Measure. Analyze. Learn.
Hello, I'm John Melville, and I'm going to talk to you today
about one of my favorite devices that we have
here at Vernier, the SpectroVis Plus.
This is our little array spectrometer,
which can also be used as a fluorometer.
And, I'm going to be talking to you today about
how you can actually use it as a fluorometer.
Now, what a fluorometer is, is a way to use a device
to look at what we call fluorescence.
And, there are several different compounds that fluoresce.
Chlorophyll is one compound that fluoresces,
it glows a nice bright red.
You may not know this, but all scorpions fluoresce
when they are, when you shine a UV light on them.
So, I have a little extraction from some scorpion cuticle.
And then, you may have heard of this other compound,
it's actually a protein called GFP,
which stands for green fluorescent protein.
And, this is derived from a protein from jellyfish that
is now used, it's a really really important protein that's
used biotechnology, the biomedical industry, as a marker.
And, you can actually show that GFP also fluoresces
bright green using our little spectrometer.
Now, the way that you actually use this device to show
fluorescence, is instead of using the normal light source,
this normal white light source for absorbance.
We're going to be using one of two LEDs.
On the device there is this purple LED,
which means it's at about 405 nanometers.
That's where it's centered at.
And then, we also have a green LED up here,
which is centered at around 505.
I'm going to be using this little purple LED at 405,
because that's the best to make chlorophyll fluoresce,
or basically glow; scorpion cuticle glow; and then this
special type of GFP called uvGFP.
Now, to set the device up to measure fluorescence,
I have to change the units on the device.
I actually have to change the 'Mode'.
So, I'm going to tap here, on the meter screen.
And, I'm going to go to "Change Units".
And, I'm going to select "Fluorescence 405 {nm}".
And now, if you were to look down inside this
little spectrometer, you would see
that there's a little purple LED that's on.
Now, one of the reasons why I love this little spectrometer
so much is that I can actually see
the entire fluorescent spectra of a compound.
That means I can see whether it's emitting light
in the purple, all the way out to the red.
So, let's take a look here at chlorophyll.
I'm going to put the chlorophyll in here.
And if you were to look down into the device,
you could see that it's glowing a little bit red.
So, we should see some fluorescence in the red.
Now, another nice thing about fluorescence is
you don't have to do any type of calibration.
You just take the sample, put it into the spectrometer,
and then just hit 'collect'.
And there is the action spectra,
the fluorescent spectra, from chlorophyll.
And, you can see that there's this nice peak here in the red.
Now, there's a really nice trick here with our spectrometer,
if you don't want to see this rainbow background,
or you just want to see this little strip.
All you have to do is double click near the top of the screen.
That's what I'm going to do right here.
So, now you can see there's this nice peak.
There's actually, it's not emitting any light in the blue.
It's only in emitting light in the red, right in there.
Now, you can see that this is at about 0.6,
what we call relative fluorescence units.
I can actually change the size of this peak,
by going back to the meter screen,
and changing something that we call the sample time.
Now, you only want to change the sample time if
you are in fluorescence mode.
You would never want to do this if
you were using this device as an absorbance spectrometer.
To change the sample time;
you just go over here where it says "Mode";
and you change this number here, that says "Sample Time".
And, you can increase it, to make the peak larger,
or decrease it to make the peak smaller.
To really see a nice peak, you want the
fluorescence peak to be at about 0.8 or 0.9 relative units.
So, I'm just going to increase this to "100".
I'm going to select 'OK'.
Then I'm going to hit 'play'.
And, I'm going to 'Store' my latest run.
And there we go, I'm going to hit 'stop'.
And now you see we have a nice peak.
It's at about 0.804, nice shape.
And, if I wanted to make this peak smaller;
Let's say I had too much chlorophyll in this little cuvette,
and it was just overriding the spectrometer,
I could decrease the sample time to make the peak smaller,
so I could actually see what it actually really looks like.
Now, there are lots of other compounds that fluoresce.
So, I'm just going to 'Store' this run.
Let's take a look at some scorpion cuticle.
Now, all scorpions fluoresce.
