Because we know that most of you want to find out more about how to take care of your microscopes, we have decided to go into more detail about how to clean one, which is an essential practice that will help extend the optical instrument’s lifespan. After you read this article, you will become a pro in cleaning your microscope and you will not need outside help.
What can cause the lack of clarity of my microscope?
What you need to take into account before starting to clean your microscope is that dirt is not always what causes a lack of clarity. Therefore, before beginning the cleaning process, you should check all the other variables that could be responsible for the unclear image, like diaphragm settings, or illumination settings.
If everything seems to be in order, then you should consider cleaning the microscope, but remember that you have to be very careful during the process because it might be a little bit tricky and you have to make sure that you will not damage it, which can happen very easily.
Am I allowed to clean every component of the microscope?
Well, you are allowed to do anything you want in terms of cleaning, but it is recommended that you don’t touch some of its parts and leave those to a specialist. Your microscope comes with a manual and, before you begin the cleaning process, you should read it to find out more about how to do it and what is accessible and what is not.
The parts that you can clean are all the glass pieces that you have access to which include the protective glass of the sensor, the eyepiece lenses, the objective lenses in the front, and also the condenser lenses. Some manufacturers also recommend that you clean the actual surface of the camera sensor.
Tips to consider before you start:
If you have to move your microscope in order to clean it, you have to be very careful not to bump it or shake it, because it is very possible that you will cause damage to its construction.
Avoid touching the lenses with your bare hands and make sure that what you use to clean them is not dirty and does not have dust particles that could damage the lenses.
The best choice is to clean it indoors in order to avoid dust, dirt, and exposure to sunlight, which could cause damage.
Never forget to unplug the microscope before you start cleaning it because you might get hurt and the microscope could be affected as well.
Gloves that do not have talc powder
Lint-free cloth pieces
Small vacuum cleaner
Lint-free cotton swabs
Steps to take in order to clean the mechanical parts of the microscope
When you clean your microscope, it is important to remember to clean the mechanical parts as well, not just the optical lenses because the mechanical parts can gather dust which can get stuck on the lenses as well. This is an easy part because you will not have to do much.
Step 1: Clean the casing of the microscope with a moist tissue.
Step 2: Mix the distilled water with the detergent and clean the mechanical parts.
Step 3: Wipe everything down with a dry cloth that is lint-free.
Steps to take in order to clean the optical parts of the microscope
Before you start cleaning the optical parts of your microscope, you should take into account some important facts that can influence the process.
Try to avoid solvents
This is a very important aspect that you should consider because your purpose is to clean the lens, not to damage the coating. Most lenses, including the ones you use for your eyeglasses, have coatings in order to improve their performance. The problem is that these coatings are made of chemical substances that can be damaged if you use solvents.
Some manufacturers apply coatings that are not that sensitive when it comes to solvents, but most of them will get damaged. This is why the only liquids you should use are distilled water and isopropyl alcohol. Some people say that using acetone is ok, but we encourage you not to do so because if you don’t know how to use it properly, you might wipe off the coating.
Never wipe if there is dust or dirt on the lens
You might ask ‘how to clean the lens then if you are not allowed to wipe the dust and the dirt off?’. Well, wiping the lens if there is still dust on it can be compared to using sandpaper on it. The dust has the same effect because the coatings are very sensitive and the clarity of the lens can be affected by this process.
Therefore, you should first use a vacuum or oil-free compressed gas in order to remove dust, but we are going to talk about that later in this article.
So, what are the steps that you need to take?
Step 1: Identify where the dirt is located
Dirt can have a place on the camera or on the other lenses of the microscope. If impurities are located on the camera, when you move the camera the impurities will have the same positioning as before. If they don’t, then the camera is not the location that you are looking for.
Step 2: Remove dust
We have mentioned before that you are going to need a vacuum in order to properly clean your microscope and now is the time to use it. Before doing anything, you should use the vacuum to remove all dust and impurities. Not every vacuum is appropriate and you will have to buy a vacuum made specially to clean microscopes.
After you have done that, use a compressed gas sprayer in order to make sure that all the dust particles are removed. You can either use a vacuum or a gas sprayer; however, a good practice is to use them both to be sure.
What you have to consider before buying a compressed air sprayer is that it shouldn’t contain any oils or solvents. Many people think that using oil will diminish the chances of scratching the lens but, the truth is, using oil will only leave residue on the lens that will be very difficult to remove without solvents and all you will do is to damage the lens at the end.
