Advance microscopic techniques and nanotechnology

FLUORESCENCE

For any particular molecule, several different electronic states exist,

depending on the total electron energy. Each electronic state is further

subdivided into a number of vibrational energy levels

associated with the atomic nuclei and bonding

orbitals. At room temperature, in the liquid phase,

very few molecules have enough internal energy to

exist in any state other than the lowest vibrational

level of the ground state, and thus, excitation

processes usually originate from this energy level.

Fluorescence is the gain of absorption by the molecule.

1. Excitation of a susceptible molecule by an incoming photon

2. Vibrational relaxation of excited state electrons to the lowest energy

level → Then the vibration energy is lost because of collisions

3. Return of the molecule to the ground state and emission of a photon

The energy emitted is lower than the energy absorbed: lower energy, lower

frequency and longer wavelength.

When we talk about fluorescence, we usually talk about fluorescence

intensity (of emission). However, there are other features of fluorescence

that are “invisible”, meaning that they are not the straight emission:

- the lifetime of fluorescence → how long does the molecule stay in the

excited state, so the delay between the absorption of the photon and

the release of the other one

- fluorescence anisotropy → fluorescence anisotropy or fluorescence

polarization is the phenomenon where the light emitted by a

fluorophore has unequal intensities along different axes of

polarization

So fluorescence can be observed as intensity of light, or we can look at these

other 2 things (lifetime and anisotropy). FLIM uses the lifetime.

POLARIZATION

We have 2 characteristic times:

- lifetime of the fluorescent excited state (tF)

- lifetime for rotational diffusion (tD)

→Both in the range 1-50 ns

Because of their anisotropic chemical structure, all fluorophores have a

preferential direction of absorption. Each molecule has an axis such that

light polarized along it is more efficiently absorbed than light polarized

perpendicularly to it. Analogously, emitted fluorescence light is polarized

along the same axis.

By illuminating a system with polarized excitation light, the fluorophores

with their axis along the polarization are more probably excited than those

perpendicular to it.

Thus, if the fluorophores rotates slowly (fluorescence lifetime larger than

rotational diffusion) fluorescent emission is polarized in the same direction

of the excitation light. While if it rotates fast (fluorescence lifetime smaller

than rotational diffusion) the polarization is lost

How is a molecule with a good absorption made? We need delocalized

electrons, so we need aromatic groups (like phenyl rings) and double

bounds. These are the situations where we have resonance, so the electrons

can move. This structure is not spherical but elongated in one direction.

The light comes: the electric field is oscillating vertically and if the

polarization of the light is vertical and the position of the molecule is

vertical → I am absorbing more light. So If I have vertically polarized light,

the molecule will absorb more if it is in a vertical position rather than in a

horizontal one. What happens? The molecules that are by chance vertical

are absorbing more than the one that by chance are horizontal.

In our sample we can’t control the direction of the molecules. When I have a

vertical polarized light, only the molecules that are by chance vertical

absorb more than the molecules that are horizontal. So I am making a

selection in the direction of fluorophores. If a vertical molecule is excited,

the electrons are moving vertically and they acquire energy in the vertical

axis, and they emit light in all direction, but this light is polarized in the

axis of the molecule (vertical).

We have a system of molecules in every direction and assume that they don’t

move. If vertical light comes, I excite more the vertical molecule and I have

mostly light from these. So if the molecule don’t move, when I shine

polarized light, I obtain a polarized fluorescence (actually, not completely).

Now let’s leave the molecule free to move. What will happen? Would I still

have a polarized fluorescence or not? And what could it depend on? The

fluorescence will not be polarized. So If the molecules are very slow the

fluorescence is polarized, while if they are free to move very fast we don’t

have polarized fluorescence. By that, I can know something about the

molecule, for example if it is moving faster or slower than the fluorescence

lifetime: if the molecule moves faster than the fluorescence lifetime, it’s not

possible for the molecule to emit polarized fluorescence.

The fluid is illuminated with a polarized excitation light with the use of a

linear polarized. Fluorescence emission is measured after having been

selected by a second polarizer, that can be parallel (||) or perpendicular (⊥)

to the one of the excitation light. The fluorescence intensity measured in

the two conditions (I and I ) can be either the same, meaning that the

|| ⊥

emission is not polarized (P = 0) or I can be larger than I , which indicates

|| ⊥

polarized fluorescence emission (P > 0). If P = 0, the emission is not

polarized and the intensity parallel and perpendicular are the same. If P = 1

there will be a total polarization, but usually it won’t reach the maximum of

polarization, so if there is polarization in the emission, P > 0. So P can go

from 0 to 1.

