Advanced microscopic techniques and nanotechnology, inglese, Microscopy, Medical biotechnology

I

ass 0

are two, the amount of energy absorbed is no longer proportional to the

intensity but to the intensity squared (I ), because you need two photons

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I

ass

to be at the same time and hit the same molecule. This means that you need

more photons because they have to be in the

same place at the same moment.

In linear absorption the intensity of excited

fluorophores is proportional to the intensity of

shining light while their number is proportional

to the intensity of energy: the total energy is

but the intensity varies

always the same,

through the hourglass.

number of fluorophores excited is always the same the density is

So the but

greater in the middle. linear absorption.

This is what happens with

The profile of the intensity of the beam is like a

Gaussian and this is also the profile of the intensity of

the excited fluorophores, if the probability is

proportional to the intensity (as a single photon).

In two photons, if the probability is proportional to

the squared of the intensity, the probability of

much larger in the centre

excitation changes: it is

and less in the side, much more

so it becomes

probable to excite the fluorophores in the centre of

the beam.

With double photons emission I’m concentrating the excitation in the centre of

the beam,: with this technique only molecules in the centre of the beam get

excited and so I narrowed the region where I can excite molecules!

lose resolution because I use a bigger wavelength,

However using this method I

using a smaller portion of a larger beam.

What do we gain? When I shine light with linear excitation, I’m exciting photons

everywhere where the beam goes, while if I have two photons I’m just exciting

where the beam is minimum without the

need of having a pinhole (I’m doing a sort of

scanning microscopy!).

The other thing I gain is that I have much

less photobleaching because I’m exciting

only the centre of the beam so I’m not

wasting fluorophores below and upper, so

the fluorescence I produce is just where I’m

looking at. Moreover I use longer

wavelengths so I can penetrate better the

sample.

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much powerful sources,

For this method we also need to use like a million

times more powerful. It is also possible to use pulsed sources that don’t shine

photons continuously but accumulate photons and the shine them all together

in order to have a very concentrate energy within the pulses.

STED (Stimulated Emission Depletion fluorescence)

In order to have absorption I have to shine light, while to have an emission I

don’t have to shine light.

molecule to emit energy if I shine it whit the

I can convince a

same light that it emits: for example if I shine my molecule in

green but also in red (that is my emission light, as in figure),

the energy decay become faster. Not only I release my light, I

release it in the same direction and with the same maximum

stimulated emission is the phenomenon by

and minimum:

which the rate of emission of an excited molecule increases

proportionally to the intensity of light that it is illuminated by, if

the light is the same wavelength of the fluorescence emission

(1’).

STED uses two lights:

excitation

- One for the excites the fluorophores



stimulated emission

- One for the makes the fluorophores de-excited.

 The number of molecules which

are on level 2 (dn ), the energy

2

from which we started to get

fluorescence, change in time by

virtue some terms one of that is

proportional to the intensity of

light that shines (h ) times the

STED

difference between level n and

3

level n .

2

If level n is more populated than level n light comes and goes down.

2 3

If level n is more populated than level n the light can also go back.

3 2

(formula riquadro rosso).

I can de-excite the fluorophores with light.

So in STED I’m using two beams of two different shapes:

excitation pulse normal

- The is a focus beam



de-excitation pulse donut-shape

- The is a beam darker in the centre and



bright on the side.

So I excite molecules were the beam is, then I turn down the fluorophores

around with the de-excitation beam. 16

Non linearity is used in de-excitation and

to do that you have to shine stronger and

stronger.

Increasing the power of light, the hole

become smaller and smaller because the

probability to getting de-excitated is not

anymore proportional.

Since the stimulated emission is brought

in a non-linear regime when you increase

a lot the power, the region where the

fluorophores are not de-excited becomes

really small: increasing intensity the light areas become saturated and dark

areas are contracted. We gain much if the turning off is proportional to

the intensity: I have to use non linearity in de-

excitation, so in turning fluorophores off and I can

do this shining stronger, with super powerful pulse.

With this method of increasing power the hole

becomes smaller and smaller: the probability of

being de-excited is proportional to the de-

excitation pulse when this pulse is not very

powerful; because the probability of being de-

excited cannot be more than a limit, when the de-

exciting pulse goes over this limit, all the

molecules become de-excited.