Step 3: Clean the lens using a cotton swab
In order to properly clean the lens, you have to use a cotton swab that is attached to a stick. A wooden stick is preferred and the cotton should be lint-free. You can do this either using distilled water or isopropyl alcohol. The essential thing to remember is that you have to follow this step only if there is solid dirt on the lens. If not, just removing the dust should be enough.
If you want to effectively remove impurities using a cotton swab, you should make circular moves because otherwise, you will not be able to leave the lens dirt-free. Do not use the cotton swab without submerging it in liquid first because you will damage the lens coating. We can not emphasize enough how sensitive to scratching lens coatings are.
Step 4: Prevention
This is the number one thing that you have to do in order to protect your microscope from getting dirty, increasing its lifespan in this way. You can do this by cleaning the outside parts after every use, which is a great practice if you think about it because it will take less time than doing it only once in a while.
Also, a great practice is to cover the microscope after every use and leave it covered when you are not using it. This will prevent dust from gathering on the lenses inside the microscope which means that you will not have to perform general cleaning that often. This will reduce the chances of you scratching the lenses.
Microscopes are very sensitive optical instruments and you should take care of them if you want to enjoy exploring the microscopic world for a long time from now on. This includes periodically cleaning yours in order to keep its clarity. After reading our article, we are 100 percent sure that you are going to do it effectively if you follow our tips.
Optical instruments like the microscope have made a huge impact on the evolution of science and if you want to know what the best ones are you can find more info here. Even if scientists were able to make a lot of progress using a conventional microscope, the fluorescent microscope made a huge difference.
In this article, we are going to tell you all about how a fluorescent microscope works, what the advantages of using one are, and in what areas you will be able to use one. So, let’s begin your journey in finding out all about the fluorescent microscope.
What is a fluorescent microscope?
If you are not familiar with how a conventional microscope works, you should know that it uses visible light between 400 and 700 nanometers in order to illuminate an image and then to magnify it so that the person examining the image will be able to see it clearly. This method has been used for a long time, but it is a little bit limiting.
So in order to overcome these limitations, scientists have come up with another type of microscope, the fluorescent microscope. This microscope, in addition to the magnification of the image, uses phosphorescence and fluorescence.
Fluorescence is a term used to name the light produced by a substance when another light source is stimulating it. Practically, a fluorescent microscope uses a laser along with reflected light in order to offer a clear image. In this case, the light does not simply travel through a sample to make the image visible.
Phosphorescence is similar to fluorescence, but the light is not reflected right away by the substance. There are many types of fluorescence microscopes. In essence, any type of microscope that uses fluorescence is a fluorescent microscope, like the confocal microscope, for example.
How does a fluorescent microscope work?
The principles behind fluorescent microscopy are very simple and after reading them, you will clearly understand why a fluorescent microscope is superior to a conventional one.
Principle no.1: You have to use a dye in order to make some components of the cell visible because most parts of the cell have no color and are not easily distinguished.
Principle no.2: Dyes that are fluorescent have the ability to reflect light when they are excited by an external light source. These dyes are called fluorophores.
Principle no.3: The light emitted by these substances can be separated from the light source that excites the fluorophores in order to be able to have a clear image.
Reading about these principles has made it all very clear. The simple way that this microscope uses light in order to illuminate certain parts of a cell is no longer a mystery. However, what dyes do you have to use?
There are a lot of dyes (stains) specially designed for different types of molecules. The way they work is that some of them are made of molecules that are fluorescent by themselves and, when applied to a sample, they will quickly bind to certain types of molecules in order to make them visible.
The most common stain is DAPI, which is a fluorescent stain that can bind to DNA. DAPI is a very interesting stain that can pass through the membrane of a cell. Live cells’ membranes are not that easy to penetrate, but DAPI can do it, allowing scientists to examine a lot of interesting things that happen inside a cell.
What are the advantages of using a fluorescent microscope?
Fluorescent microscopy helps scientists study the internal parts of cells and helps them observe structures and behaviors that conventional microscopy is not able to identify. This is why the study of different bacteria types is possible. Therefore, fluorescent microscopy comes with a lot of advantages compared to conventional microscopy.
People who want to observe different types of molecules at the same time are able to do so by using different stains for each of them in order to identify them inside cells.
The precision of fluorescent microscopy is very high, this is why while observing a cell using a fluorescent microscope, someone can determine as many as 50 molecules in an area measuring 1 cubic micrometer.
