Autoradiography and RIA - Biotechnology Engineering Video Lecture - Biotechnology Engineering (BT)

in this lecture we'll continue our
discussion on liquid scintillation
counting if you could recall in previous
lecture we were discussing about the
various aspects of liquid scintillation
counting like we were discussing about
how energy transfer takes place from a
radioisotope to the solid earth to solve
it from solvent to primary floor and
secondary floor and then finally
electrical signal generating through the
photomultiplier tube we have seen
through the different advantages and
disadvantages of liquid scintillation
counting if you could recall where we
have seen that how the liquid
scintillation counting method is very
accurate efficient and it's a versatile
technique particularly for weak beta
emitters it has advantage of counting
more than one isotope at a time if they
have sufficiently different energy
spectrum so you had lot of advantages of
liquid scintillation counter counting
but there were disadvantages also like
quenching was a major problem or photo
multiplier noise was a problem which has
been solved to a certain accident so if
you even if there are certain
disadvantages like cost also but they
could be overlooked because of lot of
advantages of liquid scintillation
counting now today we are going to
discuss about the sample preparation in
liquid scintillation counting so will in
sample preparation you require a lot of
things like sample Wyles what kind of
like scintillation cocktail has to be
taken what is the volume of the
scintillation cocktail how sample needs
to be prepared for proper counting all
these things we are going to discuss in
this lecture now let's start with sample
while sexually as far as solid
scintillation counting is concerned
sample preparation is easy and only
volts transferring the sample to a glass
or plastic while compatible with the
counter but as far as liquid
scintillation counting is concerned
sample preparation is complex and it
starts with selection of suitable while
to be used now types of wiles which can
be used which has a sample wise they
could be glass they could be low
potassium glass with low levels of 40k
and they could be plastic or
polyethylene Wyles
now glass wise provide unparalleled
optical clarity good visibility and they
are chemically inert so making it
suitable for use with aggressive
reagents and where many solvents are
used actually now glass wires can be
reused many times if thoroughly cleaned
and low potassium glass wires could be
used where you can reduce the background
count so you have lot of advantages of
glass while in here now apart from glass
wires you can use plastic wires also now
plastic wires exhibits lower background
label then glass it is compared to
combustible and therefore easier for
waste disposal it is shatterproof and
therefore safer in the laboratory it is
cheaper but problem with this is you
cannot reuse it so polyethylene wires
you better transfer that is better light
transfer and results in slightly higher
counting efficiencies but are inclined
to exhibit more phosphorescence then do
the glass wise so you can choose which
while has to be used it could be plastic
while or it could be glass while now
what size of the boils needs to be used
here now most of the time it could range
from the size of the wires available
could range from somewhere less than 4
ml up to 20 ml in many photomultiplier
tubes it is a kind of 20 ml is the
maximum amount which is fixed due to the
dimensions of certain photo multiplier
oops now many wires may be used to
reduce the cost and in terms of
environmental issues where scintillation
fluids are toxic so you can use a
smaller amount of sample and the
scintillation cocktail now some counters
are designed to accept very small
samples in special polyethylene bags
spins into array of many compartments
for example in pharmaceutical industries
for doing lot of different kinds of
assays you can use these kinds of simple
carriers of wild section then very
important part of sample preparation is
scintillation cocktails now
scintillation cocktails are mostly
solution of floors and there could be
different types of installation
cocktails and this will involve a
solvent as well as primary and secondary
floors now two types of solutions which
are one is which is miscible with a
quest solution and one which are not
miscible with equestrianism now the
majority of radioactive species are
present in a quest for and as such are
not miscible with aromatic solvents
which are your scintillators now the
most commonly used water accepting
cocktail is dioxin and the most common
water immiscible ones are Halloween
based cocktails now problem with this it
will not take much of the Equus
samples as solvent and water are
immiscible and massive quenching might
result a second solvent mixed with 12in
can somewhat solve this problem but not
to a great extent now cocktails based on
dioxin that is one for dioxin and left
naphthalene can accommodate up to 20%
volume to volume water and can be used
but are they have been phased out due to
toxicity for most purposes less
frequently used xylene based cocktails
are suitable as they have greater