*Dashed lines indicate the percentage of quenched

fluorophores.Upon entering the non- linear regime

the region where we can still have excited

fluorophores shrinks!

The much I increase the power of the de-excitation light, the narrower becomes

the region where I still have fluorophores emitting light

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*the dashed blue line represents the 100% quenching level.

This technology is made up of a first green excitation laser and

then a second read beam that has a donut-shape; the size of the

hole is the resolution; the red pulse is much stronger and a little

delayed compared to the green beam. After I’ve de-excited my

fluorophores on the side, I’m able to look the remaining ones at

the centre. scanning technique

At the end what I have is a where I have a

smaller region but with very concentrate fluorophores.

10.10.2016

Excitation is intrinsically a stimulation, because electrons absorb energy and

move to the next orbital.

Stimulated emission is instead a new concept: fluorescence life time (the time

the molecule stays excited) can be reduced when the molecule is shined at the

same wavelength it would emit.

This technique uses two beams:

Excitation beam

- normal shape



Quenching beam

- donut shape, with a hole in the centre



The excitation pulse has the same usual shape of a light beam.

Light beam is usually a Gaussian, a peak. It’s

more intense in the centre, less intense in the

sides. The intensity walking along x will start

with low intensity, than it reaches the

maximum at the centre, than it goes off on the

other side. This is a linear excitation because

the number of fluorescent molecules excited is

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proportional to the intensity of light. The density, the number of molecule over

volume (for example the number of molecules in a cube of 1μm volume) that

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are excited after this pulse, will have the same shape so there will be more

excited molecules in the centre of the area.

If I cut in half the intensity of the beam, the density of the molecules will be

half.

The second beam has the opposite shape:

from zero, it goes up to the maximum, goes

down to zero in the centre and then it goes

up again to maximum and down again. This is

the quenching beam, the stimulated

emission.

If it is linear, the fraction of excited molecules

which are quenched is proportional to the

intensity, so it will de-excite more where

there are the peaks at the edge and less where it goes down in the centre.

For example: we have some flourescent molecules and we excite a fraction of

them and then with the de-excitation pulse we quench a fraction of the excited

molecules proportionally to the intensity.

If we double the intensity we also double the number of quenched molecules.

If both beams are linear we don’t gain anything because the width of the

excitation beam peak is the same of the hole in te quenching beam.

If I increase the intensity of the second beam, I increase the fraction of the

excited molecules that are quenched, and if I increase the intesity a lot I can

reach the 100% of the excited molecules quenched. But if from this intensity I

double the power, I won’t double the effect because I can’t go over 100%!

So the pattern of quenched molecules has the same pattern of light as long as

they are proportional: when I have a quench beam that de-excite 100% of the

molecule, if I augment the intensity I will shrink the hole, so I will shrink the

area where the molecules are still excited and the only fluorophores that

survive are the ones in the centre. This is non-linearity.

The blue dash is the line where the 100% of

fluorophores are quenched.

Look at the four profile patterns each one

stronger than the precedent from blue to

violet: assuming that the smallest (1) at the

maximum of the intensity de-excites 100% of

the molecule in that area, if I increase the

intensity (same wavelenght) I can quench

molecules proportionally to the intensity, and

so in a bigger area (because it’s not linear).

In this way I can shrink the area where

molecules are still excited: for each pattern the area where molecules are still

excited is smaller than the area of lower intensity pattern.

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Summarize: In order to perform STED I use two beams:

Excitation beam is a pulsed laser with an

 ordinary Gaussian shape

Quenching beam is a pulsed laser that is higher

 in intensity and has a donut shape; the size of

the hole is usual 0,5 m.

The first pulse excites the fluorophores, the second

beam quenches them: if the STED beam has a power

zero, I can see the signal from all the fluorophores

excited. The larger the intensity of the second pulse,

the fewer are the fluorophores that remain excited.

Usually this technique allows to see the 10% of the

fluorophores, while 90% are de-excited: the 10% that

are left are the ones in the centre of the hole.

The shape of the light coming from my fluorophores is

much more narrow so I can move my resolution from

0,5µm (black spike) to 100nm (red spike).

Also you can stain your molecules with two different fluorescence proteins (GFP

and YFP) and you can excite them with the same beam and then quench them

by shooting very hard staying on the same wavelength, then looking at the

emission using a dichroic mirror to recognize the two different emission (paper

– two colours STED...).

So STED is a super-resolved op