Dark field and phase contrast microscopy Video Lecture - Biotechnology Engineering (BT)

and previous two lectures we have
discussed about the various concepts in
the basic microscopy and these concepts
will be encountered in the coming
lectures those were if you can recall
they were like absorption refraction
diffraction magnification contrast
resolving power and so on we discussed
about geometric optics and physical
optics and how the formation of image
takes place was explained also we have
discussed about the compound microscope
various components of compound
microscope like if you could recall
there is a source of illumination the
condenser lens which condenses the light
the objective lens through which an
intermediate image formation takes place
and finally ocular or eyepiece which
forms the final image which will which
will be finally captured by the eye now
both in here in subsequent lectures we
are going to discuss about specific
microscopy techniques in both light
microscopy and electron microscopy now
light microscopy an electron microscope
if they are compared the basic concepts
remains the same but there are certain
specific differences between the two
techniques in light microscopy for
example light source is simple halogen
lamp or simple white light is utilized
in contrast in electron microscopy you
have a beam of electrons which is
generated from electron gun as we will
discuss in later then lenses the light
microscopy the lenses are glass lenses
or optical lenses as we call them
whereas an electron microscope you have
the electromagnetic lenses which are all
we will be utilizing all like condenser
objective but their electromagnetic
lenses
light microscopy we do not have an
enclosed system you do not need vacuum
but in electron microscopy you need
vacuum so it's like enclosed chamber
which is evacuated so there are a whole
lot of differences between light
microscopy and electron microscope
microscopy in terms of resolution also
the you get much higher resolution many
fold higher resolution in electron
microscopy because the wavelength of
electrons can be modulated and is much
lower than the light microscopy in light
microscopy at best you can go is
slightly less than the point 2
micrometer which will reveal only
certain details but in electron
microscopy you can go too much higher
details another important part or
compare if you compare light microscopy
in electron microscopy is the specimen
preparation electron microscopy requires
extensive specimen preparation where you
cannot use live cells and they have to
be dehydrated so then you have to add
the mass to the specimen so that
electrons can be deflected
whereas in light microscopy both stained
and live cells could be utilized for
example certain techniques uses stem
cells but certain other techniques
contrast can be created by changing or
creating certain optical phenomena where
you can visualize the object as we will
discuss later on now in subsequent
lectures we are going to discuss a
specific techniques and let's introduce
you to these techniques here first in
light microscopy we are going to discuss
bright field microscopy dark field
microscopy phase contrast microscopy
polarization microscopy fluorescence
microscopy and an electron microscope we
are going to discuss the transmission
electron microscopy and scanning
electron microscopy now bright field
microscopy as it says that the
background is bright
and the image which is formed is
relatively darker than the background
now if you could recall when the light
from condenser passes to specimen a
specimen there are two kinds of lights
which will come out of a specimen into
the objective one is refracted or
altered light another will be unaltered
or direct light or undeviated like now
non altered or direct light forms the
background which is bright and the image
formation is relatively dark now here
many biological specimens are
transparent so staining is a must in
many cases and but only problem is that
the staining you cannot use live cells
because staining kills the cells and
also a lot of processing is required now
other techniques which we are going to
discuss in the in the lectures will not
be requiring the staining of the
specimen rather there will be contrast
will be created with other methods which
we are going to discuss dark field
microscopy is first such technique where
as it says the the background will be
dark and a bright image will be formed
against a dark background we will
discuss in detail how this works out how
the image formation takes place in dark
field microscopy the next one will be
phase contrast microscopy which is a
very valuable technique for cell and
molecular biology people now here a
simple optical trick is utilized to
create contrast I think all of you know
that when the rays of light passes
through a specimen they encounter
different components of the specimen and
depending on the optical density of the
components the light will be deflected
accordingly and it will be retarded
because of that phenomenon now this
creates or this leads to the phase
differences that is the waves are now in
comparison to the original wave
there are face changes have been taken
place now this phase change cannot be
seen by our eyes and because these are
not intensity differences so in this our
simple optical trick leads to the
changes in phase to the intensity
differences that is phase change is
translated into intensity differences
and we will discuss in detail how it is
done in a brief like the scientist or
physicist who did that he used one
direct beam that is reference beam and
the diffracted beam which were later
combined to form three contrast and
we'll see how that is done in detail
next technique which also does not