efficiency of detection than toluene and
with lower toxicity
but Calvin is more common because of low
cost so the problem of this where it
cannot take a lot of water this could be
solved by presence of surfactants in the
cocktail which enables an aqueous ample
to come into the intimate contact with
the aromatic solvent by forming a stable
micro-emulsion so this could be one
method to accept their quest solutions
so therefore emulsified cocktails are
most frequently used for counting a
quest samples and they are composed of
solvent scintillators and surfactants
which could be like for example triton x
hundred could be used as a detergent
here now these can accept up to 50
percent water but many times problem
occurs which is of phase transition and
they might as water content increases
they might result into two phases
appearing and this could hamper the
accurate counting actually so lot of
ready-made cocktails are available in
market with very precise instructions
that could be used for preparing your
samples with particular scintillation
cocktails volume of cocktail is a very
critical and measurements or counting
efficiencies one has to use the same
amount of volume every time like for the
sample and instruments need to be
calibrated with the same value as for
the experimental samples so this is very
important for efficient scintillation
counting and which should vary with the
sample volume so volume of the cocktail
has to be like same for both standards
as well as like instrument needs to be
calibrated with the same volume as you
are going to use in experimental samples
now another problem which comes when you
are preparing your sample how to
overcome quenching problem now the
quenching problem could be solved but
different methods are there and they
have certain advantages and
disadvantages and the user has to
side and come to particular way to solve
this problem the samples can be bleached
before counting if there is a color
quenching now bleaching agents there are
a whole lot of bleaching agents like
hydrogen peroxide can be used but this
gives chemiluminescence so care has to
be taken by using this hydrogen peroxide
there are solubilization systems like
for example there are alkaline systems
there are acidic systems in alkaline
systems the alkaline hydrolysis and
quaternary ammonium hydroxide or NaOH
could be used in acidic system its
acidic oxidation and you can use
perchloric acid nitric acid or their
literature's could be utilized for
soluble addition there could be lot of
other systems also for solubilization
there are tissue soluble ideas like
solid samples such as plant and animal
tissues are best counted after
solubilization by quaternary amines such
as NCS soluble Iser or solving now
solving 350 is widely used it's a
organic based soluble Iser formulated in
toluene it's a classical industry
standard soluble Iser and it is used for
biological and plant samples it's a
corrosive and flammable so these all
these solutions are highly toxic so they
great care has to be taken while using
them how do you stall you belies it so
simple like you add sample to the glass
while you add sample a small amount of
soluble Iser and allow digestion to
proceed and after digestion is complete
scintillation cocktail is added and the
sample is computed problems like
chemiluminescence and others has to be
needs to be taken care of they will be
because of these materials they might
result or show some chemiluminescence
now there are combustion methods which
are a suitable alternative to bleaching
or solubalization or digestion of
tissues now these combustion methods in
how
there used is that samples are combusted
in an atmosphere of oxygen in a
combustion apparatus now samples
containing say 14 C would be combusted
to 14 co2 or carbon dioxide now which
could be collected in a trapped agent
trapping agent such as NH and then
counted samples containing say 3h could
be converted to 3 H 2 O 4 counting so
likewise you can use lot of different
methods where combustion could be an
alternative there could be lot of other
methods for preparing sample for LS C
which could be like cutting of paper
chromatography
it could be membrane filters could be
used gels which contain radioisotope TLC
scrapings etcetera could be used for as
a sample actually now all this sample
preparation like I said it involves it's
a complex procedure it involves lot of
different like materials now there is a
technique where you don't really have to
go through the sample preparation and
this is called surrender cope counting
now surrender cope counting is one of
like upcoming it is going to be used and
being used in combination quite a lot
now surrender cope radiation is
electromagnetic radiation emitted when a
charged particle passes through a
dielectric medium at a speed greater
than the phase velocity of light in that
medium that is useful for a saying
medium high energy emitters in occur
solution without using cocktail so
that's the very important part that you
really don't have to use a cocktail and
floors and all those things if has a
decay energy like if certain
radioisotope a beta particle has a decay
energy which is an excess of 0.5 million
