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Introduction to Fluorescence Microscopy

00:00:11;24 So much of the microscopy that we
00:00:13;09 are doing now a days is fluorescence microscopy
00:00:16;16 and i'll be explaining what that is in this lecture
00:00:22;03 So why do we use fluorescence in the microscope?
00:00:26;28 As you probably seen so far, there are two
00:00:31;05 important aspects in making
00:00:34;23 a great picture in your microscope
00:00:36;27 One is resolution, you want high resolution,
00:00:41;04 and the second thing is contrast,
00:00:43;26 you want high contrast
00:00:45;21 Otherwise you wouldn't see anything
00:00:47;28 And what fluorescence offers, is this high contrast
00:00:54;15 So, its demonstrated in this image here
00:00:57;12 We have part of the cell, labeled in green,
00:01:02;17 in green, we are looking at microtubules,
00:01:04;23 so we label microtubules with the fluorescent dye
00:01:08;13 and we have part of the cell labeled in red,
00:01:11;25 and that part is actually the uh, nucleus
00:01:15;02 Its DNA, its a red label attached to the histone protein
00:01:19;10 and what you see here,
00:01:20;21 is that these labels stand out on a black background
00:01:26;19 So we have a very high contrast,
00:01:28;26 we see what we want to look at
00:01:30;24 whereas our background is totally absent
00:01:34;11 so it gives an actually extremely high contrast
00:01:37;27 and what we already see in this specific image,
00:01:41;05 is that you get specificity
00:01:43;19 we can attach these fluorescent labels
00:01:46;27 to specific proteins, to specific molecules,
00:01:51;27 to specific dyes, to specific places in the cell
00:01:56;07 or wherever we want them,
00:01:57;16 so that we can target them,
00:01:59;06 that they show what we want to see
00:02:01;27 then fluorescence is quantitative,
00:02:06;00 so if I calibrate my system correctly,
00:02:09;15 areas where we have higher signal, like here
00:02:15;00 those are areas where I have more of my,
00:02:18;18 the protein that I am interested in
00:02:21;00 so in this case, there is a lot more tubulin here
00:02:23;18 in this bright area, than here in these dimmer areas
00:02:27;20 and then lastly, and very importantly,
00:02:31;12 fluorescence is compatible with imaging of living cells
00:02:36;21 so this image here is not only, a still image,
00:02:41;15 but its actually part of a sequence
00:02:43;24 of images that I took
00:02:45;16 and when we now start playing those faster,
00:02:49;05 we see that it's actually a dividing cell
00:02:52;16 the microtubules assemble in this spindle,
00:02:56;25 and the spindle then pulls apart
00:02:59;10 the chromosomes into daughter nuclei
00:03:03;21 so fluorescence is a great thing,
00:03:06;05 a great thing for microscopy
00:03:08;11 but what is it really?
00:03:11;27 and what's kind of surprising to me still,
00:03:15;10 is that human kind has been aware
00:03:17;19 of fluorescence
00:03:19;07 for only about a hundred and fifty years
00:03:21;08 so one of the first descriptions
00:03:23;12 of fluorescence was by Sir William Herschel
00:03:27;29 He was studying the aspect of
00:03:31;16 an extract of the bark of the cinchona tree
00:03:36;21 And he held up this extract in the light
00:03:41;03 of the sun that came through his window
00:03:43;10 and he noted that the light of the sun,
00:03:46;27 induced a celestial blue light
00:03:51;03 coming out of that extract of the
00:03:53;21 bark of the cinchona tree
00:03:58;28 I will now try to repeat that one hundred