Using this special technique, fluorescent microscopy allows you to observe cells both in vitro as well as in vivo, which is actually pretty amazing.
A fluorescent microscope can isolate a single protein among other parts of the cell that are not illuminated.
A fluorescent microscope will help you observe the behavior inside living cells and also the dynamics and is one of the most popular methods to do that.
What are the uses of a fluorescent microscope?
Observing the structure of a cell
As we have said before, using a conventional microscope makes it hard to observe certain things that would help researchers in their quest for solving health issues or for identifying and solving mysteries that have been present in the minds of humans for a long time. A cell is practically without color and all its components are the same.
A lot of things happen in a cell because most cells have different components and each has a role, so in order to be able to observe the dynamics inside cells or to examine the structure of one of its components, it is best to use a fluorescent microscope.
Studying cell populations
Cell populations are usually a group of cells that have similar characteristics. You can observe cell populations in order to see how they interact with one another and how the internal structures of these cells interact. You can also determine if the cell population is alive or dead.
Observing DNA and RNA within a cell
Fluorescence microscopy is very often used to observe DNA and RNA present in cells and scientists are able to measure the DNA present in certain cells. You can also see how DNA and RNA molecules interact with other components of the cell using DAPI, the fluorescent substance that we have talked about earlier in this article.
Spinning disk confocal imaging
This technique uses a confocal microscope, which is also a fluorescence microscope, in order to use 2D images to construct 3D images. This is amazing because you will be able to create a 3D image of a certain cell or molecule. Confocal microscopes are very complex and use very precise techniques to do that.
Creating an image of a single molecule
A fluorescent microscope can help you localize and observe the activity of just one molecule inside a cell. This is such a big deal especially because you can observe this without damaging its natural environment and you can actually analyze its activity.
Electrophysiology imaging used in neuroscience
Many new technologies have helped neuroscientists make astonishing discoveries in the last century. Fluorescent microscopes are optical instruments that have been found very helpful in this field. Using such a microscope to observe brain tissue has led scientists to even be able to find out what the function of such a cell is.
Cellular activity is examined here by analyzing the cells’ response to electrical stimulation, so you can imagine that the possibilities here are infinite. This is how scientists have been able to discover a lot about how our bodies work and about the role that our brains have in everything that is happening to us.
Are there any limitations to fluorescence microscopy?
One limitation would be the fact that when you use such an instrument to examine a sample, you will be able to analyze only the certain structures that have been previously labeled to be examined. Also, some cells might have toxic reactions to certain light wavelengths, so this is also a limitation that a fluorescent microscope has.
As technology has evolved, so has the ability of humans to observe and develop new ways of making the world a better place. To do so, we must first understand what is going on around us, and fluorescent microscopes can help us do that. Due to the technology they are using, they can help scientists observe life at its deepest levels.
There are a lot of benefits that come along with using this type of microscope even though there are some limitations as well. However, the images provided by such a microscope and the scientific discoveries that it has made possible make it indispensable if we want to move forward with making the world better.
If you are interested in figuring out what the top-rated microscopes on the market are, you can check it out here. However, if you simply want to understand a bit more about the various types of such devices that are currently being used and which one to choose based on your needs, then this article is the one for you.
Many people have in mind the image of the standard compound microscope when they think about these devices, but the truth is that there are plenty of other models out there, each one designed with specific needs in mind. For instance, if you are planning to use a microscope on the go, you are not going to use a compound one that can be difficult to transport.
On the other hand, if you are working in lab conditions, and you need to rely on the accuracy of your readings, then you are not going to use a portable microscope that has a high chance of being less clear than a standard one.
With this being said, it’s important to have an overview of the main types of microscopes, so that you’ll know exactly what you need the next time you want to purchase a new one. Of course, the prices can vary greatly as well, so it’s always a good idea to know what you are after in terms of characteristics, whether you need greater resolution or higher magnification.
If we’re going through the various types of microscopes, there’s no way not to mention the simple microscope, which is the initial device that led to everything that we have today in terms of these handy devices. Created in the 17th century, the simple microscope was an invention of Antony van Leeuwenhoek.
He essentially combined a holder for specimens with a convex lens and got what could be today seen as a steady magnifying glass. On the other hand, this device did have a magnifying power between 200 and 300 times, which proved to be enough for van Leeuwenhoek to see different shapes in red blood cells and to observe plenty of other biological specimens.
