Microscopy: Introduction to Fluorescence Microscopy (Nico Stuurman)

so much of the microscopy that we're doing nowadays is fluorescent microscopy and I'll be explaining what that is in this lecture so why do we use fluorescence in the microscope and as you have probably seen so far far there there are two important aspects uh in making a great picture in your microscope one is resolution you want a high resolution and the second thing is contrast you want high contrast otherwise you wouldn't see anything and the uh what fluorescence offers is this high contrast so it's demonstrated in this image here we have um part of

the cell labeled in Green in green we're looking at microtubes so we labeled microtubes with a fluorescent D and we have part of the cell labeled in red and that part is actually uh the nucleus it or it was DNA it's a red label attached to the histone protein and what you see here is that this label stands out on a black uh background so we have a very high contrast we see what we want to look at whereas our background is is totally absent so it gives an actually extremely high contrast then what we

also already see in this specific image is that you get specificity we can attach these fluorescent labels to specific proteins to specific molecules to specific dyes to specific places in the cell or wherever we want them so we can Target them that they show what we want to see then fluorescence is quantitative so if I calibrate my system correctly um areas where we have higher signal like here uh those are areas where I have more of my uh the protein that I'm interested in so in this case there's a lot more tubulin here in these

bride area than here in these dimmer areas and then lastly and very importantly fluorescence is compatible with imaging of living uh cells so this image here is not only a still image but it's actually part of a sequence of images that I took and when we now start playing those faster we see that it's actually a dividing cell so uh the micr tubles assemble in this H spindle and that spindle then pulls apart the chromosomes into two daughter nuclei so fluorescence is a great thing great thing for microscopy but what is it really and what's

kind of surprising to me still is that humankind has been aware of fluorescence for only about 150 years so one of the first descriptions of fluoresence was by Sir William herel he was studying the aspect of an extract of the bark of the Kona tree and he held up this extract in the light of the Sun and that came through his window and he noted that the light of the sun um induced a Celestial blue light coming out of that extract of the bark of the Kona tree and I will now try to repeat that

150 year old experiment here in the lab and be showing you that now so the bark of the kinona tree is uh hard to come by but luckily the active ingredient can be found in every supermarket in the form of uh contained in tonic water and so I'll take this tonic water pour it in a glass and then the other part is that we need uh UV light to illuminate it and that you can buy in the form of what's called the urine tracker in the pet store and so when we now shine this UV

light in the quinine what in tonic water and you see that uh you get a blue light emanating from within the tonic water it's a another wavelength it's a longer wavelength than this light with which I illuminate and that's why we see it a lot better our eyes are not very sensitive to this UV but they are highly sensitive to the blue light in the tonic water quinine is not the only compound that fluoresces of course otherwise it will be quite interesting already in the uh 19th century uh scientists synthesized this die called fluorescene that

is highly fluorescent and when I shine blue light in that we get yellow fluorescence coming out and another very well-known dye is rodamine uh and when you shine green light in that you get this orangey yellowish fluoresence coming out and so this is fluorescent okay so what we have seen is that we use light of a lower wavelength so in this case of the quinine from the extract of the bark of the Kona tree we use UV light and then the Dy responds to that light by emitting light of higher wavelength um so in this

case blue light so in general uh except for very special cases emission light is of a longer wavelength and it means lower energy so there's some energy loss going on in this process than the excitation light now this aspect of fluorescence is very important and we often uh show that uh by measuring the Spectra so we measure an excitation Spectrum which is often very similar to an absorption spectrum and we measure an emission spectrum so here that's this curve here um which is the fluorescent light emitted by the Dy so we characterize these dyes by

their excitation maximum and by their uh emission maximum those numbers tell you immediately uh a lot about how to use that die in your actual experiments the difference between the excitation and emission maximum is called The Stoke shift after sir Stokes who first realized that there was this difference and you can have dyes that have a small Stoke shift where the emission is very close to the excitation or you have dies with a large Stoke shift where the emission is very far away from the excitation light now so there's energy loss going on and that

causes this difference in wavelength but what how does that happen and one of the most useful ways of thinking about that is through these so-called uh jablonsky diagrams jablonsky was a Polish scientist who did this work at the beginning of the 20th century and in these jablonsky diagrams we use um we're we're looking at the uh the orbitals that electrons can be in when an electron now absorb or when a Dy absorbs light that electron is going to be kicked in an higher orbital in a higher energy State this absorption goes very very fast so

