welcome to lecture number forty in the last lecture we have discussed about the total
internal reflection geometry to achieve very tiny observation volume and we were
also discussing about this confocal geometry to create the small observation volume
in the confocal geometry i was showing you this uh diagram with this animation that if
i have this excitation light and this is my lens so these molecule is actually defusing
and when this molecule will enter this observation volume it will going to show the fluorescence
right and when it will go out right then we will not going to see the florescence
so this is given here right you see in this case this is my excitation laser beam right
when it will reach this sample position let let me define this samples are present throughout
lets say this is your sample position starting from here everywhere is your sample everywhere
is your sample and when so all are sample right all are sample so if you just follow
this excitation light i will just take this which colour is this blue one so
i will just take the same colour and i will redraw this excitation light please follow
so the excitation light is coming like this way this way this way and it is exciting throughout
this this is the other side of this excitation light beam and then it will excite throughout
that means whatever the molecules are present within these shaded region let me shade it
nicely so these shaded region will be excited will
be excited whole shaded region will be excited but obviously these volume is much more right
it is not a like a femtoliter one femtoliter so to get this one femtoliter i must restrict
my observation to some part of that the tiniest part in the width of this excitation beam
as i told you in the last class will be somewhere in the focal plane that is the tiniest one
right if you remember i showed you right so this is the spot size right and so the
observation volume if i can restrict around this region then that will be the tiniest
right so if you restrict your observation around this region then the size the volume
will be more than this thats for sure so i need to do something over here so that
right although the molecules which are present here or here or here or here everywhere the
molecule will give fluorescence but i will not going to detect the fluorescence from
everywhere i will going to detect the fluorescence only from a specific position right within
this observation volume how to achieve that that is the question right
it is simple that if you just put a pinhole over here right i just put a pinhole now let
me erase all those thing and you follow this line as i told you that this blue one is your
excitation [safe divide] excited that means the molecules which are present at these position
that is also going to be excited the molecule which is present at these position that will
also be excited and the molecule which is present at these position that will also
to be excited now you you follow this line this line the
molecules which are present below the focal plane right after you putting the pinhole
lets say first you follow the line the molecules which are present at the focal plane so if
you follow this line with a basic knowledge of your optics you follow and you see this
rayed actually passing through this pinhole and reaching the detector that means the fluorescence
which is originating from this position this focal plane actually is reaching the detector
because this pinhole pinhole position is over here right
now keeping this pinhole position same however dont move it now keep the pinhole position
same keeping this pinhole position same now if you follow the line the ray diagram
of this light originating from this particular position you see the follow it here it will
be something like this and then it will it will go like this way this way and it is blocked
this part is blocked over this part is blocked over there right so it cannot reach the director
now if you consider this point right if the fluorescence originates from this particular
point you follow this line what you will going to see it is now blocked at this position
and this is now blocked at this position so the placing a pinhole at this position ensures
me that the fluorescence which is originating very near to the focal plane only will be
pass through this pinhole and that light only going to be detected by this detector
that same figure that same concept i have displayed over here in this case see here
that this is your focal plane so when the signal is originating this is your focal plane
right here this plane when the signal is originating from lets say
here at this position it is not passing through this pinhole it is blocked when the signal
is originating from this position it is not passing through this pinhole it is blocked
over here is blocked over here is blocked over here but when the signal is originating
from this focal plane this signal is passing through right see signal is passing through
this pinhole right and this is called this confocal geometry that means it is very simple
we have objective we have this lens and exciting you can excite the sample throughout but just
by putting this pinhole you can actually restrict your observation volume
now i will go back to my this animation now you see here you once you excite your
molecule we will start moving because the molecular motions is random right this is
the brownian motion so once it enter over here then this signal is actually passing
through this pinhole where with this pinhole and you will reaching the detector but when
the signal is like this when the molecule is present over here then that signal is blocked
when the molecule is present over here then the signal is also blocked over here
so you will going to get the signal only when the molecule is present within these volume
and the height of this will be guided by the width of this pinhole right there is a direct
relationship from this width of the pinhole and height and if you now consider that is
a gaussian shift of this laser beam then with using some mathematics you can actually
calculate the volume of this observation right so it is called the detection volume or observation
volume this region or observation volume so thats it
if i can achieve this observation volume then i can see the single molecule when the molecule
will present inside this observation volume then it is visible when the molecule is present
outside outside if the molecule is present here it will be excited it will going to give
you fluorescence but it is not visible because of the because of this pinhole right once
the molecule is here obviously it will give the fluorescence then only it is visible
if the molecule is present here right then it will not going to fluoresce if the molecule
is present here it will fluoresce but it is not visible if the molecule is present here
will not going to fluoresce so i can restrict my observation and i can achieve this femtoliter
type of observation volume now consider that you have some matrix let
me draw this matrix over here you have some matrix and you have all the molecules are
present right throughout this matrix lets say this is your matrix lets say this is a
polymer matrix and in this polymer matrix the molecules are present right and you have