electron volt then this will cause water
to emit blue white light and which is
referred to as ceramic of light after
its Discoverer
this light can be detected using typical
liquid scintillation counter so what you
see is here that you really do not have
to do any sample preparation in terms of
using scintillation cocktails but it
could be directly used for a measurement
and what are the advantages of this
technique one is that it's a very simple
sample preparation no requirement for
organic solvents and floors so it's a
relatively cheap or technique no problem
of chemical quenching it can handle
large volumes of
excess solution for counting and it
allows for analyzing beta emitting
isotopes or liquids and scintillation
counting without using any cocktail so
most work on this senator Cove counting
has been done on 32 P which has 80
percent of its energy spectrum above the
Tsarina cope threshold and it can be can
be detected around 40 percent efficiency
so as the proportion of energy spectrum
above 0.5 million electron volt
increases the detection efficiency will
also increase so if you could look at
this table here if you see here there
are radio isotopes which has energy
spectrum above 0.5 million electron volt
like your percent of a spectrum which is
60 above 0.5 million electron volt is
60% for any 80 percent for 32 P then 30
percent for 35 CL and 90 percent for 42
K and they have counting efficiencies
which is 30 40 10 and 80 percent
respectively so what you see is here
these isotopes could be used for
scintillation liquid scintillation
counting or surrender cope counting in
liquid scintillation counters this
completes our section on the liquid
scintillation counting now if we can
just go through we have learned how
liquid scintillation counter works it
has it is a very versatile technique
very accurate and very efficient
technique
particularly for 3h isotopes which are
very weak there is a particular way of
energy transfer from radioisotope to
solvent to primary floor to secondary
floor and photomultiplier tube and then
you have lot of advantages in this
particular technique in terms of like
efficient counting which is efficient
efficiency is almost 50% more in terms
in for 3h in terms of measuring more
than two isotopes at a time through
different channels as we have seen dual
labeled samples could be measured easily
so there are a whole lot of advantages
and a lot of disadvantages has been a
partly or completely solved also so it's
a very versatile very useful technique
it is much widely used in comparison to
say GM counters or GM counting which are
very handy but and routinely used but
mostly for routine purpose and not for
like accurate quantification actually so
we have discussed two methods of
detection of radio activity now let's
move on to the third method of detection
that is autoradiography now
autoradiography is a technique to
localized or to detect or to visualize
radioactivity radioactively-labeled
samples using x-ray film or nuclear
emulsion now it utilizes the action of
ionizing radiation on a photographic
emulsion for locating a particular
radioactive material in a specimen now
when we say Auto this prefix indicates
that radioactive material is within the
sample or object which is going to be
imaged imaged here in contrast to
radiography now recording medium which
makes visible the resultant image is
usually nuclear or photographic emulsion
now after chemical development the
resulting pattern of grains so the
distribution of radioactivity substance
in a specimen and microscopy technique
it could be light or electron microscope
it could be used for observation so this
is another technique which is based on
exposure to photographic or nuclear
emulsion now here there are certain
differences from a standard photographic
film emulsion which is that nuclear
emulsion differs from a standard one in
a high ratio of silver halide to gelatin
roughly equal volumes at least in
nuclear emulsion or maybe higher and
also in the size of the grain so you
will have a small size of the grains and
the nuclear emulsions as compared to
photographic emulsion
now although autoradiography is used to
locate rather than to quantify but you
can use density o meters which could be
used for quantification as they will
record the intensity of the image so
that could be done in biology this
technique has lot of different
applications it may be used to determine
tissue localization of a radioactive
substance either introduced in to other
introduced through a metabolic pathway
or it is bound to a receptor or
hybridize to a nucleic acid and a lot of
other ways it could be localized or and
detected one example could be let's say
if you want to determine the site of
location of a particular drug which
could be radioactively-labeled
then where is the location throughout
the body then what could be done done is
that the whole body section of animal
could be kept in close contact with a
sensitive emulsion which could be extra
plate or other nuclear nuclear emulsion
after an exposure for certain period of
time the development of x-ray plate will
exhibit an image of tissue or organs
where radioactivity was present and
