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Phase contrast microscopy is a powerful tool for the characterization of materials at the nanoscale. In this technique, a focused beam of electrons is scanned across a sample to produce a high-resolution image of the surface.
It can be used to study various materials, including semiconductors, metals, and insulators. Plus, it has many advantages over other methods of surface characterization, such as atomic force microscopy (AFM) and scanning electron microscopy (SEM).
Here’s an overview of phase contrast microscopy and its several uses.
The principle of phase contrast microscopy is based on the wave nature of electrons. When an electron beam is an incident on a sample, it interacts with the atoms and molecules in the material. As a result, the beam is scattered in all directions. A portion of the scattered electrons will be transmitted through the specimen. The transmitted electrons will interfere with each other, producing contrast in the image. Phase contrast microscopy presents the phase changes into amplitude changes.
To understand this well, you must know what a phase object is. It’s an unstained specimen that does not absorb light. That’s because it diffracts the slight light phase change. The light phase shifts nearly 1/4 wavelength in comparison to background light. Human eyes cannot detect phase differences. Our eyes only notice a change in the intensity or frequency of light.
Phase contrast microscopy produces high contrast images by increasing the light phase differences. In this way, it separates the background light from the light the specimen diffract. However, not all light passing through a specimen will diffract. The light waves that do not diffract create a bright image on the objective’s rear aperture.
Meanwhile, the waves that diffract are focused on the image plane, separating the diffracted and background lights. The contrast is further increased by using a condenser placed between the specimen and the objective. The condenser has a unique lens that produces a converging beam of light. It increases the amount of light diffracting through the specimen, resulting in a higher contrast image.
Phase contrast microscopy has many applications in cell biology, microbiology, immunology, and medicine. Here’s how this technique helps researchers in each of these fields:
In cell biology, phase contrast microscopy helps scientists study the internal structures of living cells. Likewise, a phase contrast microscope can help you look at living cells in culture.
It means you can study the cells without killing them first. Doing this is crucial because it allows you to observe how the cells change over time.
For example, suppose you’re studying the effects of a new drug on cancer cells. You can use phase contrast microscopy to see how the medication affects the structure of the cells.
Phase contrast microscopy is also applicable in microbiology. Researchers use it to study the structure and function of bacteria, viruses, and other microorganisms.
It helps us understand how these organisms cause disease and how we can develop new treatments to combat them.
Many immunologists use phase contrast microscopy to study the structure and function of immune cells. For example, they might use the technique to study how these cells fight off infections.
They can also use it to study the side effects of new drugs or treatments. For example, if you’re studying a new cancer treatment, you can use phase contrast microscopy to see how the treatment affects the structure of immune cells.
Organelles are subcellular particles that have specific functions. They include the nucleus, mitochondria, and chloroplasts.
Phase contrast microscopy helps scientists study the structure and function of these organelles. For example, you can use the technique to study how organelles change in response to a new drug.
You can adapt any upright or inverted light microscope for phase contrast microscopy. However, you must incorporate the following components.
A phase contrast condenser is an add-on condenser (not a replacement) that contains a particular phase annulus. The purpose of the phase annulus is to introduce a phase shift in light passing through it.
The amount of phase shift is a function of the medium’s refractive index and the annulus’s thickness.
Suppose a phase contrast condenser has a refractive index of 1.515 and a thickness of 4.5 millimeters (mm). The result is a light field shifted 180 degrees from the surrounding medium.
The shift creates a contrast between the light and dark areas in the specimen.
A phase contrast objective is an optical element that contains a phase plate. The phase plate is responsible for creating the contrast in the image.
The thickness of the phase plate is carefully designed to create a phase shift that is a function of the wavelength of light. The result is an image that has increased contrast and resolution.
The phase plate is in the rear focal plane of the objective. It is made of clear material with a refractive index different from the surrounding medium.
The difference in refractive index creates a phase shift in the light passing through it. Thus, the phase plate is responsible for the contrast in the image.
Phase contrast microscopy has various advantages over other microscopes. For example, it offers high-contrast images of living cells and can be used to observe cells in their natural environment. Here are some of these advantages.
Previously, scientists could only study dead cells under a microscope. But with phase contrast microscopy, they can now study living cells in their natural environment.
Using this technique, it is possible to study the behavior of cells and how they react to their surroundings. Moreover, this method can help analyze the development of cells and how they change over time.
One of the significant advantages of a phase contrast microscope is that it offers high-contrast images because this technique uses light interference to create the image.
The images are usually more precise and have more contrast than those produced by other microscopes. High-contrast images allow for better observation and analysis.
If you’re viewing a live cell, phase contrast microscopy can help you see the structure and movement that might be otherwise impossible to observe.