00:04:00;27 and fifty year old experiment here in the lab
00:04:03;06 and i'll be showing you that now
00:04:06;22 so the bark of the cinchona tree is
00:04:08;00 hard to come by, but luckily the active ingredient
00:04:10;25 can be found in every super market in form, of,
00:04:14;26 contained in tonic water
00:04:18;13 i'll take the tonic water, pour it in a glass,
00:04:21;17 and the other part is that we need
00:04:24;02 UV light to illuminate it
00:04:27;04 and that we can buy in a form
00:04:29;20 of whats called a urine tracker in a pet store
00:04:32;08 so when we now shine this UV light
00:04:35;05 in the quinine, in tonic water, you see that
00:04:39;00 you get a blue light eminating
00:04:44;03 from within the tonic water
00:04:47;01 its another wavelength, its a longer wavelength,
00:04:51;03 than this light with which I illuminate,
00:04:54;16 and that is why we see it a lot better
00:04:57;06 our eyes are not very sensitive to this UV,
00:04:59;02 but they are highly sensitive to the blue light
00:05:01;18 in tonic water
00:05:02;29 Quinine is not the only compound that fluoresces,
00:05:05;15 of course,
00:05:06;05 otherwise it would be quite uninteresting
00:05:08;14 already in the nineteenth century,
00:05:11;10 scientists synthesized this dye
00:05:13;18 called fluorescein that is highly fluorescent
00:05:16;19 and when i shine blue light in that,
00:05:19;07 we get yellow fluorescence coming out
00:05:22;11 and another very well known dye is rhodamine,
00:05:27;28 and when you shine green light in that,
00:05:31;19 you get this orange-yellowish
00:05:34;09 fluorescence coming out
00:05:36;13 and so this is fluorescence
00:05:39;26 okay so what we have seen, is that
00:05:42;00 we use light of a lower wavelength,
00:05:45;02 so in this case
00:05:46;25 of the quinine from the extract of the
00:05:49;16 bark of the cinchona tree,
00:05:51;06 we use UV light
00:05:53;26 and then the dye responds to that light,
00:05:56;25 by emitting light of higher wavelength,
00:06:00;15 so in this case, blue light
00:06:03;29 so in general, except for very special cases,
00:06:07;11 the emission light is of a longer wavelenth,
00:06:10;25 means lower energy
00:06:13;03 so there is some energy loss going on
00:06:15;04 this process, than the excitation light
00:06:19;07 this aspect of fluorescence is very important,
00:06:23;17 and we often show that
00:06:27;11 by measuring the spectra,
00:06:31;01 so we measure the excitation spectrum
00:06:34;21 which is often very similar to an absorbtion spectrum
00:06:38;12 and we measure an emission spectrum,
00:06:41;25 so here that is this curve here,
00:06:43;20 which is the fluorescent light
00:06:46;19 emitted by the dye
00:06:48;14 so we characterize these dyes
00:06:50;18 by their excitation maximum,
00:06:52;19 and by their emission maximum
00:06:55;24 those numbers tell you immediately
00:06:59;13 a lot about
00:07:01;09 how to use the dye in your actual experiments
00:07:05;00 the difference between the excitation
00:07:08;15 and emission maximum is called the Stokes Shift,
00:07:11;24 after Sir Stokes
00:07:14;08 who first realized that there was this difference
00:07:18;08 you can have dyes that have a small Stokes Shift,
00:07:21;00 where the emission is very close to the excitation
00:07:24;10 or you have dyes with a very large Stokes Shift
00:07:26;21 where the emission is very far away
00:07:29;02 from the excitation light.
00:07:31;07 Now, so there is energy loss going on,
00:07:34;23 and this causes the difference in wavelenght
00:07:38;11 but what, how, does that happen?