require staining is the polarization
microscopy now polarization microscopy
is a very useful technique for certain
samples which cannot be stained and even
they sometimes cannot be visualized in
phase contrast microscopy because the
phase increments are not large enough to
create intensity differences now these
are polarization microscopy utilizes
accessories like polarizer and analyzer
here you require a sample which has a
particular structural arrangement like
either the individual particles are
arranged in a parallel array array or in
form of state disks now this leads to a
particular phenomenon called form
birefringence by these arranged
particles where a polarized light will
be allowed to pass only if it is
parallel to their individual arrangement
so this particular technique is very
useful for certain applications and
utilized for those samples which cannot
be visualized by staining or by other
techniques like phase contrast
microscopy
next one which we are going to discuss
is fluorescence microscopy fluorescence
microscopy utilizes the phenomenon of
fluorescence where a florist
fluorescent substance absorbs the
smaller wavelength particular
wavelengths and then emits the longer
wavelengths which could be which are
invisible region and this phenomenon
could be utilized for lot of different
applications like locating a particular
substance like protein or DNA or other
macromolecules or other components
various components at different times in
vivo so this is a very useful technique
now fluorescence microscopy requires the
use of certain fluorescing substances
because not many biological substances
are fluorescent in itself so you have to
have external fluorophores to be
utilized in this other extension of
fluorescence microscopy is can focus
microscopy where you can use thick
specimen or tissues now remember all
these techniques right from light
microscopy and electron microscopy
require thin slices of a specimen which
are in micrometers and nanometers in
electron microscopy and because
otherwise absorption of light or other
problems will occur in image formation
so but in confocal microscopy you can
use take a specimen and it utilizes
fluorescence optics now here you can
focus and at different times or you can
rester the whole specimen in depth or in
sides and you can take various pictures
which are umpteen number of images which
can be later combined to give
three-dimensional look or
three-dimensional picture of the whole
specimen so that's a very good technique
for visualizing three-dimensional
structures
also in phase contrast microscopy there
will be extension of phase contrast
which we are going to discuss is
differential interference contrast
microscopy where you have a three
dimensional quality images are obtained
because of certain interference
techniques being involved in here where
you use lot of prisms and other
accessories to make that happen so these
were the techniques which will be
utilized in electron in the light
microscopy now the two techniques which
we are going to discuss an electron
microscope is Tam and Sam that is
transmission electron microscopy and
scanning electron microscopy now as it
says transmission electron microscopy
the image will be formed by transmitted
electrons through specimen and the image
is formed due to the transmitted
electrons from the specimen hitting the
fluorescent screen now here very thin
slices like I said in nanometer has to
be there and there should be a
deflecting material in the specimen in
forms a form of heavy atoms which are
added during staining and sample
preparation there is extensive sample
preparation for electron microscopy in
scanning electron microscopy rather
looking for the details internal details
of details of the specimen here surface
details are visualized or recorded so
here rather than a transmitted electron
here backscattered electrons which will
be elastic or known as elastic
scattering those electrons will be
collected for image formation and again
for retaining the surface there are
special specimen preparation techniques
so this was a little introduction to the
different techniques of microscopy in
light and electron both which we are
going to discuss subsequently so let us
start with some of the light microscopy
techniques we will start with the bright
field
microscopy now this is one of the
simplest optical microscopy illumination
techniques and mostly used in compound
microscopes like I said earlier the term
brightfield is derived from the fact
that a specimen is relatively dark
contrasted by bright surrounding because
of the DA because of the direct light
which forms a bright background now
sample is illuminated from below that is
it is transmitted light and observed
from above like we I have shown you in
the compound microscope that will be the
particular arrangement or in the
microscope now contrast is created by
the absorbance of some of the
transmitted light in dense area of
sample now like I said as few biological
samples absorb light staining is
required to enhance the contrast for
example there might be certain colored
substances or which might absorb light
but otherwise the it is very hard to
visualize or make visible the details of
the specimen in bright field microscopy
and for staining there are a lot of
different materials dyes and other
things which are there to create
contrast and we'll be discussing this in
the end of the light microscopy section
in a specimen preparation now bright
field microscopy may use either critical
or cooler illumination and it consists
of a light source which is a simple
halogen lamp condenser objective and
ocular lens or it could be camera to
view the samples so this is we are not
going to much orbit tail because it is a