it happens at a time scale of ftocs and um fto seconds to me don't mean much so I like to express this in the distance that light uh travels during that time period And since light goes extremely fast to me it's pretty amazing that light travels only like3 microns during this time period once that electron finds itself into these higher orbitals through a process called internal converion version it quickly falls back to kind of what you could call the ground level of the excited state and that internal conversion is a tiny little bit of heat

that dissipates this way so we have a bit of energy loss here and that happens on the level of uh time scales of Picos seconds and light would travel only3 mm during that time period now then the electron can hang out here for what is uh in comparison to this previous time skills almost uh eternity uh something on the order of many uh nanoseconds and then it will fall back to the real ground state and emit a photon and that Photon will be of a wavelength um uh less than the wavelength that was sorry of

an energy that is less than the energy that was put in uh initially and that means that that Photon that's coming out is going to be of a higher wavelength now uh not only uh this return to the ground state is not the only process that can take place when a die is in the excited state it's also possible that the energy is transferred to other molecules or even it can return to um the electron can fall back to the ground state but not uh emit a photon so there's also nonr non-radiative Decay so the

we have a uh a total um number of events some of which result in light coming out some of which that don't and we express The Rao of those two as the quantum efficiency and the quantum efficiency is uh a very important parameter of the die it not only depends on the D itself it also depends on its environment for instance what other molecules are uh uh around the Dy but nevertheless Quantum efficiency is something you want to know and that you want to measure or that you want to be told what it is another

important aspect is the brightness and the brightness in the end is the uh uh uh is determined by how well a Dye absorbs the excitation light and what fraction um of the uh uh excited state returns to the ground state under emission of fluorescent so it's it's determined by both the absorption coefficient and the quantum efficiency now um another important aspect here is what we already talked about how long does it take between absorbing a Dy and for the Dy to return to the ground state and that is what we call the lifetime and so

in it's mainly determined by how long it takes before we return to the ground state out of the excited state so usually this is on the order of nanoseconds nevertheless it's also um a parameter that is DP specific that is also dependent on the environment of the dice and there's a whole form of fluorescence microscopy called lifetime microscopy that actually reads out lifetime instead of only reading out the fluorescence uh uh itself nevertheless what we're now going to talk about is microscopes that discriminate between the exitation light and the emission light so how do we

do that in a microscope well the main thing is that we use filters so we use filters that filter the exciting light so that we get out of our light source only the light that is of the wavelength that will excite our die that light will bounce off the dichroic mirror and then excite uh our sample after it went through the objective the sample will start fluorescing and that fluorescence then travels back through the same objective it will now pass through the dichroic mirror and then there's an emission filter that will let through the fluoresence

but that will completely block any scattered excitation light so that we will only see the fluorescence on our detector which I symbolized here with an i but that could also be a CCD camera or whatnot and so this is the simple basic principle of a fluorescent microscope so the most important aspect here and the thing that makes it different from a normal microscope is filters and these filters need to be very good because they really need to discriminate this excitation light from the emitted light so what options do we have there well you can have

uh filters that are based on absorption that absorb C certain wavelengths of light um those uh are not that great in prus because their Spectra are very broad so they will also uh absorb a bit of the emission light if they um so they they will not exactly do what we want them to do and the type of filters that we mainly use these days are interference filters so what are those interference filters are basically thin alternating layers that have different um refractive index so that we get um interfaces there that can reflect the light

so you can almost think about them as thin transparent layers that are about the wavelength of the light or um half the wavelength of the light with semi-reflective coatings in between so what now happens when light hits this filter is that part of it will be reflected reflected part of it will move through the light that reflect will interfere with itself and when the wavelength is such that it uh matches the wavelength of the light you will get constructive interference and that constructive interference will then in effect let the uh light pass through this filter

whereas when the wavelengths are different from the two um the distance between the layers we get destructive interference and it will be reflected out now the filter manufacturers can completely compute how these layers will behave so they can design in the computer what wavelengths this filter should pass through and what wavelength should be reflected Ed and you can basically order any type of filter that you may possibly need now just to make this a little bit more clear there's one example of interference that you're highly aware of and that every one of us have seen

