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Microscopy School Lesson 5 - Principles of Fluorescence Light Microscopy

Using transmitted light microscopy researchers can have an overview of the morphology of the specimen. However, the localization of specific structures and proteins inside the cell is not possible. Fluorescence microscopy came to fill this gap introducing the use of chemical dyes that allow the localization of intracellular structures. 

These dyes can be coupled to antibodies to recognize specific proteins, or have affinity for specific cellular structures such as DNA, mitochondria, etc. The discovery of fluorescent proteins pushed even further the development of fluorescence microscopy techniques and applications. Fluorescent proteins opened the door to live-cell fluorescence microscopy and its derivatives, such as FRAP, FRET, Photoactivation/Optogenetics, etc. Fluorescent proteins are also commonly used in other technologies that require more complex hardware such as TIRF.  

To be able to understand the full potential of fluorescence microscopy techniques and possible applications, it is crucial to have background knowledge of what is fluorescence and fluorescence microscopy. In this webinar, Dr Florindo will present the concept of fluorescence, and how is this physical propriety is applied to microscopy. We will give a brief overview of the hardware required for a fluorescent microscope, as well as discuss some concerns that should be considered when starting a fluorescence imaging protocol.

Overall, the goal is that individuals that attend this webinar gain (refresh) their knowledge about fluorescence microscopy potential. We hope to arm researchers with better tools to plan their work.

Learning Objectives:

  • Understand the principles of fluorescence.
  • Interpret the spectra of a given fluorophore.
  • Acquire an overview of the hardware  required for fluorescence microscopy.

Questions Answered:

  • How to choose a fluorochromes to avoid spectrum overlap?
  • Which filter should be chosen to image a given fluorophore?
  • What is photobleaching?

I am Claudia Florindo, product specialist for Life Sciences at Andor. On today's webinar, I will explain how fluorescence works, what is the hardware required to do a fluorescence microscopy experiment and to acquire the images, what are the caveats in fluorescence microscopy, and then how to set up and design your own experiments. So let's start from the beginning. What is fluorescence?

Fluorescence is the emission of photons by atoms or molecules whose electron were transiently stimulated to a higher energy state and went from the ground energy level to a higher energy level. Once they go back to their ground state, they will emit a photon and this is fluorescence. We have here the representation of an atom and you have the nucleus and the electrons going around. And once there's external radiation coming in, the electrons go to a higher energy state, and then they go down to the lower energy state, and they will emit photons. The energy released when the electrons go back to the ground level is always lower energy than the energy that came in to excite the electrons. And for this reason, this photon will have a wavelength, a corresponding wavelength longer than the incoming wavelength of light.

So, when the incoming light ceases to exist, fluorescence emission will automatically cease to exist. Molecules that fluoresce are called the fluorochromes. And in fact, in microscopy, we already in fluorescence microscopy use fluorochromes in which their absorption and emission spectrum are extremely well characterized. So, for a given fluorochrome, we know that it will absorb light at a certain wavelength. So, we know which light should we excite it with, and we know also what is the emission wavelength for the specific fluorochrome so we know which filter should we use to capture the light that comes from this fluorochrome. And these are the maximum absorption and emission peaks of a given fluorochrome.

The stokes shift is the difference between in nanometers the absorption and emission peaks of a given fluorochrome. As for the fluorochromes, there are several features that will actually affect the brightness of the fluorochrome such as the chemical environment, the pH, the redox potential, the ionic strength, etc. And there are several features that are extremely important in the fluorochromes. One of such is the quantum efficiency, which is the ratio of the photons absorbed versus the ratio of the emitted photons. It means that the highest the quantum efficiency, the better this fluorochrome, this given fluorochrome is in to convert the absorbed light into emitted light, so the brighter it will be.

The resistance to quenching. So, quenching is a process that decreases the fluorescence intensity of a sample. Several molecular interactions can result in quenching such as molecular rearrangements, energy transfer and ground state complex formation. So, if the fluorochrome becomes quenched, he will decrease a lot the emitted fluorescence and also the resistance to photo bleaching. Photo bleaching is the irreversible damage of the fluorescence irradiation by a given fluorochrome. So, when it's photo bleached, it will emit fluoresce, it will no longer emit fluorescence.

Here's an overview of commonly used fluorochromes in life sciences. We have chemical dyes such as Alexa, Hylite, and cyanine dyes. And the examples of such are here is DAPI, Acridine orange, and Oregon green. And the biological dyes that most of them derived from GFP, although currently there are other organisms who produce these fluorescent proteins that have evolved to supply us with a palette of rainbow colors. Talking a bit more on fluorescent proteins, as I just mentioned, there is a palette of colors for fluorescent proteins that are available for researchers from which they can do multiple color experiments using those fluorescent proteins.