thereby location of the drug could be
fine found out likewise radioactive
metabolites can be located and recovered
on a chromatogram or electrophoretogram
in various metabolic studies so that's
how you could it could
we are very useful technique
photographic film and autoradiography
technique has come a long way from the
time in it where in 1867 it was observed
that uranium salts were responsible for
blackening of a photographic film - now
where a lot of progress has taken place
where different kinds of emulsions with
liquid photographic emulsions has been
developed for autoradiography stripping
film technique has come up and finally
it's like imaging plate technology has
come in in 1990s so a lot of progress
has taken place in in this field of
autoradiography now let's little bit
deal about the principle of
autoradiography now video.i active
isotope have ability to blacken
photographic emulsion and let's see how
the whole thing occurs actually now for
an auto radiograph or radiation source
that is radioactivity is required which
is emanating from within the material to
be imaged that is object or sample then
you require a sensitive emulsion so
which will be exposed here now Emerson
consists of a large number of silver
halide crystals as we have discussed
which are embedded in a solid phase
gelatin so what happens beta particles
which are emitted by a radionuclide
or a dia or a source radioactive source
they will penetrate the film emulsion to
a depth which would be proportional to
its energy we are going to discuss that
in a little detail now as particle
passes through the film they will
activate the individual silver halide
drains in the emulsion now this is
because of the interaction which could
be electron-electron interaction with
electrons or nuclei and once these
individual silver halide drains are
activated they are rendered or we call
them it renders them too susceptible to
conversion into a metallic silver that
is their exposed and if
former latent image now they could be
then developed by a photographic
developer which can show them as the
blackening of film and fixtures to
remove any remaining silver halides that
image could be formed or auto
radiographic image could be formed
now each emission converts silver on to
a silver atom to produce a latent image
and when in the development stage these
silver atoms catalyze the reduction of
the entire silver halide crystal to a
metallic silver which produces an auto
radiographic image of the radioisotope
distribution the distribution of
radioactive material may be investigated
as function of time after the injection
of the radio labeled compound and like
for example in pulse labeling in place
of photographic film
like I said image plates have come with
imaging plate scanners which could be
utilized which you can manipulate the
film digitally also now this figure lets
little bit see get into this figure here
which kind of explains what we have
discussed now now you can see here there
are few things one is this is your
sample and this sample contains the
radioactive material this is your red
color this radioactive material now this
sample with radioisotope is what it is
done
you have covered it with nuclear or
photographic emulsion and as the
radioactive source emits the radiation
or particles this that could be like say
beta particle it will strike it will
interact with these grains here which
are shown around grains here now these
grains once it interacts they are
activated on exposure and they are
sensitive it means they are sensitive
for to be developed and then when you
develop the blackening of the film is
seen in here so what you see is here
that how the from exposure to black knee
or to develop
of the film where you can see them it
occurs now we call this one is called
emitter which is this an emulsion this
is and which is this
so emitter to emulsion relationship is
very crucial let me show you in this let
me show you on your screen actually this
whole thing that how the emitter and
immersion is important there could be
lot of different arrangements like one I
have shown you now these arrangement
Arrangements could be many different
kinds as you see you know the screen if
this is your glass slide then if your
emitter could be this could be your
emitter which is if I say this
arrangement this emitter and emulsion
arrangement that emitter is under so
emitter is under the emulsion actually
there could be another arrangement where
you have you have this is your emulsion
like this was your emulsion here so and
emitter could be above or on surface of
the emulsion so this will be called
condition which is on the surface of the
emulsion there could be a third
condition where it could be like you
have emulsion and your radioisotope is
distributed here and we can say this is
in Exley this your emitter and emulsion
relationship is that your emitter is in
the emulsion and there could be third
one where what you could have is you
have emulsion here we have emulsion on
the top and your emitter is in between
that is it's sandwiched between the
emulsion so you could have either of
these conditions which is it could be
under
it could be on the surface it could be