simple illumination technique and it is
a very direct technique which is widely
used in observing the stained specimens
now let's move on to the next technique
which where staining of the sample is
not required and rather what you do is
there are certain optical tricks you
apply to
yet contrast now in dark field
microscopy the bright image of object is
produced against a dark background many
objects now having refractive indices
very very close to their surrounding are
very difficult to image in bright field
microscopy and to view these things
there are certain technical techniques
have been developed dark field
microscopy is very useful for counting
small particles and observing living
unstained preparations
how does image formation takes place in
dark field microscopy so before we
explain it on the figure let us
understand little bit here so in
critical illumination if you could
recall light forms a very solid cone at
object plane that is light coming from
condenser forms a solid cone at the
object plane as we have discussed
earlier now supposing if there is no
object in the object plane so an absence
of an object causes field to be brightly
illuminated uniformly now here what is
done is there is an accessory which is
an opaque disc with transparent annulus
is inserted just below the objective or
in a condenser which causes the cone of
light to become Hollow so what you do is
annular like disk or another aperture
opaque disk containing another aperture
inserted below the condenser lens and so
what will happen a hollow cone of light
will be illuminating the object plane
now if there is no object then it will
emerges another hollow cone through
objective now analyst is made
sufficiently that is apertures where
apertures are present in the practice it
is sufficiently large that the hollow
cone of light which is surrounding the
object will not enter the objectives so
objective is made smaller so to
understand this no direct light is and
the objective now what will happen if
there is an object placed in the object
plane if there is an object which is
placed in or a specimen placed in the
object plane then certain diffraction is
bound to happen
and some of the diffracted rays of
certain orders will enter the objective
so what will happen a bright image
because these are light which is
entering the objective and will be
formed against a dark background because
direct light does not enter so there
will be dark background but bright light
and certain orders of diffraction and
twist objective and they will form a
bright image against a dark background
so the outcome is that you get
exceptional contrast but very limited
details here and mainly many details are
lost because you have avoided zero order
light which is direct light now
depending on light intensity and degree
to which this internal reflections have
been eliminated the quality of the
picture will be or image will be better
now in dark field microscopy if you see
the the optics of this this figure shows
you the optics of dark field microscopy
you can see here the light from the
light source is allowed to pass to the
condenser but you have inserted a
annular aperture here now these here not
shown here in this but if you see see
here there is a hole or there is a area
the this is like aperture is there in
this particular disc now the light
passes from only this area and not from
the other part which is opaque here so
what you get you get a hollow cone where
this is part is not illuminated here and
you get a hollow cone of light
illuminating the object plane now this
hollow cone of light in absence of an
object will directly go and will not
enter objective because you can see the
size of the objective or numerical
aperture of the objective is smaller now
if object is present there will be a
diffracted light so
diffracted light this is just one light
ray we have made but there will be
certain diffracted light which will
enter the objective and will form an
image in the image plane now this will
be the bright image against a dark
background so you will get very good
contrast here now this particular
technique is used for this is ideal for
objects which are minut living and they
have very close refractive index with
the surrounding for example living
aquatic organisms die atoms small
insects bones fibers bacteria and cells
in animal tissue culture can be used
here non biological specimens could also
be utilized here which include semi
chemical crystals or thin sections of
polymers ceramics having small
inclusions or other in our different
specimens could be realized so dark
field microscopy is quite good for
particular applications but not good to
get too much of details here
all right like for example it is very
good technique to count maybe number of
particles like say number of viruses or
other particles but if you want details
then you have to use other techniques so
this was about dark field microscopy now
let's move on to the next one that is
phase contrast microscopy now phase
contrast microscopy is one of the most
valuable techniques in the field of cell
and molecular biology and here the
contrast the way contrast has been
created is is very fascinating to
observe now this particular technique
was first described in 1934 by the
Dutchman Fritz journey and for which he
was awarded awarded Nobel Prize for
Physics and 53 now this is an contrast
enhancing optical technique for
transparent unstained specimen like
living
else sub cellular organelles
microorganisms etcetera remember one
thing has to be taken into account that
thick specimens are not suitable for
this technique another thing is the
samples or a specimen must be non
absorbing because that will create
intensity differences and will lead to a