Most of them are derived from the original GFP which stands for green fluorescent protein, but some of them actually are derived from new proteins discovered in other organisms. But there are more to fluorescent proteins than just, which is already a lot, shine in all the colors of the rainbow. There are photo convertible fluorescent proteins. And these are proteins that start to radiate in light in a given wavelength. But once excited, change to another wavelength of emitting light. And that allow to, in a given experiment, specifically track a super population of cells at a single time point.

Other examples are photoactivatable fluorescent proteins that do not shine any light up until the moment they are irradiated and activated. Or even photoswitchable fluorescent proteins in which the protein starts to have one emission wavelength such as green. Once irradiated, change its emission wavelength and turns into red, and further irradiated, it will become green again. And these are several examples of fluorescence proteins that can be used to multiple applications, such as CRISPR technologies, optogenetic tools, and so on.

And now, going on to the hardware of the EP fluorescence microscope. We start with the light source. There's a light source that will emit light of several wavelengths that are appropriate for fluorescence microscopy. But then, we will need a filter cube to select the appropriate wavelength. How does the filter group works? It will have an excitation barrier, a dichroic mirror, and an emission filter, the excitation barrier or excitation filter. Then we have the objective, the two blends and the detector that can be most of the times is a camera. And here we have an example of an Andor camera. So we have the light source. The light source will emit light of multiple wavelengths, but we only want to excite with the blue light.

We have an excitation filter that will select only the appropriate wavelength to reach the sample. It goes into the objective, which is the sample, and is reflected back with another wavelength, a longer wavelength, less energetic light. The dichroic who reflected blue light will now allow the passage of the green light, and the emission filter works as a further barrier to further select the wavelength that we will want to acquire. As for light sources that can be used in fluorescence microscopy, we have mercury arc lamps, xenon arc lamps, metal halide lamps, or LEDs, which are currently much in use due to their lower energetic radiation. They are very useful for fluorescence microscopy. Or if you use a multimodal system such as a Dragonfly you can use the Integrated Laser Engine, the ILE, for laser widefield illumination.

Looking a bit further inside of a filter cube. The main components of the filter cube are, as I explained previously, the excitation filter, the first barrier that will select the desired wavelengths from the light source, the dichroic mirror, which will act as a mirror that will reflect the desired wavelengths to the sample but then will allow the passage of the emission light to the excitation filter and to the detector. And then the excitation filter works as a further barrier to select the exact wavelengths that we want to target with that specific excitation. So, explaining a bit more in detail and in terms of graphics and wavelength, what is the filter set and how does it work?

The filter set as I just previously mentioned and will stress again will allow the excitation of the sample with specific wavelength, and also will allow the detection of specific wavelengths that were emitted from the sample after the excitation. In graphic terms, we have the excitation filter who selects the passage of a certain wavelength. And we have here the dichroic mirror, who reflects the wavelengths that are below this line. We imagine this would be 400 nanometers, below 400 nanometers, but allow the passage of wavelengths that are longer than these ones. And here the emission filter, which will work as a further barrier to select from the emitted wavelengths.

In summary, the excitation filter specifically allows the passage of the excitation wavelength light. The dichroic mirror will direct the excitation wavelength light to the sample and will specifically allow the passage of the emission wavelength light, and the emission filter will further stringe the passage, the...of the light that will reach the detector. So, different types of filters are named differently according to their capacity to discriminate between different wavelengths. Excitation filters are generally short pass filters because they allow the passage of shorter wavelength. Emission filters are generally long pass filters allowing the passage of longer wavelengths and blocking shorter wavelengths. And dichroic filters or splitters reflect some wavelengths, while allow the passage of other wavelengths.

There are also narrow band pass filters that will block shorter and longer wavelengths, allowing only the passage of a narrow band of wavelengths. And by using the characteristics of different filters and dichroic mirrors, we can create a specific set of filter sets that is adequate to perform multicolor fluorescence imaging experiments. Two basic types of filters can be used, single and multiple color filter sets. Single fluorescence filter sets will enable higher signal to noise ratio but slower imaging, whereas multicolor filter sets will enable faster imaging but lower signal to noise ratio.

And now talking about an extremely important part of the microscope, which is the objectives. It is essential to bear in mind that all things in the microscope matter. And for example, if you plan to use DAPI and you have a UV filter, a filter that is specific to visualize DAPI and UV dyes in your microscope, and you stained your sample in DAPI and then you go to the microscope and you are unable to see. Maybe your staining didn't work. Okay? But it could be another thing. What if your objective is unable to transmit UV dyes, UV wavelengths? If that's the case, you will never see DAPI with that objective. So you need to be aware of the hardware you're using and adjust your experimental settings to the specific hardware.