in the emulsion and it could be
sandwiched between like in the emulsion
now if you see here to go a little bit
if it is in the emulsion which is first
place under the emulsion then half the
radiation goes in one direction and half
the radiation goes towards the emulsion
if it is on the surface again all these
half which will go on here all the
emulsions and half or less than 50% will
reach to the emitter emulsion actually
here also that will be like going they
will go in all different directions here
and this one here will go it will be
exposing on all sides actually so you
will see you can see that depending on
the emulsion and emitter relationship
they will be exposed to a certain extent
as not all the particles emitted will be
hitting the emulsion alright so this was
a little bit to show you the
relationship between the emulsion and
the emitter and which is very important
in terms of lot of factors which which
we are going to discuss in a little
while
now autoradiography can be done on
either microscopic scale or macroscopic
scheme so when you say microscopic
autoradiography it's a resolution is
down to 0.5 micrometer or so and it is
done for localization of a tracer at
this tissue or cellular label if it's a
macroscopic autoradiography then
resolution could be down to 50
micrometer and localization is at the
organ label so now what are the most
suitable isotopes which are used in
biological systems for autoradiography
most commonly used Radio isotopes are of
three types which could be high-energy
32p it could be medium energy like 14c
and 35 s beta emitters and could be
low-energy like
three H beta emitters very not so
frequently alpha emitters like polonium
or thorium can also be used so we are
going to discuss about like how they
will be exposing a particular emulsion
now first thing when we are talking
about the exposure of a photographic
film there is when these radio particles
are emitted particles when they exposed
there are certain factors to be
considered one is that is track length
of various emitted particles now when a
particle emitted by a radioactive source
passes through our nuclear emulsion it
loses energy by collisions with nuclei
and other orbital electrons now this
energy produces defects in the silver
halide crystals and renders renders them
developable as we have discussed earlier
and they are exposed so the resulting
patterns of the grain in the Mulsanne is
called
which you will see finally is called
track and it is characterized by three
parameters which is which is length
which is green density that is greens
per unit length or grains per total
tract length and shape which could be
linear curved and angled so the
parameters are determined by the mass
and energy of the particles emulsion and
development of emulsion let me show you
a little bit what does that means like
track length and all those things say
this is your
emulsion actually which is server
hydride in gelatin now when we say that
a radio isotope or beta particle emitted
from a radioisotope exposes or interacts
with the silver halide crystals here and
finally you get a track what his track
means track means a pattern which is
obtained after the exposure which could
be a simple line it could be a straight
line and most of the time the density of
the track as we'll discuss is more at
the end of the track actually like if it
starts from here the density will be
higher in this particular area then it
could be like it could have different
kinds of like it could be straight
it could be curved or it could be very
small actually like for 3/8 it would be
very like it doesn't have so much of
energy to have a longer tracks so you
have our track has a particular
direction it has a particular density
which is grains per unit length exposed
and it has a particular kind of like you
know how many grains it is going to
expose so this is like what we were
talking about here let's move back here
so what we were talking about is that
three parameters that is length grain
density and the shape so length will be
higher if the higher energy is there the
green density per unit length will be
mostly again by energy but it is the
track length will be or density will be
higher at the end of the track and the
shape which could be linear it could be
curved angle as per the particular kind
of the emitter actually so let's see
each of these emitters and their effect
on the particular or effect on nuclear
emulsion actually all right let's
discuss with alpha particles now alpha
particle as we have discussed earlier
also it's a heavy doubly positively
charged and less penetrating
so these massive particles are not
affected by collision with electrons and
usually maintain a straight path so like
I showed you earlier that they will
maintain a straight path after collision
and will have a tremendous disrupting
effect on orbital electrons as they pass
through emulsion now this results in two
excitation of almost every silver halide
crystals they interact with and
therefore generate a very high green
density or and alpha particle will lose
energy rapidly as it interacts with very
large number of electrons per unit
distance so therefore it will have a
