity and complexity of life. Requirements for students majoring in biology have been designed for both breadth and depth of training.

Class sizes are small and almost every course has weekly laboratories, taught by faculty members, where students learn to become biologists by making observations, asking questions, and designing and testing hypotheses. Our students read and evaluate primary research articles, write laboratory reports, and are given opportunities to make oral presentations.

The curriculum prepares students to pursue careers in research and the health sciences or to apply their biology interests to careers as diverse as science education and public health. Moreover, our undergraduates have an excellent record of acceptance into medical, dental and allied health professional schools. Faculty members are committed to helping students investigate career opportunities and pursue careers which most clearly match their interests and abilities.

Biology at PLU operates within the Rieke Science Center, which also houses the Departments of Chemistry, Geosciences and Physics. Thirteen teachers-scholars make up the biology faculty. All are full-time and all have a doctorate. Their research ranges from the responses of the host to bacterial infections and physiological processes and signaling pathways in the plant, to the evolutionary biology of North American catfishes and song divergence in North American Red Crossbills. Hands-on laboratory experience is central to any biology curriculum, and we invite students to use departmental facilities for independent study and work with faculty members in ongoing research.

Education in Microscopy and Digital Imaging

A transmitted light microscope will typically be of little use to anyone wanting to examine the structure of metallic samples, the surface of ceramics, integrated circuits, or printed paper documents. As a result, the reflected light microscope has been developed for these purposes. Reflected light microscopy is often referred to as incident light, epi-illumination, or metallurgical microscopy, and is the method of choice for fluorescence and for imaging specimens that remain opaque even when ground to a thickness of 30 micrometers. Much like the fluorescence microscope, in reflected brightfield microscopy the sample is illuminated from above through the objective. The Köhler illumination principle applies in cases where the objective with its pupil plane is also utilized as the condenser.

The range of specimens falling into this category is enormous and includes most metals, ores, ceramics, many polymers, semiconductors (unprocessed silicon, wafers, and integrated circuits), slag, coal, plastics, paint, paper, wood, leather, glass inclusions, and a wide variety of specialized materials. Because light is unable to pass through these specimens, it must be directed onto the surface and eventually returned to the microscope objective by either specular or diffused reflection. As mentioned above, such illumination is most often referred to as episcopic illumination, epi-illumination, or vertical illumination (essentially originating from above), in contrast to diascopic (transmitted) illumination that passes through a specimen. Several reflected light specimens are presented in Figure 1. The surface of an integrated circuit is shown using reflected light differential interference contrast ( DIC ) in Figure 1(a), while the jewel bearing of a watch mechanism captured in brightfield is presented in Figure 1(b). Darkfield is another useful reflected light technique, as evidenced by the image revealing surface structure of a superconducting wire cable in Figure 1(c). Finally, a magnetic thin film (Figure 1(d)) can be imaged using polarized reflected light microscopy to examine surface defects (blisters) that affect the homogeneity of the film.

Back to top ^ The Reflected Light Microscope

Today, many microscope manufacturers offer advanced models that permit the user to alternate or simultaneously conduct investigations using both vertical and transmitted illumination. A typical microscope configured for both types of illumination is illustrated in Figure 2 (the transmitted light source and optical pathway is not shown in this illustration). The optical pathway for reflected light begins with illuminating rays originating in the lamp housing for reflected light (the upper housing in Figure 2). This light next passes through the collector lens and into the vertical illuminator where it is controlled by the aperture and field diaphragms. After passing through the vertical illuminator, the light is then reflected by a beamsplitter (a half mirror or elliptically shaped first-surface mirror) through the objective to illuminate the specimen. Light reflected from the surface of the specimen re-enters the objective and passes into the binocular head where it is directed either to the eyepieces or to a port for photomicrography. Reflected light microscopy is frequently the domain of industrial applications, especially in the rapidly growing semiconductor arena, and thus represents a most important segment of microscopical studies.

A typical upright compound reflected light microscope has a viewing tube with two eyepieces (Figure 2) and often a trinocular tube head for mounting a conventional or digital/video camera system (not illustrated). Standard equipment eyepieces are usually of 10x magnification, and most microscopes are equipped with a nosepiece capable of holding four to six objectives. The stage is mechanically controlled with a specimen holder that can be translated in the x- and y- directions and the entire stage unit is capable of precise up and down movement with a coarse and fine focusing mechanism. Built-in light sources range from 20 and 100 watt tungsten-halogen bulbs to higher energy mercury vapor or xenon lamps that are used in fluorescence microscopy. Light passes from the lamphouse through a vertical illuminator interposed above the nosepiece but below the underside of the viewing tube head. The specimen's top surface is upright (usually without a coverslip) on the stage facing the objective, which has been rotated into the microscope's optical axis. The vertical illuminator is horizontally oriented at a 90-degree angle to the optical axis of the microscope and parallel to the table top, with the lamp housing attached to the back of the illuminator. The coarse and fine adjustment knobs raise or lower the stage in large or small increments to bring the specimen into sharp focus.