It's one of the ways that you can actually
see scorpions out in the desert,
is you can actually take a UV light and shine it on them,
and they will glow a bright green.
And we still don't really know what the compound is
that actually makes them glow this color,
but if you look down into this little cuvette,
you can see that there's this nice little green emanating from it.
So, I'm going to hit 'collect'.
And, let's just see what the
fluorescent spectra from this scorpion is.
See, there's very very little fluorescence there,
so I'm going to hit 'stop'.
And, I can see a series of peaks.
But remember, this is actually quite low.
So, what I'm going to do is,
is I'm going to increase the sample time.
So, I'm going to go back here.
I'm going to go to "Full Spectrum".
And, I'm going to increase the sample time quite a bit.
I'm going to say, let's take a look at about 800 milliseconds.
What I'm doing when I'm changing that sample time is,
I'm telling the detector in the device
to just gather much more light.
Say, gather light for at least 800 milliseconds.
So, now I'm going to hit collect.
Let's 'Discard' that last run.
And it's going to take longer to collect a spectra.
And there we go.
Now you can see I've actually maxed out the detector.
It's actually well over one.
So what I would need to do then is just
adjust the sample time down a bit.
But, you can see, we can actually see this
fluorescence spectra much better now.
Instead of having a little teeny peak, we have a nice big peak.
So, I'm going to go back here.
Let's change it to, how about, 600.
There we go, that's very nice.
So, if you look now across, right here,
you can see that this scorpion cuticle,
the peak fluorescence is, it's mostly right here in the green.
And, that's the color that a scorpion would look like
if you hit it with UV light. It would have this nice green color.
Now, there's many different compounds that are
in scorpion cuticle that make it fluoresce this color.
But, I just wanted to show you that you can actually
look at the fluorescence of other things.
Now, I also talked about green fluorescent protein.
Green fluorescent protein is a protein that was originally
found in jellyfish, that when hit with blue light,
or in this case, it also can be close to UV light, around 405 nm,
it glows bright green.
Why this compound is so important is because it can be used
as a tracker to look at gene expression,
to label different proteins,
to even label cells in mice or bacteria.
It's revolutionized how we do biology today.
And, you can actually demonstrate to your students that
green fluorescent protein fluoresces; just by taking it
and placing an extract of it here into the cuvette holder.
And, you can already see it's glowing this nice blue color.
It's actually is glowing greenish-blue, and we'll be able to
see that when we collect a spectra.
Now, what you may notice is that this is
much brighter than anything else that I've put in here,
so I'm probably going to have to
dramatically reduce the sample time.
So, I'm going to go here.
And, I'm going to decrease this back to "100".
And then, I'll hit 'OK'.
Then, I'll hit 'collect'.
There we go.
So, the peak from the green fluorescent protein
is right here, in the blue-green.
You can see that there's this other fluorescence
that's peaking up here. That's just actually from the purple LED
that's actually shinning through it, there's a little bit of scatter.
But, what we're really interested in
is this nice bright green-blue peak.
And, once again, I could increase the sample time
or decrease the sample time to show that peak.
Now, if I wanted to get rid of this peak from the actual LED,
there is a way to change that.
I could just go back over here.
Go to 'Mode'.
And I could say, "Well, you know, I don’t' really want to look
at anything below where the LED is centered."
And, I know it's at 405.
So, why don't we just take a look at
from about 415 nm to 900 nm instead.
And, I can hit 'OK'.
Hit 'collect'.
I'll 'Discard' that last run.
There we go, I've still got a little bit of it in there.
See, I could cut it off at around 450.
So, let's go back.
Let's just say,
"No, I just want to look at things from around 460."
There.
So now, I'm only going to be looking at
wavelengths from 460 to 900.
Once again, I'll hit 'collect'.
Hit 'Discard'.
And, there we go.
And so, we can see that this green fluorescent protein,
its peak emission from its florescence is right at around
505 nm, which is, well, green.
So, if you have any other questions about fluorescence,
or about spectroscopy, or about anything at Vernier,
just visit our website at
www.vernier.com.