short and straight track length which is
usually 15 to 40 micrometer so and it
like because it loses energy very fast
too it has a lot of energy but because
of its bigger size it loses energy very
fast and it has straight and short and
dense track length as far as beta
particles are concerned there are three
kinds of beta particles which is
high-energy medium energy and low-energy
so when these beta particles these are
electrons only and these are scattered
easily by orbital electrons now as they
collide with other electrons they
rapidly lose energy and sharply
deflected at each collision so the
magnitude of this will depend on their
energy now at a very high energy like
say 432 P particles have a tendency to
move in a straight line with minimal
deflection due to great momentum now
energy will be lost after each collision
with an orbital electron and probability
of deflection and subsequent
interactions increases because you lose
energy and I the straight path may not
be maintained for short distance the
track remains fairly straight as these
deflections are balanced out because of
high electron density of matter that is
silver iodide through which they are
passing so the grain density increases
as the particle loses energy that is at
the end of the track like I said meaning
that grain density will always be
greater at the end of the track then
at the beginning so that's a very
important factor here so like I said if
you have three kinds of beta particles
that is high-energy medium energy and
low energy we will see how the effect as
we discuss now weak leeta emitters like
3h 14c and 35s are most suitable and
these ionizing tracts of these are short
and give discreet images as compared to
32p so they have a short range of the
particle this permits accurate
localization particularly th is the best
radioisotope like I said it has less
energy it will form short tracks as
compared to 14c 35s and particularly 32p
witches which will form longer tracks
and to really localize it will be
difficult
now microscopy can be used for to
allocating the mid in developed film now
for localization of DNA bands in an
electro phoretic gel through 3h cannot
be used there 32p is used
why because low energy 3/8 Megatron's
will disappeared then their energy
within the gel and in the wrapping
around the gel thus reducing the
sensitivity to a very low level so more
energetic 32p Negatron sleeve the gel
and produce a strong image so depends on
type of application here which we are
talking about now radioisotopes if you
can see this table there are a whole lot
of radio tube could be used in here for
this particular application which are 3
H 14 C 35 s 32 P and 125 I and which has
mostly beta and 125 I gives gamma
radiation all right so that now we have
talked about types of particles or
radioisotope particles which are being
used in here like mostly beta particles
now let's little bit discuss about types
of emulsions actually there are three
types of commonly used emulsions one is
called
pre-mounted emulsion another is called
liquid emulsion and third one is
stripping film now pre-mounted emulsion
is a relatively thick maybe 50 to 200
micrometer thick layer of emulsion
mounted on a glass glass microscopic
slide as far as liquid emulsion is
concerned it is used in dipping method
so what is done is it is supplied as a
shredded gel which could be melted a
sample mounted on a glass microscope a
slide will be dipped into a molten gel
and taken out and the emulsion hardens
and forms a film of a particular
thickness depending on the concentration
of gelatinous liquid then third one is
stripping film it is supplied as a thin
5 micrometer film mounted on a glass and
can be removed from the glass with a
razor blade and then can be placed on
the water surface
now the pre-mounted sample is placed
under the floating film and lifted and
then this is allowed to dry and thin
emulsion adheres tightly to the slide
now let's see each of them in these
figures here so if you can see here and
appended which is used for liquid
emulsions what is done is there is a
liquid emulsion filled in the speaker
and you have a slide which contains the
specimen or the sample you dip the slide
into the liquid emulsion which is melted
and then take it off and then it is
allowed to dry the liquid emulsion will
form a covering around the sample and on
the and all other parts of the slide and
then once it is ready this could be used
for this could be exposing and it could
be then finally used will be allowed to
form the radiographic image actually so
this is how a dipping method is
performed then there is a stripping
method and a stripping method like I
said a sheet of commercially available
available stripping stripping film is
utilized which could be removed by the
razor blade and like let's see like how
this works out so what you have is you
have a cross section of film here
it looks something like this there's a
rectangular or you can say square strips
here which are on a glass or plastic
surface now with a razor you can remove
each of these strips here one by one and
could be used what is done is this will
be put into the water and it is allowed
to swell then as it spreads out what you
