1. L.N. PARAMEDICAL COLLEGE BHOPAL (M.P.)
Kolar Rd, Sarvadharam C Sector, Shirdipuram,
Bhopal, Madhya Pradesh 462042
Session-2019-2020
Project On
PHASE CONTRAST MICROSCOPY
CLASS-BMLT 3RD
YEAR
SUBJECT- INSTRUMENTATION
Submitted By:
2. JAYBARDHAN BAIN
L.N.PARAMEDICAL COLLEGE BHOPAL
Kolar Rd, Sarvadharam C Sector, Shirdipuram,
Bhopal, Madhya Pradesh 462042
☎️:0755-4049600
Date
CERTIFICATE
This is to certify that Mr. JAYBARDHAN BAIN is the student
of B.M.L.T.III YEAR (L.N.Paramedical College, Bhopal
(M.P.) he submitted his project on the titled “PHASE
CONTRAST MICROSCOPY” During the session 2019-
2020.
YAAUOHC AJOOP
HOD
DepartmentParamedical
L.N.ParamedicalCollegeBhopal MP
3. ACKNOWLEDGMENTS
My Project was only possible in the guidance and
supervision of Miss. POOJA CHOUBEY HOD of
department. I am thankful to her for support and discussion on
the work done.
I am sincerely thankful to Mrs. SHAILJA KURELE Mr.
ANURAG YADAV and Mr. RAJKUMAR TIWARI Faculty
of Department of Paramedical, L.N.Paramedical College
Bhopal for their co-operation and blessings to complete this
work.
I am thankfull to my family members for their blessings and
support.
Last but not the least I am thankful to almighty.
4. JAYBARDHAN BAIN
YEARRD
BMLT 3
Table of Contents
Introduction
History
Definition
Principle
How Phase Contrast Works
Parts of Phase contrast Microscopy
Advantages
Disadvantages
Reference
5. INTRODUCTION
The human eye perceives only differences in wavelength (as colour) and amplitude
(as brightness) of the light reaching it. The eye cannot see differences in the phase
relationship between different beams of light. Classically, biological specimens have
usually been viewed as stained slices of material by bright-field transmitted-light
microscopy(Figure 1f). Stains are used to alter both the colour and brightness of the light
passing through the specimen, and so increase contrastin the image. Cellular function is
understood better by studying the actual motion, growth, reproduction and exchange of
cell constituents of living cells than it is by drawing conclusions from artificially
manipulated dead ones. Since cells are generally transparent structures, they are almost
invisible to the eye by bright-field microscopy(Figures 1b,g).
The phase contrast microscopeexploits the interaction of the illuminating beam of light
with the specimen to convert an image of an invisible specimen (or one of very low
visibility) into an image that the eye can detect. Other forms of contrastenhancement,
such as darkground microscopy, differential interference contrast microscopyand
Hoffmann modulation contrast microscopy, have also been developed. Perhaps the most
widespread use of phase contrastmicroscopyin biology is for cytology and tissue culture,
admirably lending itself to quick checking of live cell cultures
6. HISTORY
Frits Zernike, (born July 16, 1888, Amsterdam, Neth.—died March 10, 1966,
Groningen), Dutch physicist, winner of the Nobel Prize for Physics in 1953 for his
invention of the phase-contrastmicroscope, an instrument that permits the study of
internal cell structure without the need to stain and thus kill the cells.
Zernike obtained a doctoratefrom the University of Amsterdam in 1915. He became an
assistant at the State University of Groningen in 1913 and served as a full professorthere
from 1920 to 1958. His earliest work in optics was concerned with astronomical
telescopes. While studying the flaws that occurin some diffraction gratings because of the
imperfect spacing of engraved lines, he discovered the phase-contrast principle. He noted
that he could distinguish the light rays that passed through different transparent materials.
He built a microscopeusing that principle in 1938. In 1952 Zernike was awarded the
Rumford Medal of the Royal SocietyofLondon.
