Light Microscopes that Detect Fluorescence: Videos & Practice Problems

Fluorescence microscopy utilizes ultraviolet light to excite specimens, causing them to emit visible light, creating vibrant images. Techniques like immunofluorescence allow scientists to tag specific molecules with fluorescent dyes, enhancing visibility. Advanced methods include confocal scanning laser microscopes (CSLMs) for 3D imaging, two-photon microscopes (TPMs) for deep tissue imaging, and super-resolution microscopes (SRMs) that achieve resolutions of 0.01 micrometers, enabling the observation of individual molecules. These technologies significantly enhance our understanding of cellular structures and functions.

Light Microscopes that Detect Fluorescence

In this video, we're going to begin our lesson on light microscopes that detect fluorescence. In certain situations, light microscopes that detect fluorescence or emitted light can be really useful. Fluorescence can be more formally defined as the ability to absorb short wavelengths of light and then immediately give off longer wavelengths of visible light. Now, fluorescent molecules will stand out as bright objects against a darker background. There are several different types of microscopes that can detect fluorescence, and we'll be able to talk about some of those microscopes as we move forward in our course.

If we take a look at our image down below at our map of the lesson on light microscopy, what you'll notice is that grayed out over here on the left, we have the things that we already covered, including bright field microscopes, light microscopes that increase contrast, including dark field microscopes, phase contrast microscopes, and DIC microscopes. Here in this video, we are focusing on the light microscopes that detect fluorescence, which moving forward, we're going to be talking about the confocal scanning laser microscope or the CSL microscope, the 2 photon microscopes, as well as the super resolution microscopes. Once again, we'll be able to talk about each of these different types of microscopes moving forward in our course. But for now, this here concludes our brief introduction to light microscopes that detect fluorescence. So, I'll see you all in our next video.

Fluorescence Microscopes

In this video, we're going to begin our lesson on fluorescence microscopes. Fluorescence microscopes are light microscopes that project ultraviolet light onto the specimen, causing the specimen to fluoresce or emit some form of visible light that can be detected. Fluorescence microscopy is able to create these awesome images that are very vibrant and very colorful. Some organisms have the ability to fluoresce naturally without any modification by the scientist. However, scientists can also tag very specific molecules inside or outside of cells with fluorescent dyes, also known as fluorochromes. By tagging these very specific molecules with dyes, it allows these molecules to fluoresce when they otherwise would not. A very specific technique known as immunofluorescence is a technique that combines a fluorochrome or a fluorescent dye with an antibody, a protein that will bind to very specific molecules to tag those specific objects and allow them to fluoresce. This allows scientists to even track just one specific type of molecule inside of a cell.

Notice down below we're showing you some images of fluorescence microscopy, which is able to create really colorful and vibrant images. Over here on the far left, what we have is fluorescence microscopy of human neurons where you can see these specific molecules being tagged with yellow, purple, and red fluorescent dyes. Here we have Caenorhabditis elegans, being tagged with some kind of molecule that fluoresces in blue color. Then here in the middle, what we have is Yersinia pestis bacteria, which is the plague bacteria that causes the plague. And over here on the far right, what we have is Escherichia coli bacteria and these two different strains of E. coli being tagged with these different fluorescent dyes.

This here concludes our brief introduction to fluorescence microscopy. But as we move forward in our course, we'll be able to continue to learn more and apply these concepts. So I'll see you all in our next video.

Confocal Scanning Laser Microscopes (CSLMs)

In this video, we're going to continue to talk about light microscopes that detect fluorescence by talking about confocal scanning laser microscopes or CSLMs. These confocal scanning laser microscopes or CSLMs are computer-controlled microscopes that couple a laser to a fluorescence microscope. The laser portion is responsible for generating high contrast, three-dimensional (3D) images that allow the viewer to access several different planes of focus inside of the specimen. This can be used to look inside of cells at different layers of the cell.