can be done is a slide with sample could
be taken and this could be put under the
film and then it could be lifted like
here it is seen it is lifted here the
film wraps around the slide and the
slide contains the sample under this
particular stripping film and so finally
what you are going to get is something
this that it is a track it's a adhering
film or which is trapped around this
particular glass slide and this finally
could be utilized for developing
autoradiography image so that's how
three kinds of emulsions are utilized
now very important part is of this
method or any method is what is the
resolution efficiency and the background
problems so high resolution high
efficiency and low background are
important factors to be attained in any
experiment
so let's little bit discuss about these
factors in autoradiography now
resolution here first thing so as far as
resolution in autoradiography is
concerned it is the ability to determine
the position of emitting source to
separate the individual grains to get
accurate green count and to separate the
emitting sources so that's what
resolution is can you really like
determine the position of emitting
source and can you separate the
individual grains to get accurate grain
count that's the most important part and
if there are more emitting sources can
you locate them or can you separate
those emitting source now this depends
on various factors like we have
discussed earlier and we'll elaborate on
that
it will depend on types of radioisotope
that is energy of the radiation if you
consider energy of the radiation like I
said 32 P if you say we were talking
about the track length now 32 P has
higher energy so what will happen as it
travels through the the emulsion in
beginning there will not be any track
which or if there is a track it will be
not distinguished from the background so
what will happen that it travels to a
certain distance and the track is seen
only when it is has traveled or certain
distance in the so it's very hard to
localize it like from where unless it is
traveling in all different directions
and then all of these tracks could be
extrapolated to a origin
likewise in say many times 14 C or 35 s
gives curved surfaces a curved track
actually now these curved tracks again
it's very hard to localize if you have
very neat very high-resolution images 3
H if it is a weak 3 H weak beta particle
from 3 H then it might be very difficult
to distinguish it from the background so
all these factors like that is the type
of right radioisotope will determine the
resolution as such the distance between
the radio labeled component in the
object and the radiation sensitive
emulsion that's very important for
example if there is a very thick emitter
then maybe a lot of self absorption
occurs that might be a problem so that
that is thickness of the object or
thickness of the emulsion exposure
period all these things will be
affecting the the resolution then size
of the silver halide drain will
certainly affect resolution if it's the
size is very large it will be very
difficult to count number of grains
because like smaller grains will give
higher resolution as compared to larger
grains in here now for example if there
is a 3h then it may not be able
to expose the larger train or render it
render it developable rather than small
grains which could be counted as well
they could be distinguished from the
unexposed grains here sensitivity of the
emulsion is also important like for
example if there is a ml emitter or low
energy emitter then unless there is a
sensitive emulsion it will be very
difficult to see the image from that
particular resultant of that particular
emitter likewise
like if certain emitter has a longer
track length then it will only form like
a track when it can expose the grain and
if the sensitivity of the grain is lower
then you need more energy to expose that
so again
localization will be a problem and
tracks will be appealing only at the end
where lot of energy is lost so these
these are very important factors in
terms of determining the resolution then
a second factor is efficiency
now efficiency means every DK should
produce a track now however if the
sample is on the surface of dimension
only one half of the DK will enter the
emulsion now even if we say sample is
embedded in the emulsion self absorption
of energy by sample because of a finite
thickness may not reach or reach with a
very least energy and not suitable for
producing a track so that's another
factor which has to be considered for
high energy isotopes like 32 P self
absorption is not a severe problem as a
5 micrometer thick sample thickness of
cells or tissue sections absorbs less
than 1% energy but for low energy
isotopes like say 14 C or 35 s almost
like 82 and 70% transmission at the same
5 micrometer thickness but for 3 H if
you consider the the efficiency is very
less it's like say 16% 4 point 5
micrometer and 4% for 5 micrometer so
stills has to be considered now mercial
thickness also affects efficiency as
most grains are produced near end of the
track with 32p having long tracks
efficiency is proportional to emulsion
thickness to a certain extent extent say
for 14 c and 35 s maximum efficiencies
