Department of Biotechnology
Indian Institute Of Technology Guwahati
BT 206
Microscopy I
Dr. Sanjukta Patra
History of Microbiology
Before 17th century, study of microbiology was limited by the lack of
appropriate tools to observe microbes.
Robert Hooke: In 1665 built a compound light microscope and used it to
observe thin slices of cork. Coined the word cell.
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• Anton van Leeuwenhoeck: Father of Microbiology
• In 1673 -first person to observe live microorganisms which
he called “animalcules” (bacteria, protozoa), using
microscopes designed by him.
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Techniques to study - the
microorganisms
Microscope – Bright field, Dark field,
Fluorescence, Confocal, SEM, TEM etc
Light Microscope Phase Contrast Microscope
SEM TEM
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Microscopy
• Energy source – Light
• Magnification - Lens
Light
1. Wave nature
2. Particle nature
The nature of light: particle-wave duality
• Absorption
When light passes through an object the intensity is reduced depending upon the
color absorbed. Thus the selective absorption of white light produces colored image.
• Refraction
Direction change of a ray of light passing from one transparent medium to another with
different optical density. A ray from less to more dense medium is bent
perpendicular to the surface, with greater deviation for shorter wavelengths
• Diffraction
Light rays bend around edges - new wave fronts are generated at sharp edges - the
smaller the aperture the lower the diffraction
• Dispersion
Separation of light into its constituent wavelength when entering a transparent medium
the change of refractive index with wavelength, such as the spectrum produced by a
prism or a rainbow
Transmission the light is transmitted from a source on the
opposite side of the specimen from the objective
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Microscopy:
Magnification
A microscope consists of two lenses
• Objective-Variable – 10x, 20x, 40x, 100x
• Eyepiece -Fixed
The magnification of a microscope is the product of the factors of both lenses.
A 40x objective and a 10x eyepiece, provide a 400x magnification.
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Resolution
The light wave defines the limit
Resolution is the ability to render two closely adjacent objects distinct.
λ
• D=
2 × 𝑛 sin θ
λ=wavelength of energy source
n= refractive index of the media
• The value sin θ corresponds to the numerical aperture (NA), the measure of the
light gathering capacity of an objective
• With blue light, the resolution limit is approximately d = 0.2 μm
• With red light, around d = 0.35 μm
• UV objectives attain a resolution just under 0.2 μm. With the naked eye, can not
differentiate structures smaller than 0.2 millimetres
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θ or μ =half aperture angle
Figure 2- A series of light cones derived from objectives of varying focal length and numerical
aperture.
As the light cones change, the angle μ increases
from 7° in Figure 2(a)
to 60° in Figure 2(c)
with a resulting increase in the numerical aperture from 0.12 to 0.87, nearing the limit when
air is the imaging medium
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Empty Magnification: Magnification without resolution
Empty magnification can sometimes be quite useful for making details more easily visible
for the human eye
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Bright-field Images
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Bright-field
Image is a dark sample on a bright background
Sample illumination is transmitted (illuminated from below and observed from above)
White light and contrast in the sample is caused by absorbance of some of the
transmitted light in dense areas of the sample
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Ray Diagram
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Light path
• The light path consists of:
• Halogen lamp -transillumination light source
• Critical or Köhler illumination to illuminate the sample
• Condenser lens - focuses light from the light source onto the sample
• Objective lens - collects light from the sample and magnifies the image
• Oculars lens - to view the sample image
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Köhler illumination- minimises internal stray light, allows control
of contrast
A=Critical Illumination. Conjugate planes are the B=Köhler Illumination: Conjugate planes are the
illuminating bulb filament and sample plane (O). When illuminating bulb filament and Condenser diaphragm.
adjusted correctly, the image of the filament is seen Second conjugate planes are the Field diaphragm and the
coincident with the sample image. A diffusing glass filter (d) sample plane. When adjusted correctly, the image of the
is used to blur the filament image. FD:Fielddiaphragm field diaphragm and the sample are coincident. The
CD: Condenser diaphragm filament is out of the plane of focus, and thus uniformly
diffuse.
The Microscope Image
• Magnification in the microscope is collectively produced by two
independent optical systems — objective and eyepiece
• The image formed is inverted and reversed, i.e. it is seen upside
down, and left to right is also reversed.
Maximum Magnification = 1000X
Resolution = 0.2μ
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Performance
• Bright field microscopy has low contrast with most biological samples –
absorbtion is less
• Staining increases contrast, but prevents use on live cells
• Bright field illumination is useful for samples which have an intrinsic colour
– chloroplasts
• Magnification is limited by the resolving power possible with the
wavelength of visible light
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Sample preparation
• Smear
• Fixation – heat fixation, glutaraldehyde, formaldehyde
• Stain
• Visualisation
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Staining enhances contrast in the microscopic image
Simple staining – one stain
Simple stains -Methylene blue, Safranin, Crystal violet
Differential staining – multiple stains
Differential stains – Gram staining, Negative stains, flagellar stains, endospore stains
Use of a colored (usually blue) or polarizing filter on the light source to highlight
features not visible under white light
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• Malachite green - blue-green counterstain to safranin in the Gimenez
staining technique for bacteria. It also can be used to directly stain spores
• Methyl green - used commonly with bright-field microscopes to dye the
chromatin of cells
• Methylene blue - used to stain animal cells, such as human cheek cells, to
make their nuclei more observable
• Neutral red - or toluylene red stains Nissl substance red, is usually used as
a counterstain in combination with other dyes.