and that is what you see in soap bubbles so in soap bubbles when light hits that you will see reflection of all kinds of colors of light the soap bubble itself has a thickness that is on the order of half the wavelength of uh a lot of the visible light um and you will get this same interference effect where some of the light some of the colors will pass through and other colors will be reflected and that gives these beautiful uh colored patterns now we will um break and show you some of these filters and

we'll also show you now here how these filters are assembled in a filter Cube okay so I have an example here of uh such an interference filter this one is a called a HQ 47040 and the 470 is the center wavelength of this filter and the 40 means the bandwidth so this one will pass uh blue light and so if I have my blue LED and I hold that up I see the blue light coming through and you will will see that this led indeed passes it through now when I would have a green light

source and hold that up and go through this filter you will see that it gets a lot dimmer and not only that you may also notice that it's actually a little bit of blue coming through and that is because this green LED is not purely green but also has a slight bit of um blue uh light in it then you know the next part is this D mirror so this one is designed again such that it um reflects blue light so you will now not see coming anything through but if I reflect it to you

you may be able to see that and then the last part is this emission filter that will transmit the green light and um uh reject the blue light so in effect when we would now place these three in this configuration in the microscope illuminate from this side go up we have the objective and the sample here come back through and go through this uh emission filter that's the principle of a fluorescence microscope these filters this is exactly the way they are arranged in these cubes that were designed by Plume where we have an excitation filter

a diic mirror and then an emission filter here and so these will as a expected um reflect and I'll now turn it to you so that you can see it so the exciting blue light will be reflected to the specimen the specimen will flues green and that will make it to the eyepiece or to the camera sensor so we've seen now these filter cubes there's an excitation filter there's a dichroic mirror and there's an emission filter what's very important important to know and the information you should always have about these filters is their Spectra so

what are the wavelengths that are being transmitted and what are the wavelengths that are being reflected and those Spectra then will uh look something like as follows so we have here an excitation filter that passes through this uh this band pass these wavelengths these colors of light that dichroic mirror is made such that it will uh reflect everything that is lower than a certain cutof band and pass through everything that is higher and then the emission filter will pass through the higher wavelengths but reject absolutely reject the excitation light so when you're you're when you're

now a symol a fluorescent microscope your task will be to select filters that match well the the dice that you'll be working with and what you will be doing in that case is to try to Overlay the spectrum of the fluo force with the spectrum of your filters so I have here a spectrum of a Dy that is um probably S3 the cyanin die um it excites in the the the green area so around 550 and it has an emission maximum around 600 so if we now use a b pass filter centered around 650 nanom

that will easily uh reject the excitation light because it's far away from that but you also note that the integral of it that it only passes through a fraction of the emitted fluorescence and that means that you're losing you know something like 75% or so of the emitted fluoresence and in general you really want to get all the photons that you can so it's not a perfect situation so if you now switch to a b pass filter that is slightly lower that has that is around um centered around 610 nanometers then you're catching a much

larger fraction like 75 or 80% of the emitted photons and and you'll be much happier because your image will be much brighter now we'll now uh switch to the lab and show how we're using these filter cubes to collect actual images here we have a real fluorescent microscope so we have a light source back here this is a Mercury Arc lamp there more in a tip on light sources about that that light then passes through the main body and then hits here the dichroic mirror so and I'll show you now how this works so this

is basically a carousel that you can take out and this Carousel contains or turret contains these same filter cubes that we have been looking at in the lecture so this one is sitting here the light hits the excitation filter I can take these cubes out like this so the light hits the excitation filter was sitting in the microscope like this goes through the exitation filter hits the mirror goes up the fluorescence light comes back makes its way through the mirror and then here at the bottom we have the emission filter that goes down and in

the end to the camera or the eyepiece and now since this thing is motorized once it's sitting in the microscope it can with a push of the button put in another of these filter cubes and these cubes have been selected to uh visualize the dice that we're interested in and so usually there's one for things like dappy for things like gfp or fluorine and a cube for something like mcherry or rodamine okay so when I now put this back into the microscope and then open the shutter here in the back that um uh blocks the