When you have to study a thin specimen, such as the cross-section of a cell, phase contrast microscopy is the ideal technique. The method uses light interference to create the image and doesn’t require staining like other microscopy methods.
It means you can study the specimen without damaging it.
A notable advantage of phase contrast microscopy is that it can be used in conjunction with other microscopy techniques, such as fluorescence. The combination of methods is called dual-mode microscopy or multimodal microscopy.
It gives rise to greater flexibility in the types of experiments that can be performed and opens up new possibilities for correlative imaging.
An example would be to image living cells using phase contrast microscopy to observe the overall cell structure and then to use fluorescence microscopy to target specific proteins within the cell for further study. Such imaging would not be possible with either technique used alone.
Similarly, other techniques such as DIC (Differential Interference Contrast) microscopy can be combined with phase contrast microscopy to provide even more information about the sample.
Modern phase contrast microscopes allow for capturing photos and videos of cells and other small objects. They do this by shining a light through the specimen and then projecting it onto a sensor.
The light has a particular wavelength that can highlight the differences in refractive index between the different specimen parts. That makes it possible to see things that would otherwise be invisible to the naked eye.
Some phase contrast microscopes also can take 3D images. These microscopes use a technique called interferometry. It involves shining two beams of light through the specimen simultaneously. The beams then interfere with each other, creating a 3D specimen image.
Most modern phase contrast microscopes work with CMOS or CCD computer devices.
Despite its many functions, phase contrast microscopy has a few shortcomings. Here are some of them.
The ring or annulus inserted in the objective lens to produce the phase contrast effect limits the amount of light that can enter the microscope. As a result, the image produced is not as bright as seen in a bright field microscope.
Since light waves are bent when they pass through a specimen, thicker specimens take longer to traverse the waves. It results in a decrease in contrast and resolution.
The halo effect or shade-off seen in some images is an artifact that is caused by the refraction of light. It can be minimized using a smaller aperture but cannot be removed completely.
The halo can make it difficult to see the perimeter of a specimen.
For some cells and structures, special dyes need to be used to appear in phase contrast microscopy. However, these dyes can be toxic and may alter the structure of the specimen.
If you have the necessary components, you can set up a phase contrast microscope in minutes.
There are a few things to remember when using phase contrast microscopy. The first is that you need to use special dyes that will not alter the refractive index of the specimen.
Plus, keep the light source as dim as possible. Using low-power objectives is also essential since the higher the power, the greater the chance of distortion.
Finally, always keep the microscope well-centered. You will not get high-resolution images if it’s off to one side.
The basic principle of a phase contrast microscope is to use the difference in refractive index between the specimen and the surrounding medium to create an image. The difference in refractive index produces a phase shift in the light passing through the specimen, which is then detected by the microscope and converted into an image.
Biotechnologists use phase contrast microscopes to observe living cells, as they offer a clear image without the need for staining. It is beneficial when studying delicate cells, as staining can often damage them.
Most phase contrast microscopes come with a green absorption or interference filter. The filter produces monochromatic light, which is necessary for the proper functioning of the microscope. Green light also has a shorter wavelength than blue or red light, which makes it easier to focus.
You can see any living organism with a phase contrast microscope, from bacteria to human cells. In the right hand, a phase contrast microscope can even help observe subcellular structures.
The amount of magnification you need for phase contrast microscopy depends on the specimen you are observing. You will need at least 1,000x magnification for bacteria and other small cells. For larger cells, such as human cells, you will need at least 400x magnification.
Yes, a phase contrast microscope can be used to see Golgi bodies. The Golgi apparatus is an organelle responsible for packaging and transporting molecules within cells. It is often seen as a series of stacked discs appearing dark in phase contrast microscopy.
The resolution of a phase contrast microscope can be up to less than one angstrom. That’s smaller than 0.1 nanometers. It is the highest resolution in microscopy.
Phase contrast microscopy is used in immunology, cell biology, microbiology, and other scientific fields to study living cells without staining. The components of a phase contrast microscope include a light source, a condenser, an objective lens, and a phase ring.
The resolution of a phase contrast microscope is exceptionally high, making it possible to see subcellular structures. Thus, phase contrast microscopy is a valuable tool for all biological studies, from microorganisms to humans.
Featured Image Credit: Catalin Rusnac, Shutterstock
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Jeff is a tech professional by day, writer, and amateur photographer by night. He's had the privilege of leading software teams for startups to the Fortune 100 over the past two decades. He currently works in the data privacy space. Jeff's amateur photography interests started in 2008 when he got his first DSLR camera, the Canon Rebel. Since then, he's taken tens of thousands of photos. His favorite handheld camera these days is his Google Pixel 6 XL. He loves taking photos of nature and his kids. In 2016, he bought his first drone, the Mavic Pro. Taking photos from the air is an amazing perspective, and he loves to take his drone while traveling.
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