7. PHASE CONTRAST MICROSCOPY DEFINITION
Unstained living cells absorb practically no light. Poorlight absorption results in
extremely small differences in the intensity distribution in the image. This makes
the cells barely, or not at all, visible in a brightfield microscope. Phase-contrast
microscopyis an optical microscopytechnique that converts phase shifts in the
light passing through a transparent specimen to brightness changes in the image.
It was first described in 1934 by Dutch physicist Frits Zernike.
8. PRINCIPLE OF PHASE CONTRAST MICROSCOPY
The basic principle to making phase changes visible in phase-contrast microscopyis to
separate the illuminating (background) light from the specimen-scattered light (which
makes up the foreground details) and to manipulate these differently.
The ring-shaped illuminating light (green) that passes the condenserannulus is focused on
the specimen by the condenser. Some of the illuminating light is scattered by the
specimen (yellow). The remaining light is unaffected by the specimen and forms the
background light (red). When observing an unstained biological specimen, the scattered
light is weak and typically phase-shifted by −90° (due to both the typical thickness of
specimens and the refractive index difference between biological tissue and the
surrounding medium) relative to the background light. This leads to the foreground (blue
vector) and background (red vector) having nearly the same intensity, resulting in low
image contrast.
In a phase-contrast microscope, image contrast is increased in two ways: by generating
constructive interference between scattered and background light rays in regions of the
field of view that contain the specimen, and by reducing the amount of background light
that reaches the image plane. First, the background light is phase-shifted by −90° by
9. passing it through a phase-shift ring, which eliminates the phase difference between the
background and the scattered light rays.
Working principle of phase contrast microscopy
When the light is then focused on the image plane (where a camera or eyepiece is placed),
this phase shift causes background and scattered light rays originating from regions of the
field of view that contain the sample (i.e., the foreground) to constructively interfere,
resulting in an increase in the brightness of these areas compared to regions that do not
contain the sample. Finally, the background is dimmed ~70-90% by a gray filter ring; this
method maximizes the amount of scattered light generated by the illumination (i.e.,
background) light, while minimizing the amount of illumination light that reaches the
image plane. Some of the scattered light that illuminates the entire surface of the filter will
be phase-shifted and dimmed by the rings, but to a much lesser extent than the
background light,which only illuminates the phase-shift and gray filter rings.
The above describes negative phase contrast. In its positive form, the background light is
instead phase-shifted by +90°. The background light will thus be 180° out of phase
relative to the scattered light. The scattered light will then be subtracted from the
10. background light to form an image with a darker foreground and a lighter background, as
shown in the first figure.
How PhaseContrastWorks
Light can be considered as a wave. When light encounters glass, or other optically
transparent material denser than air, it is slowed down, and the number of waves increases
in proportion to both the density (as determined by the refractive index) and thickness of
the material. Consider a second identical beam (from the same source), which moves
wholly in air parallel to the first without entering the glass. The first beam will have
travelled a greater distance than the second. Furthermore, the two beams, which started
out in synchrony, or in phase, are now out of phase; this difference is referred to as the
phase difference between the two beams. All specimens diffract, or scatter, light, and
these diffracted beams carry the information about the structure of the
object(Oldfield,1994;Pla´ s˘ek and Reischig, 1998). An image of the object is formed at
the primary image plane owing to interference between those beams diffracted by the
specimen and the undiffracted (zero order) beam. With a stained specimen there is a half
wavelength ( l) phase difference between the undiffracted beam and those diffracted by
the specimen. Interference of the two sets of beams leads to overall differences in
amplitude, which can be detected by the eye as differences in brightness. Coloured objects
are merely amplitude specimens that selectively absorb light of certain wavelengths
within the visible spectrum.
Thin transparent objects such as cells introduce a phase difference between the two beams
of only one-quarter wavelength ( l). Fora theoretical discussionof why this is so, using
vector treatment, see Pluta (1989) or Bradbury and Evennett(1996).To render these
specimens visible, it is necessary to have an artificial means of introducing an extra l
phase difference between the diffracted and undiffracted beams. There would then be l
phase difference contributed by the specimen and a further l phase difference by the
microscope.