Inverted reflected light microscope stands incorporate the vertical illuminator within the body of the microscope. Many types of objectives can be used with inverted reflected light microscopes, and all modes of reflected light illumination may be possible: brightfield, darkfield, polarized light, differential interference contrast, and fluorescence. Some of the instruments include a magnification changer for zooming in on the image, contrast filters, and a variety of reticules. Because an inverted microscope is a favorite instrument for metallographers, it is often referred to as a metallograph . Manufacturers are largely migrating to using infinity-corrected optics in reflected light microscopes, but there are still thousands of fixed tube length microscopes in use with objectives corrected for a tube length between 160 and 210 millimeters.

On the inverted stand (similar in basic construction to the inverted tissue culture style microscope frames commonly employed in biology), the specimen is placed on the stage with its surface of interest facing downward. The primary advantage of this design is that samples can be easily examined when they are far too large to fit into the confines of an upright microscope (such as large rock samples and industrial materials). Also, only the side of the specimen facing the objectives need be perfectly flat. The objectives are mounted on a nosepiece under the stage with their front lenses facing upward towards the specimen and focusing is accomplished either by moving the nosepiece or the entire stage up and down.

In the vertical illuminator, light travels from the light source, usually a 12 volt 50 or 100 watt tungsten-halogen lamp, passes through collector lenses, through the variable aperture iris diaphragm opening and through the opening of a variable and centerable pre-focused field iris diaphragm. The light then strikes a partially silvered plane glass reflector, or strikes a fully silvered periphery of a mirror with elliptical opening for darkfield illumination. The plane glass reflector is partially silvered on the glass side facing the light source and anti-reflection coated on the glass side facing the observation tube in brightfield reflected illumination. Light is thus deflected downward into the objective. The mirrors are tilted at an angle of 45 degrees to the path of the light travelling along the vertical illuminator.

In reflected light microscopy, absorption and diffraction of the incident light rays by the specimen often lead to readily discernible variations in the image, from black through various shades of gray, or color if the specimen is colored. Such specimens are known as amplitude specimens and may not require special contrast methods or treatment to make their details visible. Other specimens show so little difference in intensity and/or color that their feature details are extremely difficult to discern and distinguish in brightfield reflected light microscopy. The latter specimens behave much like the phase specimens so familiar in transmitted light work, and are suited for darkfield and reflected light differential interference contrast applications.

Back to top ^ Objectives for Reflected Light Microscopy

The resolving power in reflected light is based on the same relationship between the wavelength of light and numerical aperture (the Abbe equation) as in transmitted light. Optical performance is achieved in reflected light illumination when the instrument is adjusted to operate under Köhler illumination. A function of Köhler illumination (aside from providing evenly dispersed illumination) is to ensure that the objective will be able to deliver excellent resolution and good contrast even if the source of light is a coiled filament lamp. In many cases, modern reflected light microscopes may also be operated using transmitted light because the parfocal length is maintained in all objectives.

Objectives for reflected light can be recognized by the Epi or similar inscription on the decorative outer barrel (see Figure 3). They differ from objectives for transmitted light in two ways. Reflected light objectives feature lens surfaces that are particularly well coated with anti-reflection layers to prevent the illuminator light from being reflected towards the eyepiece. Such reflections would be superimposed on the image and have a disturbing effect. The second difference is that these objectives are designed and optically corrected for specimens lacking a coverslip. The vast majority of samples in the materials sciences (where reflected light microscopes are most heavily used) are usually viewed without a cover slip. Therefore, higher numerical aperture objectives require a different optical computation than do transmitted light objectives.