00:07:41;09 and one of the most useful ways of
00:07:44;27 thinking about that
00:07:46;13 is with the so called Jablonski diagrams,
00:07:49;12 Jablonski was a Polish scientist
00:07:54;13 who did this work
00:07:54;17 in the beginning of the twentith century
00:07:57;05 and in this Jablonski diagrams,
00:07:59;15 we are looking at
00:08:04;20 the orbitals that electrons can be in
00:08:09;15 when a dye, observes light the electron
00:08:14;16 is going to be kicked to a higher orbital,
00:08:17;26 in a higher energy state
00:08:20;00 this absorption goes very, very fast,
00:08:23;13 so it happens in a time scale of femtoseconds
00:08:29;16 and femtoseconds to me don't mean much,
00:08:32;05 so I like to express this in the
00:08:35;17 distance that light travels
00:08:38;28 during that time period
00:08:40;24 this is like those extremely fast,
00:08:43;23 its pretty amazing,
00:08:45;07 that light travels only like
00:08:47;11 .3 microns during this time period
00:08:51;21 Once that electron finds itself into
00:08:55;00 these higher orbitals, through a
00:08:57;14 process called internal conversion
00:09:00;15 it quickly falls back to what you call,
00:09:04;00 ground level of the excited state and
00:09:08;02 that internal conversion,
00:09:10;09 is a tiny little bit of heat that dissipates this way
00:09:13;22 so we have a little bit of energy loss here,
00:09:15;13 and that happens on the level, of
00:09:17;19 time scales, of picoseconds
00:09:21;16 and light will travel only .3 mm during that time period
00:09:27;12 then the electron can hang out here,
00:09:30;18 for what is in comparison
00:09:33;10 to this previous time scales, almost eternity
00:09:37;12 something on the order of many nanoseconds,
00:09:42;06 then it will fall back into the real ground state,
00:09:45;24 and emit a photon
00:09:48;20 that photon will be of a wavelength,
00:09:51;17 less than the wavelength,
00:09:56;09 sorry of an energy that is less
00:10:00;01 than the energy that was put in initially
00:10:03;04 and that means that the photon that is coming out,
00:10:05;14 is going to be of a higher wavelength
00:10:08;14 Now, not only, this return to the ground state,
00:10:15;26 is not the only process that can take place
00:10:18;25 when a dye is in an excited state
00:10:20;28 Its also possible that the energy
00:10:22;20 is transferred to other molecules,
00:10:25;07 or even the electron can fall back to
00:10:30;18 the ground state but not emit a photon
00:10:35;10 so there is also non-radiative decay
00:10:38;11 so we have a total number of events,
00:10:46;11 some of which result in light coming out,
00:10:49;12 and some of which them that dont
00:10:52;15 and we express the ratio of those two,
00:10:54;13 as the quantum efficiency
00:10:58;00 and the quantum efficiency is a very
00:10:59;23 important parameter of the dye
00:11:01;23 It not only depends on the dye itself,
00:11:04;07 it also depends on its environment,
00:11:06;21 for instance what other molecules
00:11:08;11 are around the dye
00:11:12;03 but nevertheless, quantum efficiency
00:11:15;20 is something you want to know,
00:11:17;26 and that you want to measure,
00:11:19;07 or that you want to be told what it is
00:11:21;29 another important aspect is the brightness,
00:11:25;18 and the brightness in the end,
00:11:28;04 is determined by how well the
00:11:33;27 dye absorbs the excitation light
00:11:36;21 and what fraction of the excited
00:11:42;20 state returns to the ground state
00:11:45;09 on the emission of fluorescence
00:11:48;04 so its determined by both the absorption
00:11:51;00 coefficient and the quantum efficiency
00:11:53;15 Now, another important aspect here is
00:11:59;21 what we already talked about,
00:12:01;23 how long does it take between
00:12:05;03 absorbing a dye and for the dye
00:12:07;06 to return to ground state
00:12:09;05 and that is what we call the life time,
00:12:11;07 it is mainly determined by how long
00:12:14;06 it takes before we return to the
00:12:17;29 ground state out of the excited state
00:12:20;18 usually this is on the order of nanoseconds,
00:12:23;17 nevertheless, its also a parameter
00:12:27;05 that is dye specific, that is also dependent
00:12:30;01 on the environment of the dyes
00:12:32;02 and there's a whole form of fluorescence
00:12:34;25 microscopy, called LifeTime microscopy
00:12:37;23 that actually reads out LifeTime instead
00:12:40;16 of only reading out fluorescence itself
00:12:45;14 Nevertheless, what we are now going
00:12:48;05 to talk about is microscopes that
00:12:50;17 descriminate between the excitation
00:12:53;20 light and the emission light
00:12:57;10 so how do we do that in a microscope?
00:12:58;11 Well, the main thing is that we use filters.