If the beam illuminating the field of view is constrained by an annulus in the first focal
plane of the condenser, it will form an annular image in the backfocal plane of the
objective (Figure 2). Supposethat a circular ‘trench’ (or, alternatively, a ridge) matching
the image of the condenser annulus is placed within the back focal plane of the objective.
The optical path traversed by the undiffracted beam alone can now be selectively
advanced (or retarded) and the necessary extra l phase difference between the two beams
introduced before they recombine to form the image at the primary image plane of the
microscope. This ‘trench’ (or ridge) is called the phase ring and it is carried on the phase
plate. The phase ring carries an absorbing layer that reduces the amplitude of the
11. undiffracted zero order beam,reducing its brightness to match that of the weaker beams
diffracted by the specimen.
The Working of Phase contrast Microscopy
Partially coherent illumination produced bythe tungsten-halogen lamp is directed
through a collector lens and focused on a specialized annulus (labeled condenser
annulus) positioned in the substage condenser front focal plane.
Wavefronts passing through the annulus illuminate the specimen and either pass
through undeviated or are diffracted and retarded in phase by structures and phase
gradients present in the specimen.
Undeviated and diffracted light collected by the objective is segregated at the rear
focal plane by a phase plate and focused at the intermediate image plane to form the
final phase-contrastimage observed in the eyepieces.
12. Constructionof the PhaseContrastMicroscope
A special set of objectives, fitted with phase plates, is normally needed
for phase contrastmicroscopy. Manufacturers generally provide several different sizes of
annuli in the condenserto match objectives of differing magnification and numerical
aperture (Figure 3d). These annuli can normally be rotated within the condenserhousing,
and brought onto the optical axis of the microscopeas required (Figures 3a,b). Provision
is usually made for centring each annulus with respect to the optical axis of the
condenser.
13. PARTS OF PHASE CONTRAST MICROSCOPY
Phase-contrast microscopyis basically a specially designed light microscopewith all the
basic parts in addition to which an annular phase plate and annular diaphragm are fitted.
14. The annular diaphragm
It is situated below the condenser.
It is made up of a circular disc having a circular annular groove.
The light rays are allowed to pass through the annular groove.
Through the annular groove of the annular diaphragm, the light rays fall on the
specimen or object to be studied.
At the back focal plane of the objective develops an image.
The annular phase plate is placed at this back focal plane.
The phase plate
It is either a negative phase plate having a thick circular area or a positive phase
plate having a thin circular groove.
This thick or thin area in the phase plate is called the conjugate area.
The phase plate is a transparent disc.
With the help of the annular diaphragm and the phase plate, the phase contrast is
obtained in this microscope.
This is obtained by separating the direct rays from the diffracted rays.
The direct light rays pass through the annular groove whereas the diffracted light
rays pass through the region outside the groove.
Depending upon the different refractive indices of different cell components, the
object to be studied shows a different degree of contrast in this microscope.
15.
16. Figure 1 All parts of this figure show the same field of view of living HeLa cells (a–e)
and fixed, embedded HeLa cells in thin section (f–h).
(a) Living HeLa cells in culture by phase contrast. (b) The same cells by
transmitted-light bright-field microscopy. (c) In bright-field mode, without
phase contrast, closing the condenserdiaphragm will enhance contrast to
some degree, but at the expense of resolution in the image. This method is to
be avoided. (d) Same image as (a), but the image has been taken with the
annulus and phase plate out of alignment (see also Figures 3e,f). (e) The use
of a green filter improves the quality of the phase contrast image. (f) Stained
HeLa cells, together with the bright-field image (g) for comparison with the
phase contrast image (h).
Parts(h)and(i)areincludedforcomparisonofphasecontrastimagesoflivingcellswiththos
ethathavebeenfixed,embeddedandsectionedthinly. The manner in which cells and
tissues are fixed (if at all) and prepared will influence the resulting phase contrast
image. The living cells (h) exhibit high contrast, where there is a relatively high
difference of refractive index between the cells and the watery medium they are
contained in. The sections of cells embedded in resin in (i) exhibit lower contrast.