Of course, the simple microscope is no longer used today, as there are so many other devices that can get this job done much better. Plus, the introduction of another lens also led to the creation of the compound microscope, which is much more powerful and, thus, useful.
Since we’ve already mentioned it in this article, it’s only natural to take a look at the compound microscope next. This device includes a second lens that further magnifies the initial image obtained with the help of the first lens. The result is a much better magnification which allows its user to observe in more detail the specimen.
Compound microscopes have some particularities as well, namely that they can be either monocular or binocular. Moreover, the specimen is lit using a light placed underneath, which is why they are called bright field microscopes. In terms of images rendered, these microscopes have a magnification power of 1,000 times, so they can definitely allow identification of individual cells.
On the other hand, the image’s resolution tends to be a bit lower, so that’s one of the downsides of choosing this type of microscope. It’s true that compound models are extremely useful and relatively cheap, which is why they are used in plenty of schools and research labs, so they can definitely help in a wide range of tasks.
Botanists are also known for using compound models, as they can easily study things such as plant cells and various parasites or bacteria. Forensics labs can also put these microscopes to good use, given that the devices are able to provide help in identifying various drug structures.
If you are looking for an item that can help you see various objects up close, while still being able to manipulate them during the observation process, then you want to take a look at stereo microscopes. These are also known as dissecting devices, and they provide a magnification that goes up to 300 times.
While this may not seem very high, these units are still very helpful, if you need to take a look at objects that are too large to require slide preparation, as is the case with compound microscopes. Stereo microscopes can ensure clear viewings of surface textures, so they are successfully used for medical and biological applications.
Another category of users that can definitely enjoy the benefits of stereo microscopes consists of those who work with electronic devices, watches, or circuit boards.
What makes confocal microscopes different from their stereo or compound counterparts is that they use laser light for observation, and the samples need to have been dyed beforehand. The two former types of microscopes that we’ve looked at use regular light for viewing purposes.
In the case of confocal microscopes, the samples need to be correctly prepared, placed on slides, and then inserted, and the dichromatic mirror included in the device’s construction helps in producing a digital magnified image that appears on a computer screen.
These microscopes work great for producing 3D images, given that operators can assemble multiple scans in order to get a clear view of the subject. While these units come with a high magnification power, just as compound models, the resolution is much better, which means that clear viewings are ensured in those cases in which fine precision is required.
In terms of how they are being used, confocal microscopes are quite popular for studying cell biology, as well as for a wide range of medical applications.
Scanning Electron Microscope
Also known as SEM, the scanning electron microscope is a special type of device because it uses electrons in order to form images, as compared to using light as the previous types of devices do. Of course, there are some significant differences in terms of the way it works, such as the fact that samples are scanned in near-vacuum conditions with this type of device.
This means that they need to be specially prepared for the process, the first step being one of dehydration, followed by a coating one in which a very thin layer that consists of conductive material is added, gold being one such example.
Once the sample is properly prepared, it’s introduced in the dedicated chamber, and the SEM uses it to produce a 3D image on a computer screen, this also being black-and-white. Given that the operator has ample control over the level of magnification, which means a lot more versatility in terms of readings, SEMs are mostly used in medical, physical, and biological sciences.
Since we’ve talked about SEMs, it’s a good moment to mention transmission electron microscopes as well. Also known as TEMs, these devices work in a very similar way to SEMs, but the image they produce is a 2D one, so they work better for objects that have a certain degree of transparency as well.
Of course, TEMs also have a wide range of applications, from medical and biological ones to forensic analysis and nanotechnology.
We’ve already looked at some types of microscopes that render images directly on computer screens, but it’s a good idea to take a closer look at what digital models have to offer. Before we go into the details, it’s interesting to note that the first digital microscope was invented in 1986 in Japan.
Today, there are plenty of models that use the power of this technology to bring images directly onto computer screens so that researchers and scientists can clearly see all the necessary details. Digital units usually connect to a computer using a USB cable, and moving images can be captured without too much trouble as well.
Of course, there are plenty of applications for microscopes within scientific organizations and communities, but given that digital devices are becoming increasingly more affordable, there is a wide usage among hobbyists and within schools that should not be neglected.
For schools and learning purposes, the easiest-to-use type of microscope one can try out is the digital USB computer one, which can be connected to a laptop right away and provides very quick readings. While the specimen doesn’t need to be prepared in a specific way, the results are less clear in terms of magnification and resolution.