So, types of objectives are achromat, fluorite, and apochromat, among others, but they will differ on the type of corrections that are offered, and with such, on the type of imaging that it can be delivered. Higher NA will correspond to higher resolution. NA means numerical aperture, which is the ability of the objective to capture a wider angle of rays of light. I have discussed already transparency to UV light, but other characteristics important for objective performance that you should be aware of when you're thinking on objectives is if they are made out of low fluorescence glass.

Also, if they are able to deliver transmitted light techniques that you need, which type of corrections do those objectives have such as fluorite and apochromat is a plan which, meaning, that they are corrected for flat field and also if they are corrected for the chromatic plane shift. Objectives in a fluorescence microscope also act as a condenser.

I will now talk about cameras. I will give a brief overview of cameras. If you want to have broader insight into cameras and detectors for microscopy, please see the previous talks on this course. So, the question is, how about cameras? Are the cameras in fluorescence microscopy color or black and white? Well, you have the answer here. In fluorescence microscopy, the most common are black and white cameras. But then we have these beautiful, multicolor images. How can we have these beautiful, multicolored images if the cameras are black and white? So we have these three colored images. But in fact, what we do when we are acquiring is we acquire a micro tool grayscale image, a simple grayscale image, and the DNA grayscale image.

But then, I will, the camera, the software will transform all these grayscale images that are mapped to 65,000 shades of gray, if it's a 16-bit camera, into 65,000 shades of green, of red, and of blue, and the color will appear. And there we have our multicolor images. And why would we do all of this? Why wouldn't we acquire immediately in a color camera? So for the color cameras, imagine this is the chip of a 16-pixel color camera. And normally, to match our eyes in a color camera the...all the pixels are masked with a filter, with a color filter that can be green, red, or blue. And to better match our eyes, if this was a 16-pixel camera, from those 16 pixels, half of them, as in 8, are green, 1/4 are red, and 1/4 are blue. This will turn that when in...irradiated to detect the light from 16 possible pixels of resolution, we would only...can be able to detect eight in green and four in red, in blue respectively. Therefore, we would have less resolution on a color camera than if all the pixels would be detected for each color individually. On the other hand, if I have a black and white camera, and I have the light source, it goes to the sample, it goes into the detector, all my pixels will be used to capture the green light, the blue light, and the red light. And therefore, I will have more resolution in a single image.

So, caveats in fluorescence microscopy. Caveats in fluorescence microscopy include autofluorescence, the bleed-through effect of a fluorescence filter set available, which is the fact that the fluorochrome that emits and is captured by the green fluorescence filter set could also be captured by the red immediately adjacent filter set. And this was an example with green and red. But what I mean is that the light goes from a fluorochrome, should be captured by filter set A, is captured not only by filter set A, but also by the adjacent filter set B. Dye photobleaching is the irreversible damage of a fluorochrome and lifecell phototoxicity. Lifecell phototoxicity is exactly what the name suggests, meaning that the cells die in a live imaging experiment due to the high energy radiation that is toxic for them.

Causes of autofluorescence will include the autofluorescence of endogenous molecules, the use of a filter set that is not ideal for the given experiment, and the reactivity to the fixative used. People need to bear in mind, it's ideal to bear in mind that different fixatives will cause different autofluorescence. So, one, we'll need to choose the best fixation procedure, two, proper fix the proteins that we need to visualize, but also if possible to reduce the autofluorescence. And also scattering of the light in the optical pathway will cause autofluorescence.

Talking a bit more about the bleed-through effect. The bleed-through effect is extremely relevant when imaging multiple fluorochromes or fluorescence proteins since the signals from one specific structure or protein will be captured in the adjacent filter set and will mask signals from both proteins or structures. Causes of bleed-through include a non-ideal filter set in which the bandpass wavelengths are quite close and not optimized to the fluorochromes used, and also as related, a non-ideal fluorochrome choice for the experiment or the microscope setup.

Possible solutions are, reduce the exposure time. If you reduce the exposure time you will minimize the bleed-through effect. Use high specific filter set with quite narrow bandpasses. But there might always be some signal crossover so please perform the controls of your experiment to ensure that you do not have bleed-through or that you know the amount of bleed-through at which what exposure you will have bleed-through from one channel to the other. Talking about dye photobleaching. So, the dye photobleaching, as I mentioned before is, the reversible damage of a fluorochrome, and here you have an example of dye photobleaching of a sample that after 10 laser scans nearly stopped irradiating any light, and therefore it was virtually invisible to the detector. So, please be aware and avoid photobleaching.