poor image now in phase contrast
microscopy a minut differences in phases
will understand how that Manu difference
in phases is created which might result
due to very small differences in
refractive indices of the components of
a specimen through which light passes
these are translated into intensity
differences remember the phase
differences cannot be observed by our
eyes but intensity differences can be
observed by our eyes so this is very
important
now here Fritz Jarek he used a reference
beam to create this whole thing let's
see how this is done that's for example
consider a transparent disc or object in
a transparent medium and it is being
illuminated by a parallel light falling
perpendicular on that disk now what will
happen if the refractive indices of the
two are different width then light tends
to different phase due to the tradition
of light now disk remains still
invisible due to the inability of eyes
or the photographic film to detect
different phases so what we are seeing
here that though something is happening
but you are not able to see this now in
presence of an object diffraction
through the object will also contribute
towards image formation but again how do
we visualize this if you see this
picture this is here to show you that
how the retardation of wave will occur
if you see here this is a direct wave
which is going on when it passes through
the specimen here then the
components like this is maybe liquid
component here this component contains
certain more material or has higher
optical density so you can see
differential retardation of the wave
here like here it is more retarded now
here again another thing is that when it
passes system will be deflected in
different directions and the diffraction
will also depend on the how they are
placed how dense and optical object is
so these things will lead to the phase
differences so when this phase
difference what we have to do is it has
to be translated into intensity
difference how it is done
let's get in here so what is done is you
place an another aperture like in dark
field microscopy what we have done we
have put in a annular aperture below the
condenser lens likewise here also you
will be placing an another another
aperture or face ring in focal plane
below the condenser and which will
result in a hollow cone we cannot in
essence call it a hollow cone but to
understand that it doesn't mean that
hollow cone is completely a hollow cone
is illuminating the specimen but to
understand this we say it's a hollow
cone of light illuminating the specimen
now
when you this light passes through
condenser analyst and it will pass
through the objective it will make the
image of the condenser annulus at that
plate of particular place a face plate
is introduced that is after the
objective where the condenser analyst
will form its image a face plate is
introduced so these are conjugate
arrangement that is it has to be
completely coinciding the face plate has
to exactly coincide with the image of
the condenser annulus because then only
this optical phenomena could be observed
now let's see so what happens then once
you have
done that then all light rays or light
waves we can say passing through
condenser endless will be either and one
still I think I will just show you that
on my screen that what is phase ring and
what is phase plate before we go any
further so please pay attention to your
screen so like I was saying there is a
phase ring and there is a face plate now
phase ring or annular aperture is
something which is like this that you
have created two apertures in here
through which the light will pass which
will be a parallel light or just we are
showing it here through the through the
light source now this could be we can
say that there is another aperture that
is there is a place-- aperture through
which the light is passing now if you if
we have to say face ring has to be
formed after the objective lens the face
ring will look something like this and
it will completely coincide with this
that is these two apertures now what is
has been done in this face ring that you
have there are two kinds of face rings
could be there one is at exactly where
apertures are there you have deposited
an extra layer another at two places so
what will happen when extra layer is
there the light rays which comes from
the objective like there will be
condenser first here then there will be
if I make it like this there will be
object plane and
they'll be objective lens and through
objective lens these light rays will
pass through these two places and so
what will happen is when the wave is
going through this place here then this
direct light there will be two lights
one is diffracted light another is
undeflected light now this direct light
which is undeviated light will pass
through this faceplate if it is layered
face plate then what will happen it will
be wave will be retarded by one quarter
wavelength so layering is done in such a
way that it will be retarded by one
quarter wavelength there could be
another arrangement which could be
rather than rather than layering it it
could be grooved there could be groove
could be easton here now when this
groove is isten here then what happens
that this groove will lead to the
advancement of because you have less
material in here so the light ray
passing through this will be advanced by
one quarter wavelengths it is done in
such a way that either in layer form it
is retarded one quarter wavelength or it
is advanced one quarter wavelength so
this is just to make understand that how
phase ring or annular purger and the
face plate is playing a role in here now
let us move on again to our slides here
now here's what we have tried to
understand in this that all light waves
