Minimizing Damage from Fluorescence

00:00:11.15 Fluorescence microscopy is great, but two of the evil
00:00:16.01 side effects that one often has to manage with fluorescence
00:00:18.25 microscopy are photobleaching and phototoxicity.
00:00:22.07 And these are illustrated in this movie here, where you're watching
00:00:26.09 rhodamine-labeled microtubules moving along a kinesin-coated
00:00:30.18 surface. And over time of illumination, you can see that the
00:00:34.09 fluorescence signal begins to get dimmer, that's photobleaching,
00:00:37.07 and these microtubules have also become fragmented
00:00:40.29 into many small pieces, and this is phototoxicity.
00:00:44.27 Because illumination is actually destroying and breaking
00:00:48.24 up these microtubules. So if one tries to capture living biology,
00:00:55.08 one needs to collect the images, but one's trying not also to
00:00:59.15 destroy the sample that one's trying to observe. So in this
00:01:03.01 little tip video, I'll discuss ways that one could minimize these
00:01:07.12 unwanted side effects of fluorescence microscopy.
00:01:11.01 So, photobleaching, what is that? So this has been discussed
00:01:15.02 in other lectures in the course, it's the loss -- irreversible loss,
00:01:19.19 of fluorescence due to the excitation of the fluorophore.
00:01:22.25 And you can see this illustrated very nicely in this field
00:01:28.00 of single GFP molecules, each of these spots is a single GFP
00:01:32.07 and over time, you can see that individual spots suddenly
00:01:36.03 disappear. And the fluorescence is lost. So, this disappearance
00:01:41.26 of the fluorescence from these individual GFP molecules is
00:01:46.21 photobleaching. And this photobleaching occurs more rapidly
00:01:51.28 at higher light exposures, and it's also very dependent upon
00:01:56.23 the fluorophore that one's using. Some fluorophores bleach
00:02:00.14 faster than others. And even the environment of the fluorophore,
00:02:03.27 it may depend even on how the fluorophore is bound specifically
00:02:08.02 to your protein molecule. The other effect is phototoxicity.
00:02:14.18 So this is actually damage to your specimen.
00:02:18.01 It could be damage to your cells or molecules that you're trying
00:02:23.00 to observe in vitro, like you saw with kinesin and microtubules.
00:02:26.11 There are two types of phototoxicity. One is due to direct
00:02:30.29 absorption of light, without the fluorescence. And UV light,
00:02:37.15 in particular, the higher wavelengths of light tend to be particularly
00:02:41.02 damaging. So in many cases, if one can use longer wavelengths
00:02:47.05 light, like green light or red light, that tends to be less
00:02:52.08 absorbed and less damaging to cells.
00:02:55.05 The other type of phototoxicity is indirect, and it occurs through
00:02:59.07 the actual excitation of the fluorophore, which can give rise to
00:03:04.21 the production of reactive oxygen species. And these are
00:03:09.01 a series of highly reactive radicals that can either interact with
00:03:16.24 the fluorophore, or also with just any biomacromolecules
00:03:21.16 and damage them. And this is obviously something that one
00:03:25.09 wants to avoid, if one wants to keep one's specimen happy
00:03:28.26 and healthy during the course of observation. So there are some
00:03:32.19 things that you can do to mitigate both photobleaching and
00:03:36.15 photoxicity. If one's lucky enough to be doing microscopy in an
00:03:41.16 in vitro sample, in those cases, you can actually remove
00:03:47.00 molecular oxygen from the buffer. And there are things called
00:03:52.16 oxygen scavenger systems, which are in fact, an enzymatic mixture
00:03:57.26 that will remove the oxygen from the solution. And there are also
00:04:05.18 things that one could add that react with the reactive oxygen species
00:04:10.06 and scavenge them before they can do damage to the cell or
00:04:16.13 macromolecule. In a fixed specimen, there are also ways of
00:04:21.11 mounting your specimen, your immunofluorescent specimen,
00:04:24.07 and commercial agents called "anti-fade" that minimizes
00:04:29.19 the rate of photobleaching to allow longer observation.
00:04:34.25 Now, I'll just show you an example of depleting molecular oxygen
00:04:38.13 this is that specimen I showed you that's being all fragmented
00:04:42.27 here, due to the reactive oxygen species. And here is the exact
00:04:48.12 same type of specimen here with these oxygen scavengers