00:13:03;23 We use filters that filter the exciting light,
00:13:08;12 so that we get out of our light source
00:13:11;12 only the light that is of the wavelength
00:13:13;29 that will excite our dye
00:13:16;15 that light will bounce off of the dichroic mirror,
00:13:19;00 and then excite our sample,
00:13:21;29 after it went through the objective
00:13:25;01 the sample will start fluorescing and
00:13:28;10 that fluorescence travels back through
00:13:30;19 the same objective and will now pass
00:13:33;02 through the dichroic mirror
00:13:34;26 and then there is an emission filter that will let through fluorescence
00:13:39;07 but that will completely block
00:13:41;00 any scattered excitation light
00:13:44;15 so that we only see the fluorescence
00:13:48;18 on our detector which I
00:13:50;14 have symbolized here with an eye, but that can also be a CCD camera, or what not
00:13:55;23 so this is the simple, basic principle
00:14:00;09 of a fluorescent microscope
00:14:03;20 so the most important aspect here,
00:14:07;08 and the thing that makes it different
00:14:09;08 from a normal microscope is filters
00:14:12;14 and these filters need to be very good,
00:14:14;21 becuase they really need to
00:14:15;27 discriminate this excitation light
00:14:18;19 from the emitted light
00:14:20;12 so what options do we have there?
00:14:22;29 well you can have filters
00:14:25;04 that are based on absorption,
00:14:27;03 that absorb certain wavelenghts of light
00:14:31;07 those are not that great in practice,
00:14:37;16 because their spectra are very broad
00:14:40;20 so they will also absorb a bit of the emission light
00:14:45;13 so they will not do exactly what we want them to do
00:14:49;26 and the type of filters that we mainly use
00:14:51;29 these days are interference filters
00:14:54;18 so what are those?
00:14:56;27 Interference filters are basically,
00:15:00;13 thin alternating layers that have a
00:15:05;23 different refractive index
00:15:07;25 so that we get interfaces
00:15:11;10 there that can reflect the light
00:15:13;29 so you can almost think about them
00:15:16;03 as thin transparent layers
00:15:20;20 that are about the wavelength of the light,
00:15:22;12 or half the wavelength of the light
00:15:26;07 with semi-reflective coatings in between,
00:15:29;25 so what now happens when light hits this filter,
00:15:35;26 is that part of it will be reflected
00:15:39;10 reflected, part of it will move through,
00:15:42;08 the light that reflects will interefere with itself
00:15:47;09 and when the wavelength is such that it
00:15:50;17 matches the wavelength of the light,
00:15:53;26 you will get constructed interference
00:15:56;17 and that constructive interference
00:15:57;26 will then in effect let the light
00:16:02;07 pass through this filter
00:16:04;26 whereas, when the wavelengths are
00:16:06;12 different from the two, um,
00:16:08;22 the distance between the layers
00:16:10;25 we get destructive interference
00:16:13;02 and it will be reflected out.
00:16:15;14 The filter manufacturers can
00:16:17;22 completely compute how these layers
00:16:20;26 will behave, so
00:16:22;03 they can design in the computer
00:16:23;29 what wavelengths this filter should
00:16:26;03 pass through and what
00:16:27;05 wavelengths should be reflected
00:16:29;28 and you can basically order,
00:16:32;20 any type of filter that you may possible need.
00:16:36;16 Now, just to make this
00:16:39;05 a little bit more clear
00:16:40;23 there is one example of interference
00:16:43;04 that you are highly aware of,
00:16:44;25 and that everyone of us has seen
00:16:46;19 and that is what you see in cell bubbles,
00:16:49;00 so in cell bubbles, when light hits that,
00:16:51;16 you will see
00:16:52;27 reflection of all kinds of color of light.
00:16:55;19 The cell bubble itself has a thickness
00:16:58;22 that is on the order of half the wavelength
00:17:01;28 of a lot of the visible light
00:17:04;26 and you will get the same intereference
00:17:08;04 effect where some of the colors will
00:17:11;19 pass through and other colors will be reflected
00:17:15;22 and that gives this beautiful color patterns.
00:17:19;29 Now we will break
00:17:22;29 and show you some of these filters
00:17:27;05 and will also show you now
00:17:28;26 here how these filters are assembled
00:17:31;12 in a filter cube
00:17:33;06 Okay, so I have an example here
00:17:35;17 of such an interference filter
00:17:37;26 this one is called HQ47040
00:17:43;19 and 470 is the center wavelength of this filter
00:17:47;07 and the 40 means to bend with,
00:17:49;08 so this one will pass blue light
00:17:52;24 and so if I have my blue LED,
00:17:55;20 and I hold that up,
00:17:56;19 I see the blue light coming through
00:17:58;14 and you will see that
00:17:59;13 this LED indeed passes it through.