If we take a look at our image down below, we can get a better understanding of these confocal scanning laser microscopes. Over here on the left, notice that we're showing you that this is a complex device that is going to be controlled by a computer, and it links a laser to a fluorescence microscope. These are abbreviated as CSLMs for confocal scanning laser microscope.

Now over here, on the right, we're showing you some of the images that can be created. This is looking at different planes or different levels of the specimen. Over here on the left, we're showing you this Philampelanchi ramosa seed coat. This is showing you Arabidopsis thaliana cells, and here it's showing you rat pancreas cells. This is another form of being able to detect fluorescence using these CSLMs.

Once again, we'll be able to get some practice applying these concepts as we move forward. So I'll see you all in our next video.

Two-Photon Microscope (TPM)

In this video, we're going to briefly introduce two-photon microscopes or TPMs. And so two-photon microscopes or TPMs are similar to confocal scanning laser microscopes or CSLMs, except the two-photon microscopes, as their name implies, are going to be using two photons and less damaging light than the confocal scanning laser microscopes. Now two-photon microscopy is also known as multiphoton microscopy at times, and so those are referring to the same thing. And once again, the two-photon microscopes or multiphoton microscopes are going to be using longer, less damaging light wavelengths that allow for deeper imaging of really, really thick structures. And it also allows for a really cool feature of time-lapse imaging, which can create somewhat of a video-type effect. And so if we take a look at this image down below, we can see that the functions of the two-photon microscope, and the two-photon microscope is also abbreviated as just TPM. And it allows for deep imaging, somewhat similar to x-ray vision you can think of. And so notice here it's showing you mouse brain, blood vessels, and neurons, which can be thick structures, and it's able to do this deep imaging into those thicker structures and create these really cool-looking images. Another feature is that it's able to create these time-lapse images. And the time-lapse imaging once again allows for somewhat of a video-type effect. And so here it's showing you on the right-hand side, how the mouse brain blood vessels, and it's showing you that the black dots and lines show the red blood cell movement through the actual blood vessels. And so, ultimately, the two-photon microscopes can be used for deep imaging of thick structures and also to create time-lapse images. And so this here concludes our brief introduction to two-photon microscope, and we'll be able to apply some of these concepts as we move forward. So I'll see you all in our next video.

Which type of light microscope can show the movement of cells or structures in living specimens with time- lapse imaging?

• A. Confocal scanning laser microscope.
• B. Fluorescence microscope.
• C. Two-photon microscope.
• D. Differential interference contrast microscope.

Answer: (C)

Super-Resolution Microscope (SRM)

In this video, we're going to continue to talk about light microscopes that detect fluorescence by briefly introducing super resolution microscopes or SRMs. And so what's important to note is that up until 2014, when the super resolution microscopes were first developed, the maximum resolution for light microscopes before 2014 was only about 0.2 micrometers, which means that objects had to be at least 0.2 micrometers apart from one another in order to distinguish them as separate objects. Now, 0.2 micrometers might seem like a pretty good resolution. However, in comparison to super resolution microscopes, really 0.2 micrometers is nothing. And this is because super resolution microscopes, or SRMs, are fluorescence light microscopes with a very high resolution of about 0.01 micrometers, which means that two objects could be 0.01 micrometers apart, and you can still distinguish them as being separate objects, and so this 0.01 micrometer resolution is about 20 times greater or 20 times better resolution in comparison to other light microscopes.

Now, the super resolution microscopes, or the SRMs, use very complex mechanisms in order to visualize molecules that would otherwise be way too close together to be seen as distinct objects, and sometimes these super resolution microscopes can allow for even a single molecule to be tracked within the cell, and that is how great of a resolution these super resolution microscopes can provide, allowing us to see a lot more detail more clearly.

If we take a look at our image below, we can clear up some of these ideas. Notice on the left-hand side of our image, we're focused on the normal resolution fluorescent microscopy that would have existed before 2014, before the super resolution microscope was developed. Notice here we are essentially comparing the normal resolution fluorescent microscopy to an old television set, maybe from the '80s or '90s. The old television set is not going to provide as much detail, and notice that our cartoon character here is saying, "What am I looking at?". It's kind of difficult to see; the image is a little blurry, not so great of a resolution.