Back to top ^ Reflected Light Microscope Illuminators

In reflected light microscopy, illuminating light reaches the specimen, which may absorb some of the light and reflect some of the light, either in a specular or diffuse manner. Light that is returned upward can be captured by the objective in accordance with the objective's numerical aperture. After entering the objective, light then passes through the partially silvered mirror (or in darkfield, through the elliptical opening). In the case of infinity-corrected objectives, the light emerges from the objective in parallel (from every azimuth) wavefronts projecting an image of the specimen to infinity. The parallel rays enter the tube lens, which forms the specimen image at the plane of the fixed diaphragm opening in the eyepiece ( intermediate image plane ). It is important to note, that in these reflected light systems, the objective serves a dual function. For light waves on the way down to the specimen, the objective serves as a matching well-corrected (always properly aligned) condenser. Alternatively, for waves reflected by the specimen, the objective serves as an image-forming optical system in the customary role of an objective projecting the image-carrying rays toward the eyepiece. Optimal performance is achieved in reflected light illumination when the instrument is adjusted to produce Köhler illumination (discussed below). A function of Köhler illumination (aside from providing evenly dispersed illumination) is to ensure that the objective will be able to deliver excellent resolution and good contrast even if the source of light is a coiled filament lamp.

Several modern reflected light illuminators are described as universal illuminators because, with several additional accessories and little or no dismantling, the microscope can easily be switched from one mode of reflected light microscopy to another. Often, reflectors can be removed from the light path altogether in order to permit transmitted light observation. Universal illuminators may include a partially reflecting plane glass surface (the half-mirror) for brightfield (see Figure 4(a)), and a fully silvered reflecting surface with an elliptical, centrally located clear opening for darkfield observation (Figure 4(b)). The best-designed vertical illuminators include collector lenses to gather and control the light, an aperture iris diaphragm and a pre-focused, centerable field diaphragm to permit the desirable Köhler illumination.

The vertical illuminator should also make provision for the insertion of filters for contrast, digital imaging, and photomicrography, as well as polarizers, analyzers, and compensator plates for polarized light and differential interference contrast ( DIC ) illumination. In vertical illuminators designed for use with infinity-corrected objectives, the illuminator may also include a tube lens. Affixed to the back end of the vertical illuminator is a lamphouse (Figure 2), which usually contains a tungsten-halogen lamp. For fluorescence work, the lamphouse can be replaced with a fitting containing a mercury burner. The lamp may be powered by the electronics built into the microscope stand, or in fluorescence, by means of an external transformer or power supply.

Back to top ^ Köhler Illumination in Reflected Light Microscopy

In a reflected light microscope vertical illuminator, the light source is positioned so that the tungsten-halogen lamp filament is located near the principal focal point of the collector lens. In Köhler illumination, the lamp collector lens serves the function of a dramatically enlarged secondary light source to enhance overall illumination. One of the primary requirements of Köhler illumination is that an image of the lamp filament must ultimately be projected onto the rear focal plane of the objective, which also doubles as the (often high numerical aperture) condenser during excitation in reflected light illumination. The light source should ideally fill the entire objective aperture to both maximize the intensity of radiation and to produce an evenly illuminated field. In many cases, a ground glass filter is placed into the vertical illuminator between the lamphouse and the neutral density filters in order to increase the uniformity of illumination. However, because diffusion filters also reduce the level of illumination, they should be avoided whenever possible.

In reflected light Köhler illumination (illustrated schematically in Figure 5), an image of the light source is focused by the collector lens onto the aperture iris diaphragm located in the vertical illuminator. This diaphragm shares a conjugate plane with the rear aperture of the objective and the lamp filament, and therefore, determines the illuminated field aperture size. Together, the light source, vertical illuminator aperture diaphragm, and objective rear focal plane (pupil) form the illumination set of conjugate planes. Unlike the situation in transmitted light microscopy, the aperture iris and light source are imaged onto the objective (acting as a condenser) rear aperture plane, rather than being physically located at this position. As an added benefit to this configuration, all obstructions (such as iris diaphragms) are removed from the light path. Opening or closing the aperture diaphragm is used to control stray light and regulate the intensity (numerical aperture) of illumination without altering the size of the illuminated field. In the image, adjustment of the aperture diaphragm affects brightness and contrast.

The image-forming or field set of conjugate planes in reflected light Köhler illumination consists of the field diaphragm, the specimen surface, and the intermediate image plane. Thus, when the field diaphragm is placed in focus at the specimen plane, the image of the light source is significantly removed from focus in order to provide a uniform field of illumination. The field diaphragm controls the size of the illuminated field without affecting the illumination intensity of the area being observed. In practice, the field diaphragm opening size should be as small as possible in order to increase image contrast. Köhler illumination produces even illumination of the specimen field in spite of the uneven illumination intensity generated by most filament-based light sources. When the microscope is properly configured, the rear focal plane of the objective is fully illuminated, providing a field that is uniformly bright from edge to edge. Köhler illumination, in the ideal case, bathes the specimen with a converging set of wavefronts, each arising from separate points on the light source imaged into the condenser aperture. In a properly configured reflected light microscope, the result is optimum image contrast and resolution.