sufficiently dilute condition so that the um this is between the two molecules are
far apart one molecule is here another molecule is here like this molecules are present but
as this is solid matrix this is in this plane right in this plane and if you just take a
cross section probably you will see one molecule here another molecule here another molecule
here another molecule here here here random random distribution like this right
so now if you want to image how the molecules are distributed in this matrix what you will
going to do you will just put this slab (Refer Time:10:00) polymer slab having all these
molecules embedded inside in a confocal setup right in the confocal set up you have
this observation volume the observation volume is having certain width and certain height
right the width is guided by this deflection limited focusing which is the approximately
half of this wavelength right so you will get a spot size like this way and then
you will get a depth because of the presence of pinhole
lets say this is this is your resolution so if you scan what you will going to see either
this layer of this molecule this is this is layer one right lets say and obviously
below that there will be another layer right below to this layer there will be another
layer this is the layer by layer so depending on which one you are actually
so this is the layer by layer thing so depending on the height of this observation volume to
the sample you will be able to see one layer now if you look at one of this layer what
you will going to see you will going to see these molecules because either because you
will going to scan right this observation volume the observation volume is here then
you scan either in in x y plane you will scan in the x y direction so then we will going
to image everything so after you scan whole thing at a particular plane out of this slab
obviously you will going to see that one fluorophore is present over here another is over here
another is present over here it is random i dont know i am just writing i am just drawing
it probably you will going to see such kind of image (Refer Time:12:00) one is present
over here here please note that this spot size right
is in the order of two hundred three hundred micron obviously depends on the excitation
wavelength the light wavelength of the light you are using to get the spot size and that
is called the [average deflection] limit and in this case the spot size will be almost
the half of this wavelength right if you are using two hundred four hundred nano meter
light for this excitation this spot size will be around two hundred nano meter
obviously the molecules right they are not like two hundred nano meter they are about
one yes one nano meter but still you will get a bigger spot size because of this deflection
limited focusing but no problem if the intensity of this spot this spot is same as this spot
is same as this spot is same as this spot that means i can conclude that each molecule
sorry each spot you will have more a similar number of molecule and in this case the number
is one right because you have taken a reasonably diluted condition so number will going to
be equal to one but if you look at this real image of this
the first thing what you will going to see is this this is
these are the molecules right what you will going to see is that some of them are light
some of them are dark i mean it means that intensity is more and for some intensity is
even more so there is some distribution in the intensity does it mean that more than
one molecule is present yes i have taken sufficiently dilute concentration then it is unlikely that
in one place two molecules will be there in the other place none of them is there right
for this even distribution of this molecule in this particular medium considering that
there is no specific interaction and all the location in this matrix are equally probable
for this type of molecule which is present in the system
the reason behind such kind of difference in the fluorescence intensity from each of
the spot maybe because of the different orientation of this molecule as i said earlier during
our discussion on fluorescence anisotropy that the direction of the the polarization
of this excitation light right will excite selectively or preferentially those molecules
whose transition movements are parallel to the electric field of this excitation light
right so its possible that for this molecule the excitation is more favourable because
its transition movement is oriented in parallely to the electric field of the excitation light
in this case it is not right its possible so in this case the different molecules
are not aligned in a similar fashion right sometime if you take this kind of image
you would also see such kind of feature like this is your this is not a full circle
this is a half circle right lets say this is the full circle this is again full circle
this is full and lets say this is half right you will also see such kind of image right
so i am just considering intensity is that same just for the time being you will going
to see so what is the reason for this right because you you are going to scan your laser
right throughout this matrix here when you are scanning you are scanning like this let
me change the colour quickly you are scanning like this you are going from here to here
you are coming like this and then like this and then like this and then like this right
like this you are scanning and you are creating this image starting from here here and then
like this like this right
it means that when you are seeing here this fluorescence is on but when you are coming
back it is off that is the half circle is fluorescent not the full circle right that
means if i sit on a particular fluorophore and keep on monitoring the fluorescence intensity
right it must show the on and off behaviour and exactly the same when you see it on a
particular fluorophore in this matrix right you can just focus on this one right you just
focus on this one and monitor how the fluorescence intensity is changing with time right
then if you plot this fluorescence intensity with time what you will going to see is a
kind of fluctuation the fluorescence intensity is more and it is like this and then it become
like this and stay time sometime from here then again it will increase it will stay like
this way and then again it will come back and stay like this and ultimately it will
never come back it is zero and this is called the blinking of fluorescence this is basically
because of this conversion of the molecule from the singlet state to the triplet state
when the molecule will go to the triplet state obviously it will take some time to come back
from the singlet state and fluorescence is only from the singlet state that we already
discussed right in the triplet state florescence is typically
quenched by this different quencher present typically by the oxygen molecule dissolved
in this your system so we will not going to see the fluorescence at the room temperature
so once the molecule will go to the triplet state it needs to come back and that takes
time and that is called the my off state so here is my off state and here is my on
state and so on but if you keep on excite the molecule right many times then the