A bit more on dye photobleaching. So, dye photobleaching is also, and not surprisingly, extremely important in time lapse microscopy. And here is an example of the first time frame of a movie, and the final time frame of the movie where you can barely see the nuclei that was stained with histone GFP. So, a bit more on dye photobleaching. It is caused by an increased exposure of fluorochromes to light. This exposure will lead to the formation of radicals that will cause modification on the molecules of the fluorochromes, and they will in turn consequently fluoresce less. It will result in a transition from a singlet state to a triplet state. And extremely important, photobleaching is irreversible. So, once we bleach the fluorophore, it will not shine anymore. So avoiding photobleaching. To avoid photobleaching, please use the most photostable dye possible.

Reduce the oxygen in your sample. And how can you do that? You can use nitrogen and use oxygen scavengers. Use anti-fading reagents in the embedding media. And not surprisingly, reduce the exposure time that you use to acquire your images. Nevertheless, as anything, if used to our advantage, can be a good thing. So not all is bad in photobleaching. There's a quite famous technique that allows multiple application and the study of the dynamics of the diffusion of molecules, of vesicle transport, of transport along the microtubules, which is named FRAP. And FRAP stands for fluorescence recovery after photobleaching. It's a technique that allows to visualize how fast does a cell recover fluorescence at the specific area of the cell once it's irradiated with so much light that all the fluorochromes in there are bleached.

And this allows to determine the diffusion coefficient of molecules from one region to the other region of the cell. And here is an example of FRAP using Andor Mosaic where these specific regions were bleached. And you can see they turn black. And then you can see the diffusion of molecules from other places of the cell into the black regions. Moving on, on caveats in fluorescence microscopy, I will now talk about live cell phototoxicity. It's not surprising that the light sources that transmit UV light will cause damage to the cells that we are trying to image since we all know that we go out into the sun without our sun blockers, we will get sunburned. The cells can also get a kind of, not sunburns, but high, energetic light burns and we need to avoid this when performing live imaging.

So, what are the problems? Some of the problems are that the filters and dichroic mirrors used are not totally efficient in blocking UV-like wavelengths. And this inability to block those wavelengths will cause damages in the cell walls, cell membranes and will lead to rapid cell death. The solutions for such a problem include reduced effect of UV radiation using UV filters, shorter exposure times, and balanced the redox environment. Use laser widefield elimination which will selective illuminate the sample with only the desired, chosen wavelength line. And, importantly, use longer wavelengths that are less energetic. If possible, use near infrared wavelengths. It will avoid UV. And it's on the other end of the spectrum.

Near infrared wavelengths are longer wavelengths and less energetic. So, this is better to the cells, healthier, and it also provides the advantage of allowing more deeper penetration into the samples. And most of the molecules that cause autofluorescence do not fluoresce on the near infrared wavelength. This actually is starting to be quite a good bet to start to use this wavelength. And more recently, there are already a number of fluorescent proteins and fluorochromes chemical dyes that can be used using those near infrared wavelengths. And, importantly, if you are at the point of choose, choose a system that is compatible with live imaging experiments. One of such systems could be, for example, a dual microlens spinning disk system.

And now, talking about the setup and design. When you're setting up and designing an experiment, you need to consider what microscopes do I have in my institute? What filter sets do those microscopes have? And also, do not...do not forget, as I mentioned before, what objectives are in there? What are the laser lines available? And extremely important, what do I want to do? So, what do I want to do? Do I want to image a live sample or a fixed sample? Do I want to perform a multicolor or a single color experiment?

And when doing such experiment, how am I going to do the staining? I will use fluorescence antibodies, fluorescence proteins, or quantum dots. What am I going to use? I need to select the appropriate fluorochromes or fluorescent proteins that are able to be seen by the equipment that I'm going to use. I need to design the sample preparation protocol. And extremely important and sometimes disregarded, before going to the microscope, design the imaging acquisition protocol. How do I want to acquire my images?

In summary, in this talk, I have walked you through on how does fluorescence work, what is the hardware required for EP fluorescence microscope, the caveats in fluorescence microscopy, and things to consider to set up and design a fluorescence experiment. So now, I just have to thank you all for listening to this talk. I would actually invite you to follow up on our Andor Microscopy School series. Coming up next, you have classes on confocal microscopy, and immunohistochemistry sample preparation. Thank you very much for listening, and I hope you can join on further classes of this course.

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