Microscopes are amongst the coolest inventions as they open a gate to a whole new world, allowing people to study even the smallest lifeforms, so make sure to check various reviews of microscopes to find the perfect one, according to your requirements.
Microscopes are the best way to research everything surrounding us and to find answers to some of the most complex problems of the world, such as the origins of life and evolution. Depending on their size and complexity, they can be used by people of all ages, starting with kindergarten children who are attracted to science.
However, as we evolved, these devices also became more specific and complex, requiring a unique set of skills to use them properly and make the most of their sophisticated compounds. The most common types of microscopes existing are light and electron devices, each coming with a specific set of properties, strong points, and downsides.
So, if you want to learn more about microscopes in general and find the perfect device for your future science projects, here is what you should know.
If you’re looking at the differences between these two types of microscopes, you should start with the year, or the century when they were first made. Although there is no exact date to mark the beginning of the optical microscopes (light microscopes), it is believed that a Dutch spectacle maker, Zacharius Jansen, and his father, Hans, were the first ones to invent a rudimentary version of the microscope, back in the 16th century.
It wasn’t until three centuries later, in 1931, that physicist Ernst Ruska and a German engineer named Max Knoll assembled the first beam or electron microscope.
Throughout the centuries, scientists have looked to expand their knowledge horizon, contributing to the creation of new, better, improved microscopes with a higher magnification rate that helped them see even more details.
However, there are many other differences between these two types of microscopes, so each of them serves a specific purpose. Let’s take a closer look at these differences and identify them so that you can know which one will suit your research purposes better. But first, let’s see some similarities.
Before diving into the pool of differences, it is worth mentioning that both types of microscopes serve the same purpose – to form a larger (magnified) and more detailed picture of small objects or areas of larger objects. These lenses are more powerful than the human eye so that the purpose of a microscope is to let people see beyond their physical abilities.
Another similarity between the beam and optical microscopes is that they are both used for studying and researching purposes, mainly in the biology and medical fields. They are extremely useful when studying anatomy, biology, and even histonomy or metallurgy.
Finally, they both use specimens or samples that must be carefully prepared using appropriate techniques to preserve the quality of the samples and ensure accurate testing and research. With the help of microscopes, we are now able to see cells and molecules and improve our understanding of viruses, bacteria, human blood, and other interesting things.
What are the main differences between light and electron microscopes?
The first and most notable difference between them is their size. Optical or light microscopes are usually small and portable and don’t weigh more than 5-6 pounds, depending on their generation and features. Thus, they are easier to carry around and set up, as opposed to electron microscopes.
The first generations of electron microscopes were as big as a room and required various people to turn them on, prepare them, and use them. However, as technologies advanced, we are now seeing smaller and more powerful electron microscopes but this doesn’t mean they are anywhere near a portable size yet.
Depending on their technical features, most light microscopes cost around $200-$300. They are available for sale online and in regular stores and come with dozens of handy accessories to help you prepare the process. There are also numerous kits designed specifically for kids that include samples or specimens, tweezers, and a carrying bag to keep everything in place and secured.
Don’t forget that some light microscopes for children resemble toys more rather than actual microscopes, so seeing clearly and obtaining a good focus might be hard. They are also made of plastic and look flimsy, so they might not be suitable for science purposes. On the other hand, they are usually cheaper, at around $50.
As for electron microscopes, they are mainly designed for professional use only, so you’ll see them in testing labs and universities. Due to their high standards, they can only be handled by highly trained professionals, which means that not every person can afford them.
Depending on their size and characteristics, electron microscopes can cost up to $100,000, although many vary between $30,000-$50,000. Given that they are an investment, we suggest you contact the manufacturer personally or only buy from reputed sellers and distributors.
One of the biggest differences you’ll notice between these two types of microscopes is in terms of magnification rate or how many times the devices can enhance an image. Depending on the scope, a good light microscope can boost the image 40x-1000x times. However, a high magnification doesn’t always mean a clearer image as you’ll also need high resolution to make the most of the magnification rate.
Electron microscopes have higher resolutions and magnification rates, and they are mainly used for advanced research, and this is one of the reasons why they cost a lot more than “desk” devices. An electron microscope can magnify an image up to 300,000X with a resolution that is about 250 times higher than the one you would get from a normal optic microscope.
Source of light and optics
The main illuminating source of an optic microscope is light, whereas electron devices use beams of electrons to deliver an image. In terms of optics, the first microscope uses eyepiece lenses, a condenser, and an objective all made of quality glass, whereas the electron microscope uses electromagnetic lenses.