This is because there is a smaller difference of refractive index between the cell
constituents and the background resin. Likewise, cells fixed in methanol, an
extracting fixative, exhibit a higher contrastimage than those fixed in
paraformaldehyde, a crosslinking fixative that retains more of the cytoplasm.
Figures(a)–(e) were taken using a Zeiss Axiovert 25, inverted microscopefor tissue
culture using a 32NA 0.5 long working distance objective. Figures (f)–(h) were
17. taken using a Zeiss Axiophot microscopeequipped with a Plan Neofluar 40NA
1.30 oil immersion phase contrast objective
Interpreting the PhaseContrastImage
Provided that the undiffracted and diffracted beams are out of phase with one another
by12l overall, they will interfere to form a visible image, and it does not matter whether
the diffracted beams are retarded or advanced by14l with respectto the undiffracted beam.
Two forms of phase contrastmicroscopyare therefore possible; these are referred to as
positive and negative phase contrast. Positive phase contrast refers to the most widely
used system where the phase plate is constructed with a ‘trench’, so that the diffracted
beams (passing outside the phase ring) travel one-quarter of a wavelength further than the
zero order beams. Structures with a refractive index higher than their
Surroundings give rise to diffracted beams retarded by one quarter wavelength, and these
more highly refracting areas will thus appear darker in the final image, against a lighter
background (Figures 1a,h). Positive phase contrast is responsible for the commonly
recognized appearance of a cell, with the nucleus, lysosomal compartments and the cell
membrane appearing darker than their surroundings.
The phase contrast effect is maximal at regions of sudden change in optical path
difference (‘edges’), and is less pronounced where the change in optical path difference
between adjacent areas is not so abrupt (‘wedges’), a phenomenon known as ‘shading-
off’. As a consequence, the centre of one structure may appear the same shade of grey as
that of another of quite different refractive index. Phase contrast is better suited to
structures with an ‘edge’ rather than structures with ‘wedge’ boundarie
19. The Halo Artefact
Most beams diffracted by the specimen will not pass through the phase ring.
However, the phase ring has an
Phase Contrast Microscopy
appreciable width and some diffracted rays will inevitably pass through it,
causing the haloes that are a familiar part of phase contrast images.In positive
phase contrast objects of refractive index higher than the background form an
image in which these dark structures are surrounded by a bright halo, and lined
internally with a darker halo. In negative phase contrast, the situation is
reversed. Phase contrast is not suited for making precise linear measurements: it
is difficult to assess accurately the precise position of an edge in the image
owing to the halo artefact.
20. Setting Up the PhaseContrastMicroscope
Set the microscopeup, in proper adjustment for Ko¨ hler illumination
for bright-field microscopy, using a well stained specimen. Ensure that the
condenseris set at the correct height,and is centred.Ifi n doubt, refer to
Bradbury and Bracegirdle(1998)or Oldfield(1994). Without altering the focus,
replace the stained specimen with the transparent one. Open the condenser
aperture fully. Swing in a low power (10 or 20) phase contrastobjective; the
specimen will probably not be visible. Insert the correctannulus; an indication
of the appropriate annulus is usually marked on the barrel of the objective in
green script (e.g. Ph3).
Remove an eyepiece and insert a centring-telescope (sometimes called a ‘phase
telescope’), or insert a Bertrand lens system into the optical path to image the
back focal plane of the objective through the eyepieces. Whichever device is
used,focus onthe phase plate with in the objective. The image of the annulus in
the condenser (which is conjugate with the objective’s phase plate) will also be
in focus.
Using the centring adjustments provided for the annuli, and without disturbing
the normal centre position of the condenseritself, superimpose the image of the
condenserannulus precisely over that of the objective phase ring (Figure 3e).
The centring screws used for this superimposition (usually set at 908 or 1208 on
the condenser housing) are not those used for Ko¨ hler illumination. They are
either captive on the condenser(Figure 3d), or may be recessed hexagonal
screws at the rear of the condenser, requiring an Allen key for adjustment. If in
doubton this point, refer to the manufacturer’s instructions. Once adjusted, the
annuli in the condensershould remain centred over a lengthy period; it should
not be necessary to recentre each time the microscopeis used. Remove the
centring-telescope and replace the eyepiece, or remove the Bertrand lens. Foran
inverted microscopethe alignment procedure is usually the same.