00:04:53.04 now added to the buffer system. And you can see that
00:04:57.02 photobleaching is minimized, and the kinesin molecules
00:05:02.19 are very happily moving the microtubules, and the microtubules aren't
00:05:06.04 being fragmented. And so this just shows how powerful
00:05:10.00 removing molecular oxygen can be. So if we're working with
00:05:15.05 living cells or tissues, we can't obviously remove molecular oxygen.
00:05:18.19 So we have to think of other strategies for minimizing photobleaching
00:05:23.13 and toxicity. And the basic strategy is to minimize the light exposure
00:05:28.17 on your specimen. And naturally, there's a trade-off here.
00:05:33.11 The more photons that we illuminate the specimen with,
00:05:36.01 the more fluorescence will be produced, and we get a better
00:05:38.28 signal to noise, but if we over-illuminate, we also risk the
00:05:44.13 danger of also creating more photodamage and in that
00:05:49.28 way, actually interfering with the processes in the cell that
00:05:53.14 we're actually trying to observe. So there are several things that we
00:05:57.01 can do. First of all, shutter the light source. During times
00:06:01.11 when one's not actually collecting the image, the light
00:06:04.19 source should be turned off. And there are many ways now to
00:06:09.13 shutter a light source and control it with a computer.
00:06:12.28 Secondly, is just to minimize the light exposure.
00:06:16.24 Again, we want to collect a decent signal to noise, but
00:06:20.19 particularly, for every light sensitive processes in cells,
00:06:25.16 we want to minimize the light exposure. And that can be done
00:06:29.09 in a few ways. One is through the illumination intensity itself.
00:06:35.01 And also the exposure time that we are collecting photons
00:06:42.09 on the camera. So, between the intensity and the exposure
00:06:46.06 time, that gives you your signal. And one has to really
00:06:51.15 think about both of these parameters very carefully so one's getting
00:06:54.26 a good image, but again not over illuminating and potentially
00:06:59.04 causing photodamage. And the other thing we can control
00:07:03.02 in a time lapse movie, is the interval between exposures.
00:07:07.13 And that depends on the dynamics of your process, but
00:07:10.15 if you're working and trying to observe a very slow process
00:07:14.02 that's going on in a cell, we have the luxury then of shuttering
00:07:17.21 the light source for a longer period of time in between exposures
00:07:22.17 and that also minimizes photodamage. Another big breakthrough
00:07:27.07 is really using very sensitive cameras. And elsewhere in the course,
00:07:32.02 you can learn about these new cameras called EMCCD cameras,
00:07:35.02 and these have been a really big breakthrough in live cell imaging.
00:07:39.02 Because they're extremely sensitive, and that allows one to
00:07:43.25 use very low illumination of the specimen and still collect
00:07:49.05 very good images and create less photodamage.
00:07:53.02 And the other potential strategy is also to minimize out of focus
00:07:59.24 light exposure. If one's trying to collect a particular plane of
00:08:03.26 illumination and image that plane, but one's bathing the specimen
00:08:07.06 above and below that plane with light, you're potentially just
00:08:11.00 creating photodamage in the cell. But that out of focus light is not
00:08:17.29 contributing to the image. So there are a couple very powerful techniques,
00:08:21.02 total internal reflection fluorescence microscopy and light sheet
00:08:28.11 microscopy, are both very good ways to minimize out of focus
00:08:32.21 light exposure. So these are a few tips and I hope you
00:08:38.25 can employ them and remember, you want to create
00:08:42.27 great images, but also keep your molecules and cells happy!
00:08:47.00 Thank you.

This Talk

Speaker: Ron Vale

Audience:

Researcher

Recorded: April 2012

Talk Overview

Ron Vale illustrates how illumination of a specimen can cause photobleaching and protein damage and gives us a tip about minimizing damage from fluorescence. Ways to minimize such problems include strategies for minimizing light exposure and removing molecular oxygen from in vitro reactions.

Speaker Bio

Ron Vale

Professor of the Department of Cellular and Molecular Pharmacology; Investigator in the Howard Hughes Medical Institute
University of California, San Francisco Continue Reading

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