Another difference between the microscopes is the type of specimens you can see on them. With a light microscope, you can use all sorts of specimens, both alive or dead. The electron microscope only allows you to see dead or dried specimens.
The preparation of the specimens and their storing also requires an elaborate and time-consuming process. The main reason why light microscopes are used at school and for studying purposes is that it won’t take more than a few minutes or hours to prepare the specimens.
On the other hand, to get the most of a dead specimen on an electron microscope, you need to properly prepare and store it for a few days.
The preparation of the specimens also differs depending on the type of microscope used. For instance, light microscopes will require specimens stained by colored dyes, while the electron devices need specimens coated with heavy metals to reflect the electrons.
The image projected by a light microscope is colored and as close as possible to what you can see with the naked eye. It forms an image that includes the range of wavelengths provided by the light source. However, with stained specimens, the colors that you see are most likely due to the stains and don’t necessarily depict the real colors present in nature.
On the other hand, the images received on an electron microscope can only be black and white although the actual term used is “greyscale” images. Sometimes, you get to see false color electron micrographs which are beautiful.
Source of power
Finally, a light microscope works on its own and rarely requires the use of high voltage electricity.
Most of these devices have batteries that fuel the lamps so you can see the specimens better, whereas electron microscopes will need a generous source of power, of about 50,000 volts and above to work. This can also lead to a radiation leakage risk, whereas the radiation risk is 0 when we’re talking about light microscopes.
Light microscopes are the most common instruments used in research and science projects and can be powerful enough to help your cause. If you’re looking for high-accuracy results and lab-precision results, you should be ready to invest thousands of dollars in an electron microscope and also have the knowledge to operate it.
Good microscopes can help you learn a lot of interesting things about everything surrounding us. When used properly, these devices can help children and youngsters fall in love with science and even allow them to dream about careers in fields like medicine, biology, and research.
However, understanding how microscopes work is a little difficult, as each device contains numerous parts that should be explained separately. If you’re looking to find out more about microscopes in general and their depth of field, continue reading this article.
Before getting into technical details about the importance of the depth of field, we should first talk about the magnification rate. Most magnifying items on the market (including magnifying glasses, binoculars, telescopes, cameras, and microscopes) use the same optical principle and glass lenses to magnify an image and allow you to see far or big objects in more detail.
Depending on the type of device used, magnifying rates range from 1x to 100,000x for the professional microscopes and, sometimes, even beyond that number.
Optic microscopes usually help you magnify an object 40x to 400x times but this doesn’t mean that the image will be very clear. To make the most out of your microscope’s technical performance, you need to take a closer look at other aspects such as resolution and depth of field.
By comparison, beam microscopes use an alternative technology that allows higher resolutions and clarity.
Understanding the field of view and depth of field
The field of view (FOV) allows you to understand how much of a specimen is visible at any given moment in the lateral plane or perpendicular to the optical axis.
You can also understand it as the diameter of the circle of light visible when looking through the lens of a microscope. The field of view is inversely proportional to the magnification rate which means that a higher magnification rate will result in a narrower field of view.
On the other hand, the depth of field usually refers to the resolution in the longitudinal plane or parallel to the optical axis. You can measure it as the distance from the farthest object plane to the nearest object plane in focus and it is usually expressed in microns. Similar to the field of view, the depth of field also decreases once you enhance the magnification rate.
To better understand the concept, you can think of two hairs set in a crisscrossed position on a microscope slide. With a lower magnification rate, you can easily get to focus on both hairs at the same time but, once you increase the magnification rate, the lens will mainly focus on one hair, as the other one will be blurry.
Depth of field, depth of focus, and image depth
These three concepts are all important when trying to understand how a microscope or camera work. Any lens can transform a 3D object into a 2D image, and a person with good eye accommodation can later view this 3D image because the eyes see objects at a different distance if they are located within the accommodation range of the eye.
When you’re using a microscope, the object you observe with your eyes through the provided eyepieces is transferred into the intermediate plane of the microscope.
A simple definition of the depth of field would be that it represents the longitudinal or axial distance between the farthest and nearest parts of an object that are in sharp focus in the image of the object. In other words, it shows you exactly how big of a section you can see on the microscope from a certain object.
This depth of field is determined by the distance from the nearest sharp object plane to the farthest object plane.
The depth of focus represents the tolerance of placing the sensor in relation to the lens. To put it simply, it is determined in the image of the object which is located in the area behind the objective lens, as opposed to the depth of field that is determined in the area in front of the objective lens.