Although in practice the phase contrast system works over the full spectrum of
white light, it must necessarily be manufactured for illumination of one
wavelength,
3
phase ring in absolute alignment.
21. Generally selected as 550 nm, This is chosen because the eye is most sensitive
to green light and objectives are bestcorrected for spherical aberration at this
wavelength. Hence, for optimum contrast, a green filter should be used in the
illuminating light path (Figure 1e). If a satisfactory phase contrastimage is not
obtained (e.g. Figure 1d), first check that the microscopeis correctly set up for
Ko¨ hler illumination, and then that the condenseris correctly centred and set at
the right height .
Applications of Phase contrast Microscopy
Phase Contrast Microscopy
Figure 3 (a) and (b) show the top view of different types of phase contrast
condenser, in which the various annuli are contained within a housing. This
permits them to bechanged quickly and efficiently as required. (c) Thecommonly
encountered green inscription engraved on the barrel of a phase contrast
objective. The correct annulus to use is denoted, shown here by the designation
Ph3.(d) The under side of the condenser in(b),revealing the separate controls for
centring the condenseronto the optical axis during alignment of the microscope,
and those for independently aligning the annulus with the phase ring. The
different sizes ofannuli can also be seen. (e) and (f) showthe effects on the phase
contrast image of not having the annulus and
22. To producehigh-contrast images of transparent specimens, such as
1. living cells (usually in culture),
2. microorganisms,
3. thin tissue slices,
4. lithographic patterns,
5. fibers,
6. latex dispersions,
7. glass fragments, and
8. subcellular particles (including nuclei and other organelles).
Applications of phase-contrast microscopyin biological research are numerous.
23. ADVANTAGES
The advantages of the phase contrast microscopeinclude:
The capacity to observe living cells and, as such, the ability to examine cells in a
natural state.
Observing a living organism in its natural state and/or environment can provide far
more information than specimens that need to be killed, fixed or stain to view under a
microscope.
High-contrast, high-resolution images
Ideal for studying and interpreting thin specimens
Ability to combine with other means of observation, such as fluorescence
Modern phase contrast microscopes, with CCD or CMOS computer devices, can
capture photo and/or video images
In addition, advances to the phase contrast microscope, especially those that incorporate
technology, enable a scientist to hone in on minute internal structures of a particle and can
even detect a mere small number of protein molecules.
24. DISADVANTAGES & LIMITATIONS
Disadvantages and limitations of phase contrast:
Annuli or rings limit the aperture to some extent, which decreases
resolution
This method of observation is not ideal for thick organisms or particles
Thick specimens can appear distorted
Images may appear grey or green, if white or green lights are used,
respectively, resulting in poorphotomicrography
Shade-off and halo effect, referred to a phase artifacts
Shade-off occurs with larger particles, results in a steady reduction of
contrast moving from the center of the object toward its edges
Halo effect, where images are often surrounded by bright areas, which
obscuredetails along the perimeter of the specimen
Modern advances and techniques provide solutions to some of these confines,
such as the halo effect.
Apodized phase contrast utilizes amplitude filters that contain neutral density
films to minimize the halo effect. Essentially, this is attempting to reverse the
definition achieved through phase contrast annuli, but the halo effect can never
be eliminated completely.
The pros that phase contrast has brought to the field of microscopyfar exceed
its limitations. This is easily seen with the myriad of advances in the fields of
cellular and microbiology as well as in medical and veterinary sciences.
25. Reference
Textbook of Medical LaboratoryTechnology –
(Praful B. Godkar)
Textbook of Microbiology- by (C. K. Jayaram
Paniker and R. Ananthanarayan)
A Textbook of Microbiology- by (D. K.
Maheshwari)
Essentials of Medical Microbiology Book by
(Apurba Sankar Sastry and Bhat Sandhya)
Textbook of Microbiology Textbook by (D. R.
Arora)
Internet