00:18:02;14 Now with a green light source,
00:18:05;02 and hold that up, and go throught this filter,
00:18:08;16 you will see that it gets a lot dimmer
00:18:11;04 and not only that, you may also notice
00:18:13;01 that its actually a little bit of blue coming through,
00:18:15;17 and that is because this
00:18:16;16 green LED is not purely green,
00:18:19;22 but also has a slight bit of blue light in it
00:18:24;21 then, the next part is this dichroic mirror,
00:18:29;19 so this one is designed, again,
00:18:32;20 such that it reflects blue light
00:18:37;06 so you will not see coming anything through,
00:18:38;27 but if I reflect it to you,
00:18:41;05 you may be able to see that
00:18:43;23 and then the last part is this
00:18:46;03 emission filter that will transmit the green light,
00:18:49;19 and reject the blue light
00:18:55;05 so in effect when we would now place these
00:18:57;10 three in this configuration in the microscope,
00:18:59;27 illuminate from this side
00:19:01;16 go up, we have the objective in the sample here,
00:19:03;25 come back through,
00:19:05;03 and go through this emission filter
00:19:08;22 that's the principle of a florescence microscope.
00:19:12;21 These filters, this is exactly
00:19:15;13 the way they are arranged in these cubes,
00:19:19;04 that were designed by Ploem,
00:19:20;16 where we have an excitation filter,
00:19:22;26 a dichroic mirror, and then an emission filter here
00:19:27;00 and so these will, as expected, reflect,
00:19:32;02 and i'll now turning it to you,
00:19:33;20 so that you can see it
00:19:34;22 so that the exciting blue light,
00:19:37;03 will be reflected to the specimen,
00:19:39;14 the specimen will fluoresce green,
00:19:42;12 and that will make it to the eye piece
00:19:44;19 or to the camera sensor.
00:19:47;21 So we've seen now, these filter cubes,
00:19:49;24 there's an excitation filter, there's a dichroic mirror,
00:19:54;05 and there's an emission filter.
00:19:56;04 What's very important to know,
00:19:59;17 and the information you should always
00:20:01;00 have about this filters is their spectra
00:20:04;08 so, what are the wavelenghts
00:20:06;15 that are being transmitted,
00:20:08;05 and what are the wavelenghts
00:20:09;22 that are being reflected
00:20:12;29 and those spectra
00:20:14;20 then will look something like as follows
00:20:18;29 so we have an excitation filter,
00:20:21;11 that passes through this band-pass
00:20:26;02 these wavelenthgs, these colors of light,
00:20:28;05 the dichroic mirror, is made such that it will
00:20:31;19 reflect everything that is lower
00:20:34;09 than a certain cut of band
00:20:36;07 and pass through everything that is higher,
00:20:38;12 and then the emission filter will pass through
00:20:41;29 the higher wavelenghts
00:20:43;20 but reject absolutely reject, the excitation light
00:20:48;14 so, when you now assemble
00:20:53;05 a fluorescent microscope,
00:20:54;20 your task will be to select filters
00:20:56;29 that match well the dyes
00:20:58;29 that you'll be working with
00:21:00;24 and what you will be doing
00:21:02;10 in that case is to try to
00:21:04;14 overlay the spectrum of the fluorophores
00:21:07;23 with the specturm of your filters
00:21:10;09 so, I have here a spectrum of a dye
00:21:13;08 that is probably Cy3 the Cyanine dye
00:21:17;18 it excites in the green areas, around five-fifty,
00:21:26;03 and it has en emission maximum around six hundred
00:21:30;19 so if you know use a band-pass filter,
00:21:32;26 that is centered around six-hundred and fifty nanometers
00:21:38;12 that will easily reject the excitation light,
00:21:42;12 because its far away from that
00:21:44;21 but you also note that the integral of it,
00:21:48;05 that it only passes through a
00:21:50;16 fraction of the emitted fluorescence
00:21:53;29 and that means that you are losing
00:21:55;16 something like seventy-five percent or so
00:21:58;21 of the emitted fluorescence
00:22:00;19 and in general you really want to get all the
00:22:03;15 photons that you can,
00:22:05;06 so its not a perfect situation
00:22:07;25 so if you now switch to a band-pass
00:22:11;17 filter that is slightly lower,
00:22:13;17 that is around, centered around
00:22:15;12 six-hundred and ten nanometers
00:22:19;08 then, your catching a much larger fraction
00:22:23;28 like seventy-five or eighty percent
00:22:26;14 of the emitted photons
00:22:28;16 and you will be much happier
00:22:29;29 becuase your image will be much brighter.