On the right-hand side of our image, we're showing you the super resolution fluorescence microscopy, which we're comparing to a flat-screen HDTV that's high quality and has a great amazing resolution. The image is going to come out much more crisp and much clearer. Notice our cartoon character is saying, "Oh, now I can see.". The bovine endothelial cell that's visualized using the super resolution microscope is going to show a lot more detail, a lot more clarity, and a lot higher and better resolution.

This here concludes our brief introduction to super resolution microscopes or SRMs, and we'll be able to get some practice applying these concepts and learn more as we move forward. So, I'll see you all in our next video.

Which type of fluorescent, light microscope creates high-contrast, 3D-images that allow the viewer to access several planes of focus in the specimen?

• A. Super-resolution microscope.
• B. Confocal scanning laser microscope.
• C. Two-photon microscope.
• D. Immunofluorescent microscope.

Answer: (B)

New and innovative technology, known as ______________________ microscopes, allow scientists to fluorescently tag and track single molecules in a living cell.

• A. Two-photon microscopes.
• B. Confocal scanning laser microscopes.
• C. Super scanning laser microscopes.
• D. Super-resolution microscopes.

Answer: (D)

Here’s what students ask on this topic:

What is fluorescence microscopy and how does it work?

Fluorescence microscopy is a technique that uses ultraviolet (UV) light to excite fluorescent molecules within a specimen, causing them to emit visible light. This emitted light is then detected to create vibrant and colorful images. Some organisms naturally fluoresce, while others can be tagged with fluorescent dyes, known as fluorochromes, to highlight specific molecules. This technique allows scientists to visualize and track specific components within cells, providing detailed insights into cellular structures and functions.

What are the different types of light microscopes that detect fluorescence?

There are several types of light microscopes that detect fluorescence, including:

• Confocal Scanning Laser Microscopes (CSLMs): These use a laser to generate high-contrast, 3D images, allowing for the examination of different planes within a specimen.
• Two-Photon Microscopes (TPMs): These use two photons and less damaging light for deep tissue imaging and time-lapse imaging.
• Super-Resolution Microscopes (SRMs): These achieve extremely high resolution (about 0.01 micrometers), enabling the observation of individual molecules within cells.

What is immunofluorescence and how is it used in fluorescence microscopy?

Immunofluorescence is a technique that combines fluorescent dyes (fluorochromes) with antibodies, which are proteins that bind to specific molecules. This allows scientists to tag and visualize specific molecules within cells. By using immunofluorescence, researchers can track the location and behavior of particular proteins or other molecules, providing detailed insights into cellular processes and structures.

How do confocal scanning laser microscopes (CSLMs) differ from traditional fluorescence microscopes?

Confocal Scanning Laser Microscopes (CSLMs) differ from traditional fluorescence microscopes in that they use a laser to generate high-contrast, three-dimensional images. CSLMs are computer-controlled and allow for the examination of different planes within a specimen, providing detailed 3D views of cellular structures. This capability makes CSLMs particularly useful for studying complex, multi-layered specimens.

What are the advantages of using two-photon microscopes (TPMs) in fluorescence microscopy?

Two-Photon Microscopes (TPMs) offer several advantages in fluorescence microscopy:

• Deeper Imaging: They use longer, less damaging light wavelengths, allowing for deeper imaging of thick structures.
• Reduced Photodamage: The use of less damaging light reduces the risk of photodamage to the specimen.
• Time-Lapse Imaging: TPMs can create time-lapse images, providing a video-like effect to observe dynamic processes within cells over time.

What is the resolution of super-resolution microscopes (SRMs) and why is it significant?

Super-Resolution Microscopes (SRMs) achieve a resolution of about 0.01 micrometers, which is approximately 20 times better than traditional light microscopes (0.2 micrometers). This high resolution allows scientists to distinguish objects that are extremely close together and even track individual molecules within cells. The ability to see such fine details significantly enhances our understanding of cellular structures and functions.