The depth of field depends on a series of factors, including lens aberrations, geometrical optics, the degree of eye accommodation, microscope magnification, and others. For instance, older people will experience a shallower or smaller depth of field than people in their 20s without eye problems.
Calculating the depth of field is done after various methods proposed by researchers, each using rather complicated mathematical formulas.
How to read depth of view
By applying one of the formulas for determining a microscope’s depth of field you can also use a simple technique to increase the depth of field. If you want to decrease the collection angle and, therefore, to reduce the numerical aperture, all you have to do is to reduce the condenser aperture by reducing its diaphragm.
The procedure will help you decrease your collection angle and the lighting beam will become parallel.
In the photography field, the depth of view is a common concept that allows professionals to play with different lenses and objectives to create amazing results. For example, wide-angle lenses and slow lenses with small effective apertures have a deep depth of field, allowing for amazingly clear and detailed pictures.
The depth of field is rather large in photography and usually measured several centimeters for fast long-focus lenses but can reach up to hundreds of meters for short-focus objectives.
However, microscope objective lenses are different and don’t usually feature the diaphragm mechanisms required to do so. There are only a few variable numerical aperture objectives that come equipped with internal iris diaphragms that allow you to increase the image contrast or axial resolution.
One easy way to do so is to make a DIY diaphragm using a thin piece of metal or a black piece of plastic. Alternatively, you can find a small washer to place in front of the microscope objective lens. The result is a reduction of the numerical aperture and an increase in the depth of field.
However, the amount of light you will receive on the image sensor or through the eyepiece will also be reduced. Overall, the results provided by the DIY diaphragm are visible with the naked eye, so you can enjoy an improved depth of field that will allow you to observe more details on the specimen.
To sum up, obtaining a high imaging resolution requires a high numerical aperture objective but one of the consequences is a fairly small depth of field. This emphasizes the quality and the performance of the focusing stage but will force you to constantly slide the specimen to notice other details from the fragment you’re researching.
Understanding how the field of view, depth of field, and magnification work is mandatory if you want to get the best results using a microscope. Also, even though they are based on the same principles, camera lenses and microscope lenses work differently so you cannot expect similar results using the same techniques for both devices.
Generally speaking, the bigger the magnification rate, the narrower the field of view, which means when looking through the lenses of a microscope, you won’t be able to see too much of your specimen. The depth of field is also altered by the magnification so, as the magnification increases, it is harder to focus on the specimen.
Although these struggles can be fixed with the help of DIY diaphragms, you will never obtain a clear image with the help of a regular optic microscope. Electron microscopes use different technology and can provide high-resolution pictures even when the magnification rate exceeds 100,000x.
However, most of these products cost a small fortune and are only available for specialists and scientists. They also need highly-skilled professionals to handle them and preparing the specimens might take days.
Therefore, you need to understand the limits of an optic microscope and work around its flaws when using the device for your research. With practice and the help of the right lenses, you can still use both alive and dead specimens and see every detail. By comparison, beam or electron microscopes can only use dead or dried specimens, and, as we previously mentioned, preparing them will take hours or even days.
Whether you’re simply passionate about observing microorganisms or you do that for a living, you need to use the right equipment and employ the proper techniques to get the best out of those specimens you want to observe under the microscope.
If you’re on the lookout for a quality lab microscope for sale, rest assured that the market is generous enough when it comes to such instruments yet if you’re new to this and you need to learn more about getting your sample ready and, especially, about staining, this post might be of help.
In order to observe microorganisms under the microscope, they need to undergo some processes. Growing the bacteria you want to observe is the first step to take and there are several ways and media used to do that.
Depending on the type of bacteria you’re interested in and your observation purposes, you can use one of the following: basal, selective, transport, or enriched media. These techniques will help you grow specific bacteria and/or prohibit the growth of others.
Once you’ve done that and made sure you took this step properly, you will have to get your microorganisms ready for observation and here is where the staining part comes into sight. Its main purpose is to highlight cells and their parts. Just as it happens with the growth techniques, staining can be done using various types of stain.
Bacteria and water have almost the same refractive index and they cannot be seen with the naked eye when under the microscope because they are almost invisible or opaque. Staining thus makes the cells and their components visible. The various substances used for staining adhere to the cell and give it color.
Without staining the bacteria, the microscope will be of little use. Therefore, the specimen to be examined must be fixed and stained properly in order to be visible. By staining them, their morphological features are highlighted for observation.