molecule may destroy and it is the very common feature and this part this part here the molecule
will never come back to give you fluorescence that means the molecule is destroyed and this
is called the photo bleaching right ok
so these are the typical behaviour of this molecular fluorescence but single molecule
fluorescence now if you just sit over on a particular molecule right and you just
keep on monitoring until and unless this molecule will go back that means photo bleached right
then you can get several information about this molecule for example you will going to
see the single molecule spectra you will be able to see single molecule
lifetime you can see the application of rate in the
single molecular level and many many many other application is possible
but i would like to tell you one important application which is not in the solid matrix
but in the solution state right and if this is my observation volume if this is my
observation volume now you have a clear idea about this observation volume right when the
molecule will come within this observation volume is my my molecular trajectory here
the molecule is nonfluorescence the molecule fluorescence will be detected only when it
is present within this observation volume throughout this path right throughout this
path the molecular fluorescence will be detected and the molecule right will take some time
to cross this observation volume if the molecule is entering from this side and taking such
kind of path then this fluorescence will be on for longer time than this if the molecule
is coming from this side and exists from this side then the molecule then the fluorescence
is on from for a shorter time right so here the fluorescence is on for a short time on
here the fluorescence is on for this time and in this case the fluorescence is on for
a much longer time if the molecule will come like this way then the fluorescence is on
for this time right so if you now monitor the fluorescence right
as a function of time then what you will going to see you will see the there is no fluorescence
this is the fluorescence intensity this f t is the fluorescence intensity from the observation
volume right so in this case the fluorescence intensity is on for when it is present like
this so fluorescence intensity is on and then fluorescence intensity off that means none
of this molecules are present here if none of them is present although the molecule here
is getting excited because your excitation light is like this right it is excited it
is excited but you will get the fluorescence only when this is not excited right this is
excited but you will only get the fluorescence only when it is present within this observation
volume that height is determined by the width of the pinhole the size of the pinhole right
so then again one the once the molecule will enter it will be like this and then like this
like this like this and so on right now so so there will be a fluorescence intensity
fluctuation right i can simply write this as intensity fluctuation now you consider
the diffusion of this molecule in and out of this observation volume is slow right then
this self-similarity of this florescence fluctuation will be more because the molecule is present
for the longer time within this observation volume the florescence is on and if it fluctuate
rapidly then the self-similarity of this fluorescence fluctuation will be less so that depends on
how quickly the molecule is coming in and going in and coming out of this observation
volume right so if i consider a short time period for very
short time period right obviously there will be lot of self similarity right if i consider
this this fluctuation now and this fluctuation after a very small time that means the fluctuation
should be very similar right then there is a lot of similarity but if i now consider
this fluctuation and same fluctuation after certain time after sometime then the selfsimilarity
will be lost but if these fluctuation widths are more right
if this is on for longer time lets say if it is on for this time this whole time region
then it is off and then it is on for this whole time region then even for this case
compared to the black one in this case after a certain time that self-similarity will remain
very high right and that self-similarity is expressed by this fluorescence autocorrelation
function and which is denoted as g tau and defined as fluorescence intensity at time
t fluorescence intensity at time t plus tau average divided by average fluorescence intensity
square right now if you change this tau value you change
this tau value right so the fluorescence intensity at time t and time t plus tau will be very
very similar if the tau equal to zero that means it is the fluctuation will have very
very similar fluctuation because it is same right f t and f t is same but when i change
this tau from one tau to two tau and sometime if i give one microsecond two microsecond
ten microsecond then the self similarity will be lost right and you will get a lower value
of g tau so g tau will start from a high value and g and it will ultimately will going to
be zero when the value of tau is very very large and in this case these g k of this
g tau should be related to the how quickly the molecule is passing through this observation
volume in other words it should be related with the
how this what is the size of this fluorophore if the size of the fluorophore is big then
it will take some time to pass through this observation volume and that can be correlated
easily if the viscosity of the medium is high then the molecule will pass through right
this observation volume the longer time period and it will be it will be seen
in such kind of g tau i will just show you this typical plot
of this g tau as you can see here g tau is plotted against this obviously tau right this
is tau is in the microsecond so here is your one microsecond two microsecond three like
that ten microsecond hundred microsecond thousand microsecond and so on so you see this g tau
value is very high is about one right is about one over here that means there is a lot of
similarity between the f t and f t plus tau at one microsecond right but as that tau value
is changing its going to be zero but there is no such similarity right at ten to the
power three now if whatever i said i expressed this in
mathematics right then it should be related with the diffusion time and if i show you
that particular equation now you see this g tau is related to this tau d that how fast
this molecule is (Refer Time: 28.00) passing through this observation volume and this is
guided by this particular equation right and where n is the number of molecules present
in this observation volume right and you see that this experimental data those circles
this experimental data are now fitted with this equation to get this value of tau d once
you know the value of tau d from stokes einstein relationship you will be able to calculate
what is the volume of this molecule if you know the viscosity and temperature and vice
versa so this is a very important application in
single molecule fluorescence and this particular experiment is known fluorescence correlation
spectroscopy
ok and is in short form it is known as f c s ok so we will finish here as our time is
up thank you very much