00:22:32;20 Now, we'll now switch to the lab,
00:22:38;01 and show how we are using these filter cubes
00:22:41;21 to collect actual images
00:22:44;17 Here we have a real fluorescence microscope,
00:22:48;28 so we have a light source back here,
00:22:52;16 this is a mercury arch lab
00:22:55;25 light sources about that,
00:22:57;19 that light then passes through the main body,
00:23:01;02 and then hits here, the dichroic mirror
00:23:05;26 so, and i'll show you now how this works,
00:23:07;01 so this is basically a carousel, that you can take out
00:23:11;02 and this carousel, contains, or turret,
00:23:16;14 contains these same filter cubes,
00:23:19;04 that we have been looking at in the lecture
00:23:21;15 so this one is sitting here, the light hits
00:23:25;24 the excitation filter, I can take these cubes out like this
00:23:30;15 so the light hits the excitation filter,
00:23:34;00 its sitting in the microscope like this,
00:23:36;26 goes through the excitation filter,
00:23:38;22 hits the mirror, goes up, the fluorescence light
00:23:41;11 comes back, makes its way through the mirror,
00:23:44;06 and then here at the bottom,
00:23:45;19 we have the emission filter that goes down
00:23:48;25 and in the end, so the camera or the eye piece
00:23:51;28 and now since this thing is motorized,
00:23:54;04 once its sitting in the microscope,
00:23:55;11 it can with the push of the button,
00:23:58;09 put in another one of these filter cubes
00:24:03;20 and these cubes have been selected
00:24:06;18 to visualize the dyes that we are interested in
00:24:11;06 and so usually there's one for things like DAPI,
00:24:13;13 for things like GFP, or fluorescein,
00:24:16;28 and a cube for something like mCherry or rhodamine,
00:24:21;03 okay, so when i now put this back into the microscope
00:24:29;11 and then open the shutter her in the back
00:24:34;21 that blocks the light at the moment
00:24:41;04 we get blue light coming out of the objective,
00:24:44;23 and with a push of the button,
00:24:46;19 I put now another dichroic in place
00:24:49;24 here we have green light for rhodamine or mCherry,
00:24:53;05 and if I go on, we get red light for
00:24:56;20 something like, a dye like Cyfive
00:25:00;20 and so, just by switching these,
00:25:03;00 we can select the wavelength of the light for excitation
00:25:07;07 and the filter use for emission and
00:25:10;03 select the fluorophore that we want to image
00:25:13;06 So we've now seen how we can
00:25:14;10 use the fluorescent microscope
00:25:16;11 to collect individual channels,
00:25:19;24 images of individual fluorophores.
00:25:22;15 Then what we can do in the computer
00:25:24;19 is put all those images together, give them a nice color
00:25:28;26 and end up with things like this,
00:25:30;02 so here we have a staining of mitochondria in red,
00:25:34;03 of DNA in blue, and what looks
00:25:39;04 like actin stress fibers in green
00:25:41;25 This works very, very well,
00:25:45;28 there are also alternative strategies to doing this and one is,
00:25:51;06 what you may have noticed in the lab part,
00:25:53;13 is that it actually takes quite a bit of time,
00:25:55;19 to move that turret
00:25:57;07 so going from one position to the other,
00:25:59;15 it takes you know quickly like half a second, or so
00:26:02;16 the turret is pretty big so it has a lot of vibrations,
00:26:06;01 if you want to go faster,
00:26:07;20 the whole thing starts moving more
00:26:10;07 those are all bad things,
00:26:11;18 and things you dont really want, so instead of
00:26:15;06 using these filter turrets, what you can do, is
00:26:18;20 put your excitation and possibly
00:26:20;28 also your emission filters, on a filter wheel.