passing through condenser annulus will
be either advanced or retarded by one
quarter wavelength depending on they are
passing through grooved
or layered nature of face plate now all
the waves deflected or deviated because
when you have a specimen then
diffraction is or to occur and all the
waves diffraction limited by the
specimen will not pass through the
groove or the layered area of the face
plate we'll see in the figure
later on now here diffracted rays will
be retarded to a different extent
because your specimen will contain
various kinds of things for example if
say you are observing a single cell or
thin layer of cells a cell will contain
cytosol also it will contain nuclei also
it will contain Golgi it will contain
mitochondria
it will contain fat droplets also it
will contain ribosomes also all of these
will retard because of differences in
refractive indices will retard it
differentially so you will get the raise
or wave friends with different faces
here so what happens that all the waves
that are diffracted which are retarded
toward different extents will pass
through the other part of the face plate
also mainly from other parts of the face
plate rather than grouped or layered
area but they will it doesn't mean they
will not pass through grooved or layered
area but direct rays will pass from
grouped or layered area and they be
retarded or advanced now all the waves
will combine would deviated and
undeviated to form image at the
intermediate image plane now deflected
and deviated waves will be retarded to
varying degree like i said due to
differences and this will create
particularly create different intensity
differences I will see how that is done
so what you have so what when the phase
difference of diffracted wave due to
specimen now here you have to understand
that as we have seen the direct light
passes through grooved or layer there is
a one quarter wavelength retardation or
advancement as per the weather it is
grouped or it is if it is layered
there will be retardation if it is
grouped it will be advancement likewise
when the waves pass through the specimen
and they are diffracted then there also
the retardation takes place and which is
assumed to be approximately one quarter
wavelength like I said different
refractive indices will
retarded differentially but one quarter
wavelength could be understood as a
standard thing approximately now when
this is combined with the phase
difference created by face plate the two
things will happen one the object will
appear bright against a relatively dark
background so what does that means that
due to constructive interference so what
does that mean is when undeviated light
is retarded so if you remember if it's a
layered face plate already
the undeviated light or direct light is
retarded one quarter wavelength now when
you superimpose this with the like one
quarter wavelength retarded diffracted
wave then both gives a constructive
reference and therefore due to
constructive interference you will get a
wave which will be more brighter
relatively to the background other thing
can happen that a second option could be
that object would be dark white would be
dark against a bright background because
undeviated light has been advanced by
the face plate that is grooved face
plate and that's called positive phase
contrast earlier one was called negative
phase contrast where it was layered so
when undefeated light is advanced by one
quarter wavelength and deviated light is
retarded by one quarter wavelength then
when they combine in the image plane
they are out of phase by one and half
wavelength that is the condition for
destructive interference so you will get
a dark image against a relatively bright
background so you will get a very good
contrast now let us understand these
combination of waves like I was talking
about and in this figure you'll be able
to understand this if you see here this
is if you say this is a direct wave
alright which is being going and which
is retarded one quarter wavelength and
this is the another wave which is also
retarded one quarter wavelength because
of specimen now when these two waves
combine then the amplitude is bound to
be
and so the brightness the resultant wave
will have higher amplitude and therefore
higher intensity and it will be a
constructive interference which will
show up as the as the bright image
against a dark background relatively
dark background we cannot say it's a
gray background
we cannot say it's a very dark in
contrast when there is an advancement in
the in the light ray or the wave due to
the groove nature of the phase plate
then this another deviated light which
is already one quarter wavelength
deviate retarded which is the
approximate value then both are out of
phase by 180 degrees and finally what
you get resultant wave is of lesser
amplitude and so lesser intensity and so
you get relatively dark images against a
bright background so these are two
phenomenons where you can either use eye
you can use either use negative phase
contrast or positive phase contrast now
to understand this optics of the phase
contrast microscopy like I have already
discussed but we can understand by this
figure you can see here this is a
annular aperture like it was in the dark
field microscopy and this is like area
where through the light will pass it
passes through condenser lens and
illuminates the object plane by
supposedly hollow cone of light now
deviated light will pass through other
parts of the face plate this is face
plate and the undeviated light passes
through the face they are layered or
grouped part of the face plate all right
now certain times when intensities has
to be balanced then objective could be
covered with certain like alumina film
or other things which does not change