Depending on the type of stain and the bacteria used, you can observe cell walls and components in a way that will help you visualize metabolic processes, the number of cells within specific biomass, and so on. Not to mention that stains will also help you see dead cells and live cells.
As we’ve said before, the purpose of your examination will help you decide which type of stain to use. Since there are over 20 types of stains, it won’t be difficult to find one that will match your exact needs. You can thus find stains that will help you detect proteins and lipids or highlight spores. Of course, the purposes are various, hence the many stain types available.
However, not all stains can be used for living cells. The ones that can be used to observe living organisms, though, include toluene red, Bismarck brown, Nile red and Nile blue as well as fluorescent stains for DNA observation, just to name a few.
For example, Eosin Y is used by medical practitioners interested in conducting a PAP smear. When contacting cytoplasm, red blood cells, and cell membrane, this acid fluorescent stain becomes red. This stain type is also employed for testing blood marrow. What’s great about stains is that different types can be used for the same bacteria sample.
If you use eosin and hematoxylin, you will get a better contrast between the different parts of a cell. The eosin will make the cell turn red whereas the hematoxylin will stain the cell nuclei in blue. It will thus be easier for a medical practitioner to examine blood marrow samples and PAP smears when these two stain types are used together.
By opting for differential stains, that is, for two or more stains, the cells can be categorized into various types or groups. Even if both simple and differential stains allow for the observation of cell morphology, differential stains allow the observer to get more information about the cell wall.
A common type of stain used by hospital workers is Gram’s stain. This is employed to identify harmful microorganisms. The stain involves the use of different colorants that will trigger different effects on different types of microorganisms. There are three steps to take when using the Gram stain.
The first phase involves the use of Hucker’s crystal violet, a colorant that will make all the microorganisms in the sample turn violet. The second step requires the use of iodine to make the color adhere to all cells that are Gram-positive. These are primarily Streptococcus and Staphylococcus.
The stain will then be washed away and Safranin O will be added. This will enhance the contrast between the cells that are Gram-negative and the rest of the cells in the slide. Gram staining is of great importance in medicine as examining the bacteria using this staining method will help the observer know more about a bacterium’s susceptibility to certain antibiotics.
That’s why it is so commonly used. However, there are bacteria that cannot be stained using this standard laboratory procedure (Gram stain). Your observation purpose and the specimens to be examined will help you choose the right stain and method, though.
There are the so-called acid-fast bacteria that, because of the mycolic acid on their cell wall, are resistant to staining procedures. They are called acid-fast bacteria because of their resistance to decolorization with acid alcohol. For example, Mycobacterium tuberculosis is a Gram-resistant microorganism and thus it should be stained with an acid-fast stain.
This bacterium can be colored with the Ziehl-Neelsen staining which includes the use of red colored Carbol fuchsin to stain the microorganism and Malachite Green or Methylene blue as a counterstain to ensure a contrasting background.
Thanks to the phenol in the Carbol fuchsin used, the cell walls are solubilized. Heat should be used to help the stain penetrate the bacterium better, make it visible, and thus allow for proper observation.
Since there are different types of stains used for microscopy and different purposes for examining bacteria, the staining procedures employed can be different, too. The specimen that is being prepared for examination on the slide can be either dry-mounted or wet-mounted, smeared or sliced into a thin section.
If a stain is used, then the specimen should be wet-mounted. This procedure means that you should use a clean dropper to place a bit of water on the slide (just a drop), set your bacteria sample in the water, and then use a coverslip to cover it.
Use the dropper to apply the stain but place it at the corner of the slide in order for it to be drawn to the bacteria by capillary action. Use absorbent paper on the opposite side of the slide to remove excess water. Examine the specimen only when the stain has covered the entire slide.
Sometimes, when simple staining is required, the sample is immersed (before or after it has been fixed and mounted) in the dye solution, rinsed, and then observed. Of course, there are other staining procedures and techniques and your specific purposes and the specimens to be examined will help choose the appropriate one.
Regardless of the stain type and method employed, make sure you follow the required steps carefully. Not paying utmost attention can affect what you get to see when you place the sample under the microscope. A little mistake might render your sample useless.
However, when the sample is perfect for observation, the stain did its job and highlighted the cell and its parts, and you use a quality microscope, it is quite fascinating to see these tiny organisms and examine their morphology. One can almost never cease to be awed by these organisms and the forms life can take.