00:26:25;01 Now, I brought such a filter wheel,
00:26:28;21 and I can show that to you here, so it really is a wheel
00:26:35;25 that has individual filters in them,
00:26:37;20 there is a motor, that can through this rubber band here,
00:26:41;20 moves that around
00:26:42;09 and we can put, by activating the motor,
00:26:46;12 we can switch between these different filters
00:26:49;03 and we can do that at a speed
00:26:51;02 that generally is much higher than you can do on a turret
00:26:55;05 so, we then, first take an image,
00:27:00;19 where we put the first excitation filter in place
00:27:05;05 so for instance in this case,
00:27:06;18 we have a blue excitation filter, we get blue light out
00:27:10;10 we get green fluorescence, and
00:27:13;07 when we move the filter wheel,
00:27:15;03 to the next position which has
00:27:18;02 a yellow filter, we get red fluorescence
00:27:21;20 coming out, so we collect now
00:27:23;14 the second image, and that goes much, much faster.
00:27:28;05 Now you may have noticed that there
00:27:31;23 is something funny going on,
00:27:35;02 because this dichroic filter here,
00:27:37;03 apparently, reflects both the blue light, and the yellow light
00:27:42;15 but it passes throught the green light,
00:27:45;17 and so that means that the
00:27:48;03 dichroic filter needs to have pretty special properties
00:27:53;00 and its very different from
00:27:54;01 what i've shown you so far.
00:27:56;04 and indeed that is the case,
00:27:58;04 and luckily the filter manufactureres
00:28:00;24 are able to make such interference filters
00:28:03;25 so they can make filters that
00:28:05;11 reflect at certain wavelengths
00:28:09;26 and then pass through the light
00:28:12;17 in between those wavelengths
00:28:15;12 and some of the spectra of filters
00:28:18;10 you can buy look a lot better than what i'm showing you here
00:28:21;20 it's literally amazing what the filter
00:28:24;16 manufacturers have accomplished
00:28:26;13 and that has been a also a great driving force
00:28:29;16 behind the whole progress in fluorescence microscopy
00:28:35;03 so, we had our image of our red dye, um,
00:28:40;21 and then we put them all back
00:28:43;10 together in the computer in these multi-colored images
00:28:48;01 now, also that emission filter, we could,
00:28:50;15 instead of having an emission filter, we could,
00:28:53;01 which, would have to be a
00:28:54;17 multi-band pass emission filter
00:28:57;05 we could replace it by a second filter wheel,
00:29:00;08 and then we have a full control and very fast control
00:29:05;19 of both the excitation and emission filters.
00:29:08;28 One practical aspect that
00:29:13;24 you need to be aware of is photobleaching.
00:29:17;27 So, fluorescence dyes, don't last forever,
00:29:22;29 they only have so many cycles that they can go through
00:29:27;00 and over tiime, their fluorescences,
00:29:29;12 tends to bleach out.
00:29:31;03 So for instance, in this movie
00:29:32;21 that i'm showing you here, um,
00:29:35;03 where i've coupled GFP to the
00:29:38;14 microtubule tip tracking protein EBI
00:29:41;15 we saw, that at the beginning,
00:29:43;14 we had bright fluorescence,
00:29:45;07 and then over time, it completely disappears
00:29:48;23 now this is regretfully a
00:29:51;26 common aspect of most dyes,
00:29:54;17 and that is something that we
00:29:55;22 usually really dont like and that we want to avoid.
00:30:00;04 So, what can you do about it?
00:30:02;20 First of all, you can select dyes that are
00:30:08;04 resistant against fade, or do not
00:30:10;11 bleach as fast as others. So for instance,
00:30:14;03 this dye, fluorescein, that I showed
00:30:16;26 to you before, tends to bleach extremely, extremely fast.