the phase to decrease the intensity of
the undeviated like now these ones will
combine in the image plane and they will
produce the images which are either dark
image against bright bright background
or bright image against dark background
as per the phase contrast microscopy
technique
used so now if you see here an optical
configuration this is like last time
what I have seen this is a simple
optical configuration of phase contrast
but if you are utilizing infinity
corrected optics then just to show how
in the infinity space these accessories
optical accessories are added if you can
see here a face plate is being added
between the objective and tube lens and
so parallel rays which are like focused
at infinity passes through objective and
then they are finally they are focused
by tube lens so it's the same optics as
I have shown you earlier so if we go
back and understand this whole thing
that simple optical trick has led to the
creation of contrast through which you
can see the different specimen different
components of the specimen very clearly
like I said the all different components
will be bright or dark relatively and to
a different extent because of different
retardation of the waves and as they
combine and the image plane and they
form a very highly contrasted image but
there are certain disadvantages also of
phase contrast microscopy which needs to
be looked into a major disadvantage of
phase contrast microscopy is the
unavoidable bright Hallows which are
surrounding the specimen and they are
mainly caused due to many factors one is
the diffraction by face plate now this
particular problem what happens is if
you see if you will observe the
different images from phase contrast
microscopy it's a very valuable very
good technique here cytosol or cytoplasm
will contrast with nuclei nuclei will
contrast with other
sub cellular organelles present in here
and there very nice pictures are
obtained but if you see the membranes
then the surrounding of the specimen
will certainly see bright hellos and
sometimes it's hard to distinguish
certain parts of the components of in
that particular area due to bright
Hallows or diffraction Hallows now
problem of Hallows have been solved to a
greatest extent or completely in another
interference microscopy technique which
is it's like phase contrast microscopy
technique which is called differential
interference contrast microscopy and
this uses a complex lens system with
prisms and other things to generate
diffract we are deflected and
undeflected lights are combined later on
and we are going to discuss in coming
lectures this particular technique so
apart from bright Hallows which are
formed there is a very good technique to
observe details now here not only that
there are a lot of different
applications of the this technique like
for example visualization of sub
cellular organelles or tissues or
microorganisms so details of this could
be observed by phase contrast microscopy
and you can use single cells or thin
layer of cells to observe that without
staining remember staining wear is not
allowed in this as a staining will
create intensity differences so you
require a non absorbing specimen in this
case dynamic mobility of mitochondria
and mitotic chromosomes etcetera can be
followed very nicely like you can
picture that or you can video record the
whole phenomenon as it is going on in
real time this has been used for
diagnosis of tumor cells it has been
used for many other applications in the
field of fire ology bacteriology
hematology and other areas of biological
sciences so
phase contrast microscopy is one of the
most important techniques in microscopy
to sum up the lecture this particular
lecture what we have discussed today is
three techniques one is bright field
microscopy dark field microscopy and
phase contrast microscopy now bright
field microscopy there is a bright
background and a dark images form
relatively dark image is formed staining
is required for transparent objects or
specimen and most biological specimens
are transparent in dark field microscopy
and contrast to bright field here the
background is dark and your images
bright but only thing is because of
avoiding of or because of not inclusion
non inclusion of direct light zero-order
light lot of details are lost so it is a
very good technique you get very good
contrast but has limited applications
like for example you can use it for
counting and other things so dark field
microscopy has particular used but it
cannot give you lot of details now third
technique which we have discussed
today's phase contrast microscopy phase
contrast microscopy has really
revolutionized the cell biology
observation of cells in cell biology and
this has led to observation of many
phenomenons which could be dynamic
motion of the movement of the
mitochondria or Microtech chromosomes
different different phenomenons which
are going during this particular process
diagnosis of lot of different kinds of
cells has been done and lot of other
applications have been given here the
contrast is developed by a face ring
annular aperture and a face plate which
is conjugate to the face ring and face
plate when deviated unlimited light
passes through the faceplate it could be
it is advanced or retarded and that
leads to the constructive or destructive
interference with the diffracted light
in the image plane and leads to the
image formation a very high good
contrast is obtained and you are able to
see images in vivo under in vivo
conditions without any problem only
background is bright hellos
non-absorbing specimens has to be used
and you cannot use thick specimens so we
will end this lecture today here and we
will continue our discussion in the next
lectures
thank you