light at the moment we get blue light coming out of the objective then when I with a push of the button I put now another dichroic in place here we have green light for rodamine or M cherry and if I go on we get like red light for something like uh a like s five and so just by switching these we can select the wavelength of the light for excitation and uh the filter used for emission and select the Fluor that we want to image so we've now seen how we can use the fluorescent microscope

to collect individual channels images of individual flu Force then what we can do in the computer is put all those images together give them a nice color and end up with things like this so here we have a staining of mitochondria in red of DNA in blue and what looks like actin stress fibers here in in green this works very very well there are also uh alternative strategies to doing this and one is what you may have noticed in the lab part is that it actually takes quite a bit of time to move that turret

so going from one position position to the other takes you know quickly like half a second or so the turret is pretty big so it has a lot of vibrations if you want to go faster the whole thing starts moving more those are all bad things and things you don't really want so instead of using these filter turrets what you can do is put your excitation and possibly also your emission filters on a filter wheel now I brought such a filter wheel and um can show that to you here so it really is a wheel

that has individual filters in them there's a motor that can through this rubber band here moves that around and we can put by activating the motor we can switch between these different filters and we can do that at a speed that generally is much higher than you can do on a turret so we then first take an image where we put the first excitation filter in place so for instance in this case we have a blue excitation filter we get blue light out we get green fluoresence then when we move the filter wheel to the

next position which has a yellow filter we get red fluorescence coming out so we collect now the second image and that goes much much faster now you may have noticed that there is something funny going on because this dichroic filter here apparently reflects both the blue light and the yellow light but it passed through the green light and so that means that that dichroic filter needs to have pretty special uh properties and is very different from what I've shown you so far and indeed that is the case and luckily the filter manufacturers are able to

make such interference filters so they can make filters that have reflect at certain uh wavelengths and then uh pass through the light in between those wavelengths and um some of the Spectra of filters you can buy look a lot better than what I'm showing you here it it's literally amazing what the filter manufacturers have accomplished and that has been also a great great driving force behind the whole progress of in fluorescence microscopy so we had our um image of our red D um and then we put that all back together in the computer in these

multicolored images now also that emission filter we could instead of having an emission filter we could which would have to be a multi-band pass em emission filter we could replace bed up by a second filter wheel and then we have uh full control and very fast control over both the excitation and emission filters one practical aspect that you need to be aware of is photo bleaching so fluorescent dyes don't last forever they only have so many cycles that they can go through and over time their fluoresence tends to bleach out so for instance in this

movie that I'm showing you here uh where we where I coupled gfp to the micral tip tracking protein eb1 we saw that at the beginning we had bright fluorescence and then over time it completely disappears now this is a uh regretfully a common aspect of uh most dyes and it is something that we usually really don't like and that we want to avoid so what can you do about it first of all you can select dieses that are resistant against Fay or that do not bleach as fast as others so for instance this D fluorine

that I showed to you before tends to bleach extremely extremely fast there are now other dyes on the market that have the same excitation and emission proper properties but that bleach much much slower so you would almost never want to use fluororesin anymore but use one of these Replacements you can try to label more densely so each die has a certain uh chance that it will photo bleach so if we now put a lot more labels there it will much take much longer before all my signal is gone then very importantly you want to change

the environment of the dye so that bleaching decreases so for instance um oxygen is important in this pH photo bleaching process so so a common strategy is to remove molecular oxygen from your sample as much as possible for instance by using glycerol you're already doing a lot better or by using enzimatic systems that take out the oxygen from the sample there there are also a lot of uh compounds that you can add to your mounting medium that will reduce bleaching and lastly and very importantly you really want to use all the photons that you can

get out of your dye so you don't want to leave on that excitation lamp uh when you're not looking when you're not observing your sample so always close the shutter immediately when you're not looking or when you're doing uh camera based Imaging you want to take the shortest exposure times possible using the lowest amount of excitation light that you can and you also want to synchronize your camera with the shutter as tightly as possible so that there's no dead time in which you're not actually collecting that that fluorescent light now I'd like to finish this

presentation going back to that movie that we saw in the beginning um you know in the end we're using fluoresence simply because it's a great tool to let us look at cells and let us look at individual um aspects of cells and to me it's still just amazing that we can see these living cells and components in these living cells uh while they're living and you know the beauty of these kind of movies and images is just utterly stunning and it makes it a joy to actually work with fluorescent microscopes