Fluorescence microscopy is great, but two of the evil
side effects that one often has to manage with fluorescence
microscopy are photobleaching and phototoxicity.
And these are illustrated in this movie here, where you're watching
rhodamine-labeled microtubules moving along a kinesin-coated
surface. And over time of illumination, you can see that the
fluorescence signal begins to get dimmer, that's photobleaching,
and these microtubules have also become fragmented
into many small pieces, and this is phototoxicity.
Because illumination is actually destroying and breaking
up these microtubules. So if one tries to capture living biology,
one needs to collect the images, but one's trying not also to
destroy the sample that one's trying to observe. So in this
little tip video, I'll discuss ways that one could minimize these
unwanted side effects of fluorescence microscopy.
So, photobleaching, what is that? So this has been discussed
in other lectures in the course, it's the loss -- irreversible loss,
of fluorescence due to the excitation of the fluorophore.
And you can see this illustrated very nicely in this field
of single GFP molecules, each of these spots is a single GFP
and over time, you can see that individual spots suddenly
disappear. And the fluorescence is lost. So, this disappearance
of the fluorescence from these individual GFP molecules is
photobleaching. And this photobleaching occurs more rapidly
at higher light exposures, and it's also very dependent upon
the fluorophore that one's using. Some fluorophores bleach
faster than others. And even the environment of the fluorophore,
it may depend even on how the fluorophore is bound specifically
to your protein molecule. The other effect is phototoxicity.
So this is actually damage to your specimen.
It could be damage to your cells or molecules that you're trying
to observe in vitro, like you saw with kinesin and microtubules.
There are two types of phototoxicity. One is due to direct
absorption of light, without the fluorescence. And UV light,
in particular, the higher wavelengths of light tend to be particularly
damaging. So in many cases, if one can use longer wavelengths
light, like green light or red light, that tends to be less
absorbed and less damaging to cells.
The other type of phototoxicity is indirect, and it occurs through
the actual excitation of the fluorophore, which can give rise to
the production of reactive oxygen species. And these are
a series of highly reactive radicals that can either interact with
the fluorophore, or also with just any biomacromolecules
and damage them. And this is obviously something that one
wants to avoid, if one wants to keep one's specimen happy
and healthy during the course of observation. So there are some
things that you can do to mitigate both photobleaching and
photoxicity. If one's lucky enough to be doing microscopy in an
in vitro sample, in those cases, you can actually remove
molecular oxygen from the buffer. And there are things called
oxygen scavenger systems, which are in fact, an enzymatic mixture
that will remove the oxygen from the solution. And there are also
things that one could add that react with the reactive oxygen species
and scavenge them before they can do damage to the cell or
macromolecule. In a fixed specimen, there are also ways of
mounting your specimen, your immunofluorescent specimen,
and commercial agents called "anti-fade" that minimizes
the rate of photobleaching to allow longer observation.
Now, I'll just show you an example of depleting molecular oxygen
this is that specimen I showed you that's being all fragmented
here, due to the reactive oxygen species. And here is the exact
same type of specimen here with these oxygen scavengers
now added to the buffer system. And you can see that
photobleaching is minimized, and the kinesin molecules
are very happily moving the microtubules, and the microtubules aren't
being fragmented. And so this just shows how powerful
removing molecular oxygen can be. So if we're working with
living cells or tissues, we can't obviously remove molecular oxygen.
So we have to think of other strategies for minimizing photobleaching
and toxicity. And the basic strategy is to minimize the light exposure
on your specimen. And naturally, there's a trade-off here.
The more photons that we illuminate the specimen with,
the more fluorescence will be produced, and we get a better
signal to noise, but if we over-illuminate, we also risk the
danger of also creating more photodamage and in that
way, actually interfering with the processes in the cell that
we're actually trying to observe. So there are several things that we
can do. First of all, shutter the light source. During times
when one's not actually collecting the image, the light
source should be turned off. And there are many ways now to
shutter a light source and control it with a computer.
Secondly, is just to minimize the light exposure.
Again, we want to collect a decent signal to noise, but
particularly, for every light sensitive processes in cells,
we want to minimize the light exposure. And that can be done
in a few ways. One is through the illumination intensity itself.
And also the exposure time that we are collecting photons
on the camera. So, between the intensity and the exposure
time, that gives you your signal. And one has to really
think about both of these parameters very carefully so one's getting
a good image, but again not over illuminating and potentially
causing photodamage. And the other thing we can control
in a time lapse movie, is the interval between exposures.
And that depends on the dynamics of your process, but
if you're working and trying to observe a very slow process
that's going on in a cell, we have the luxury then of shuttering
the light source for a longer period of time in between exposures
and that also minimizes photodamage. Another big breakthrough
is really using very sensitive cameras. And elsewhere in the course,
you can learn about these new cameras called EMCCD cameras,
and these have been a really big breakthrough in live cell imaging.
Because they're extremely sensitive, and that allows one to
use very low illumination of the specimen and still collect
very good images and create less photodamage.
And the other potential strategy is also to minimize out of focus
light exposure. If one's trying to collect a particular plane of
illumination and image that plane, but one's bathing the specimen
above and below that plane with light, you're potentially just
creating photodamage in the cell. But that out of focus light is not
contributing to the image. So there are a couple very powerful techniques,
total internal reflection fluorescence microscopy and light sheet
microscopy, are both very good ways to minimize out of focus
light exposure. So these are a few tips and I hope you
can employ them and remember, you want to create
great images, but also keep your molecules and cells happy!
Thank you.