Back to top ^ Brief Overview Contrast Modes in Reflected Light Microscopy

Due to the fact that the objective serves a dual purpose (also performing as a condenser) in reflected light microscopy (refer to Figure 6(a)), there is sufficient room to introduce auxiliary components into the infinity space occupied by the parallel bundle of light wavefronts traveling from the objective rear aperture to the tube lens (termed the observation side of the optical train see Figure 6(a)). In addition, polarizing or filter components can be inserted into the vertical illuminator before light enters the objective (termed the optical train illumination side ). Many modern microscopes also provide additional space for components that affect both light paths. This space is usually built as a slot in the objective nosepiece where a slider containing either a filter or polarizer can be easily inserted.

Several techniques are commonly employed to introduce contrast in reflected light microscopy, including darkfield illumination, polarized light, and differential interference contrast. In reflected darkfield microscopy, which is an ideal methodology for exploring the relief in surfaces of materials, wavefronts from the vertical illuminator are directed toward the objective using a specialized mirror assembly that contains an oval opening (see Figure 4 and Figure 6(b)). This light passes through an outer sleeve in the microscope objective and impacts on a ring-shaped concave mirror, which directs the wavefronts at a highly incident angle onto the specimen surface. In cases where the specimen acts as a perfect mirror (in effect, there are no relief features on the surface), there is no light reflected back into the objective from the specimen and the image remains dark. Areas where relief contours exist, however, direct light back into the objective front lens and are observed as being bright features against a very dark background. Note that in darkfield reflected light microscopy, the field and aperture diaphragms in the vertical illuminator should be opened to their widest points so that the light beam illuminating the mirror assembly is not partially blocked.

Polarized reflected light microscopy (Figure 6(c)) is a technique that is suitable for examining surfaces containing structures that alter the state of polarization during the reflection process. For example, structural grains in ore samples and a number of metallic alloys and thin films can be readily examined using this method. In the optical configuration outlined in Figure 6(c), the illuminating wavefronts encounter a polarizer that is placed in the vertical illuminator before the mirror unit that directs light into the objective. The linearly polarized light waves are focused onto the specimen surface and reflected back into the objective. After leaving the objective aperture as a parallel bundle of wavefronts, the light is then projected onto a second polarizer (the analyzer ) oriented at 90 degrees with respect to the polarizer. Only the depolarized wavefronts are able to pass through the analyzer to reach the tube lens. An auxiliary lambda plate can also be inserted just prior to the analyzer in the optical train to examine the sign of birefringence (changing gray to color contrast). This method is sometimes referred to as sensitive tint . In cases where objectives of very low magnification are used in reflected polarized light, a rotatable optical plate (termed an Antiflex cap ) consisting of a one-quarter wavelength lambda plate is placed on the objective front lens element to block reflections from the objective itself. The Antiflex method is also particularly useful when the specimen has very low reflectivity, such as would be observed in coal samples.

One of the most powerful techniques for introducing contrast into reflected light imaging is differential interference contrast, which allows the visualization of minute elevation differences in surfaces. In the optical configuration (Figure 6(d)), a birefringent prism (also known as a Wollaston or Nomarski prism, depending upon design) is placed in the infinity space just above the objective and a polarizer is installed in the vertical illuminator (similar to polarized light). The prism splits the polarized light wavefronts into two orthogonal polarized beams on their way to the specimen. These perpendicular light beams impact the specimen to create a lateral displacement in regions where surface relief exists. If the surface is completely flat, no features are observed. However, if there is, for example, a small step (see Figure 6(d)) between the two wavefronts, one of the beams must travel a path that is longer and is assigned this path difference. Once the parallel beams have returned to the microscope after passing back through the objective and prism, they pass through a second polarizer (the analyzer) where interference produces an intermediate image where path differences are translated into gray values that can be seen by the eye. Similar to polarized light microscopy, a lambda plate can be positioned beneath the analyzer to shift gray values into colored hues.

Contributing Authors

Rudi Rottenfusser - Zeiss Microscopy Consultant, 46 Landfall, Falmouth, Massachusetts, 02540.

Erin E. Wilson and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.