If a fluorophore is bound to a large molecule, or its fluorescence lifetime is

short, fluorescence emission is polarized as the excitation light. When a

fluorophore is free or bound to a small molecule, or when its fluorescence

lifetime is long, the fluorescence emission is depolarized.

DIFFUSION

Diffusion is the process by which since we have thermal motion (the

temperature is not 0), the molecules can move around. These movements are

random and they are the movement by which for example an mRNA can

reach a ribosome. Diffusion has a certain speed, direction and rotation.

With normal (linear) diffusion, the larger the molecule, the slower the

diffusion. The same happens with rotational diffusion: the larger the

molecule, the slower is the rotational diffusion. So we have a linear diffusion

and a rotational diffusion, in fact molecules can move back and forth, but

also in their axis. Upon interacting with other molecules, or because of

chemical reactions, the rotational diffusion can be modified, and thus

detected by depolarized fluorescence

We can use this property to study ligand-receptor interaction. We have a

ligand with a fluorescent group, and it rotates pretty fast with rotational

diffusion. If we excite the fluorescent group, by the time the fluorescence is

emitted (so in the duration of fluorescent lifetime), the molecule is free to

rotate, so the polarization is lost. But if the ligand interacts with the

receptor (forming a larger complex), the speed of rotation is slower, so the

light remains polarized (because the molecule does not have the time to

rotate much during the lifetime). So by just looking at the light with a

polarizer, we can see something that happens in nanoseconds. So anisotropy

is based on the rotational diffusion, that is a kinetic property of the

molecule.

We care about how fast molecules are moving in order to understand if the

molecules are in complexes.

Anisotropy is a property based on the kinetic of the molecule. Similar to

normal diffusion, we have rotational diffusion: because of collisions,

molecules change their direction. If we fix the center of the molecule, it

doesn’t move but it rotates. The laws of orientational diffusion are the same

as linear diffusion. The rotation depends on the rotation coefficient, that

depends on the temperature (the larger the T, the more the molecules

rotate) and it is inversely proportional to viscosity and size.

P is how polarized the

fluorescence emission is.

As the molecule is larger, it rotates slower, so the polarization increases,

because during the lifetime the molecule has no time to rotate much. The

curves are separated by 4 examples of 4 different fluorescent lifetime. If the

fluorescent lifetime is 16 ns and the molecular weight is 50, we have some

polarization. If the molecule is smaller, the polarization is smaller because

the molecule rotates more. The shorter the fluorescent lifetime, the more

polarization we have. The longer the fluorescent lifetime, the more

molecules have time to rotate so we have less polarization.

On the right: we have a kinase that produce ADP. We have a fluorescent tag

attached to anti-ADP antibody, so it is big and slow. Then the kinase

produces other ADP, but this is without the fluorescent tag. The antibody

now binds to this ADP without the fluorescent molecule (it’s a competition

assay) and the ADP with the fluorescent tag remains without Ab, so it is

smaller, and we get less polarization because it can rotate faster.

We can have commercial beads that are selective for the phosphate

produced by phosphatase. If we have a solution with a fluorescent peptide

and it is added with a phosphate group, the size is bigger, so the

polarization goes down.

In a microscope we have to put the first polarizer after the excitation lamp

and the second polarizer where we observe. We can have some fluorescent

drug molecule and measure the intensity in the cell. We can do it in 3d (it is

confocal). So we can measure intensity, or anisotropy: you can show the level

of polarization and we can see that in that region the fluorescent is weaker

and more polarized, so we can say that the fluorescent molecule reached the

target and now it is stuck. So if we have a small fluorescent molecule, it

doesn’t have anisotropy alone because it rotates fast. But when it binds to a

target, it is bigger and can’t rotate as before, so we get polarization.

FRET

FRET: fluorescent resonant energy transfer. The donor is excited and gives

the energy to the acceptor, that emits fluorescence → transfer of excitation

energy from one fluorophore to another one in close proximity → the

emission energy of A excites B, without emission of light.

To have FRET, the emission spectrum of the donor and the absorption

spectrum of the acceptor must overlap. This doesn’t happen with the

emission of light, but with the oscillation of the magnetic field.

What distances can we measure with FRET? Nanometers. FRET becomes

impossible when the molecules are too far from each other, in particular 10

nm.

The resonance leaves the fluorophore bands unchanged, but turns on the

acceptor, while turning off the donor → in practice, the emission has the

wavelength of the acceptor instead of the one of the donor.

The transfer efficiency is E = intensity given out by the acceptor ratio the

intensity that would be