00:30:21;20 There are now other dyes on
00:30:24;05 the market that have the same excitation and emission properties
00:30:29;01 but that bleach much, much slower, so you would
00:30:31;27 almost never want to use
00:30:33;26 fluorescein anymore, but use one of these replacements.
00:30:38;12 You can try to label more densely,
00:30:42;08 so each dye has a certain chance that it will photobleach,
00:30:48;10 so if we now put a lot more labels there,
00:30:50;21 it will take much longer before my signal is gone.
00:30:55;22 Then, very importantly, you want to
00:30:57;08 change the environment of the dye so that bleaching decreases.
00:31:01;18 So for instance, oxygen is
00:31:04;23 important in this photobleaching process.
00:31:08;16 So a common strategy,
00:31:12;06 is to remove molceular oxygen from
00:31:15;11 your sample as much as possible,
00:31:17;13 for instance by using glycerol,
00:31:19;20 your already doing a lot better,
00:31:21;14 or by using enzymatic systems that
00:31:24;10 take out the oxygen from the sample.
00:31:27;29 There are also a lot of compounds
00:31:30;25 that you can add to your mounting
00:31:33;09 medium that will reduce bleaching.
00:31:36;05 And lastly, and every importantly,
00:31:38;20 you really want to use all of the photons
00:31:41;25 that you can get out of your dye.
00:31:44;12 So, you don't want to leave on that
00:31:47;12 excitation lamp when your not looking,
00:31:50;22 when your not observing your sample.
00:31:52;23 So always close the shutter
00:31:55;13 immediately when your not looking, or when
00:31:58;28 your doing camera based imaging,
00:32:01;17 you want to take the shortest
00:32:03;25 exposure times possible, using the lowest
00:32:07;00 amount of excitation light that you can,
00:32:10;02 and you also want to synchronize
00:32:12;19 your camera with the shutter as tightly as possible,
00:32:16;10 so that there is no dead time that
00:32:18;03 your not actually collecting that fluorescent light.
00:32:23;07 Now, I'd like to finish this presentation,
00:32:28;10 going back to that movie that we saw in the beginning,
00:32:32;03 you know in the end, your using
00:32:35;09 fluorescence simply because it's a great tool
00:32:40;28 to let us look at cells, and let us
00:32:43;23 look at individual aspects of cells
00:32:47;15 and to me, it's still just amazing,
00:32:50;00 that we can see these living cells, and
00:32:52;17 components in these living cells
00:32:54;26 while they are living, and you know,
00:32:58;14 the beauty of these kind of movies, and images,
00:33:03;10 is just utterly stunning and it makes
00:33:05;09 it a joy to actually work with fluorescence microscopes.

This Talk
Speaker: Nico Stuurman
Audience:
  • Student
  • Educators of Adv. Undergrad / Grad
  • Researcher
  • Educators
Recorded: April 2012
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Talk Overview

Fluorescence is a process in which matter absorbs light and re-emits at a different wavelength. Fluorescence is widely used in biological microscopy. This lecture describes the principles of fluorescence and fluorescence microscopy.

Questions

  1. Match the time scale to these transitions:
    1. Femtosecond
    2. Picosecond
    3. Nanosecond
    1. Transition from the excited state to the ground state
    2. Photon absorption
    3. Transition to the lowest energy excited state
  2. True or False: The transition from the excited to the ground state is always accompanied by the emission of a photon.
  3. The brightness of a dye depends most upon its
    1. Photobleaching rate
    2. Fluorescence lifetime
    3. Absorption coefficient
    4. Solubility in water
  4. Which is true of fluorescence microscopy
    1. Switching filters in an emission filter wheel is typically slower than changing the filter cubes in a turret.
    2. A 480/40 emission filter passes 440-520 nm light.
    3. The filter cube typically has two filters and one dichroic mirrror
    4. The tube lens is generally placed before the filter cube

Answers

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Speaker Bio

Nico Stuurman

Nico Stuurman

Nico Stuurman is a Research Specialist at the University of California, San Francisco, in the lab of Ron Vale. Nico combines his expertise in computer programming and microscopy to advance many projects including the Open Source software, Micro-Manager. Continue Reading

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