reached at an emulsion thickness between
3 & 5 micrometer for 3h you have a range
of 1 micrometer and nothing is gained by
using thicker emulsion so if it's a 32 P
you can use thicker emulsion but if it's
a 3h you have to use very thin emulsion
and there's no use using thicker
emulsion for 3h so these factors has to
be taken into account to increase the
efficiency of the experiment and very
important factor is background so
developed emulsion that have not been
exposed to a radioactive sample contains
dark grains and these are called
background now to identify a track like
how to distinguish it from the
background a conventional approach is to
look for at least four greens in a
straight line to define a track but in
case of 3h track may have only one or
two grains so it is important to reduce
the background to very low label if such
isotopes needs to be used there is
another thing is background for that is
latent image on the developed film can
result from say accidental exposure to
light presence of chemicals or metals in
the sample natural background
radioactivity like say from 40k a cosmic
rays mechanical pressure applied and the
way film is stored so a lot of these
things can give rise to background now
background can form before the sample is
applied or during the exposure in liquid
commercial tracks are destroyed if when
emulsion is melted so that could be one
thing in a stripping film the prior
background is reduced to the greatest
extent as film contains a latent image
fed feeder
so that could be another way to reduce
the background backgrounds will always
increase during exposure time so one has
to shorten the exposure time also the
problem of latent image fading could be
another problem because it refers to the
fact that expose drains gradually revert
to the unexposed form
hence undeveloped able so these all
these factors has to be taken into
account and they have to be considered
when autoradiography is being done now
there are certain techniques to enhance
the sensitivity factor or sensitivity in
autoradiography these are we'll discuss
them one by one one is pre flashing now
pre flashing it's like as the response
of photographic emulsion to radiation or
light is not linear and involves a slow
lag period or initial phase before a
linear phase is obtained so the
sensitivity of the film may be enhanced
by pre flashing that is it involves
exposing the film to a millisecond flash
of light prior to placing it on the
contact with the sample so track
technique is often used in high
resolution work then there is a
photograph field that means that
radioactive sample containing soft beta
emitters
such as 3h in a chromatogram or
electrophoresis gel they can be located
precisely as much of their energy is
lost in the gel so they cannot be
located precisely
however sensitivity could be enhanced
drastically by infiltrating the gel with
a scintillator or floors like PPO and
then drying the gel and placing it
against the pre flashed x-ray film so
the negatron emitted from the particular
isotope will excite the floor and
subsequently emit light so thus both
excitation and ionization effects are
used in Ciroc Rafi it great increases
greatly the sensitivity with low-energy
emitters
likewise sensitivity can also be
enhanced by adding floors to the
emulsion
so this is like shown in this figure
that there is a beta particle and there
is a flow here which can increase the
sensitivity to many fold and then the
last one is intensifying screens now
this is an opposite problem to that of
low-energy isotopes so here radiation
from high-energy isotopes like 32p and
gamma isotopes labeled samples suffering
proteins or other samples now these are
highly penetrating particles or rays and
as they penetrate through the film
causing little exposure of the film and
therefore producing a very poor image so
what happens substantial improvement in
the mid generation can be done by
placing on the other side of the film
from a sample or thick intensifying
screen made up of solid phosphorus so
Negatron penetrating the film will cause
the phosphor to fluoresce and emit light
which superimposes its image on the film
film exactly and this can enhance the
resolution many times this is shown here
this is a cassette hyper caster there is
a like particular kind of film and the
sample could be placed in here this is
the active side of the blot and there is
a force for surface so what happens when
the high-energy particles or rays cross
over and do not expose properly and the
x-ray film from the sample then image is
superimposed which is made from
fluorescence of this phosphor compound
so this is one another method which
could be utilized all right so this
completes our section on autoradiography
and also we have discussed three methods
in previous lectures which was based on
ionization of gases then second method
based on excitation of solutions and
solids and then finally on based on
exposure of photographic film that is
autoradiography now in the next lecture
we are
going to discuss one more method that is
radio amino acid and also will be
considering different safety aspects and
applications of the radioisotope
technique thank you