• Nile blue - stains nuclei blue, may be used with living cells.
• Nile red - also known as Nile blue oxazone, is formed by boiling Nile blue
with sulfuric acid Nile red is a lipophilic stain, it will accumulate in lipid
globules inside cells, staining them red.
• Osmium tetroxide - is used in optical microscopy to stain lipids, in fats,
and is reduced by organic materials to elemental osmium, an easily visible
black substance
• Rhodamine - is a protein specific fluorescent stain commonly used in
fluorescence microscopy
• Safranin - Safranin O is a nuclear stain. It produces red nuclei, and is used
primarily as staining processes which use more than one chemical stain.
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Gram staining
• determine gram status to classify bacteria broadly
• It is based on the composition of their cell wall
• Gram staining uses crystal violet to stain cell walls,
iodine as a mordant, and a fuchsin or safranin
counterstain
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Negative staining
• Smear the sample on to the slide
• Nigrosin (a black synthetic dye) or Indian ink (an aqueous
suspension of carbon particles)
• After drying, the microorganisms may be viewed in bright field
microscopy as lighter inclusions well-contrasted against the dark
environment surrounding them
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Dark field microscopy
Transmitted light microscopy
• Both diffracted (interacting rays with specimen) and non diffracted
(undeviated rays through specimen) are collected by objective and
contribute to image formation
• The component of non diffracted background light is very large
• This results in bright low contrast images with poor details
• Removal of non diffracted waves can be a solution
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Dark field
Image is bright on a dark background
Image is composed solely of diffracted wave component
Unscattered (non diffracted) beam from the image is excluded
As non diffracted light is excluded, the field around the specimen (where there is no
specimen to scatter the beam) is generally dark
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Principle
In dark field, an opaque disc is placed underneath the condenser lens
Only light that is scattered by objects on the slide can reach the eye
The light is reflected by particles on the slide
Bright image against a dark background
Pigmented objects are often seen in false colors - the reflected light is of a color
different than the color of the object
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May be false colour visualisation
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Light path
• Light enters the microscope for illumination of the sample
• The patch stop blocks some light from the light source, leaving an outer ring
of illumination
• The condenser lens focuses the light towards the sample
• The light enters the sample, some is scattered from the sample
• The scattered light enters the objective lens, while the directly transmitted
light misses the lens
• Only the scattered light produces the image, while the directly transmitted
light is omitted
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When to use dark field
• Dark field illumination is most readily set up at low magnifications, can be
used with any dry objective lens
• In a liquid sample, dark field is preferred
• Dark field is especially useful for finding cells in suspension
• For initial examination of suspensions of cells such as yeast, bacteria, small
protists, or cell and tissue fractions including cheek epithelial cells,
chloroplasts, mitochondria, even blood cells
• Initial survey and observation at low powers of pond water samples, hay or
soil infusions, protist as Paramecium or metazoan cultures
• Examination of lightly stained prepared slides
• Initial location of any specimen of very small size for later viewing at higher
power
• Determination of motility in cultures
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Advantages
• Live sampling
• Recovery of samples
• Technique well suited for live and unstained biological samples
• No sample preparation
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Disadvantages
• The main limitation is the low light levels seen in the final image
• This requires the sample to be very strongly illuminated, which damage the
sample
• Not only the specimen, but dust and other particles scatter the light and are
easily observed
• Sample materials need to be spread thinly, too much material on the slide
creates overlapping layers leading to difficult interpretation
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Hanging drop:
It is used to observe the motility germination or fission of microorganisms
a cavity slide, which has a circular concavity in the centre, is used
The periphery of the concavity on the cavity slide is smeared with Vaseline
A drop of liquid microbial culture is placed in the centre of the cover glass
If the culture is solid, it is mixed with a drop of distilled water before placing on the cover glass
The cover glass is inverted over the concavity so that the drop hangs freely and the edge of cover glass
adheres tightly to the Vaseline coated periphery of the concavity
The microorganisms present in the hanging drop are now observed under the microscope
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Wet mount method:
• A drop of liquid containing microorganism to be examined is put on a glass
slide
• A cover slip made of thin glass is placed on it
• The fluid spreads out in a thin layer between cover slip and slide
• The mount is now examined under the microscope
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Phase contrast Microscopy
PHASE CONTRAST MICROSCOPY
• Frits Zernike-1934
• Wave property of light
Principle
• Light passing through one material into another of a different
refractive index undergoes change in phase
• Differences in phase are translated into variations in brightness of
the structure, hence detectable by the eye
• Phase – invisible, Light intensity-visible
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Refraction
Short wavelengths are bent
more than long wavelengths
dispersion
Light is bent and the resultant colors separate (dispersion). Red is
least refracted, violet most refracted.
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• The necessary phase shift is introduced by rings etched accurately
onto glass plates so that they introduce the required phase shift when
inserted into the optical path of the microscope
• This technique allows phase of the light passing through the object
under study to be inferred from the intensity of the image produced
by the microscope
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(a) Organelles are nearly invisible in bright field although they have different refractive indexes
(b) Light is bent and retarded more by objects with a high refractive index
(c) In phase contrast a phase plate is placed in the light path. Barely refracted light passes through
the center of the plate and is not retarded. Highly refracted light passes through the plate
farther from center and is held back another one quarter wavelength
(d) The microscope field shows a darker background (in this case the cell cytoplasm has a higher
refractive index than the contractile vacuole), with the organelles in sharp contrast
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The phase-plate increases the
phase difference to half a
wavelength. Destructive
interference between the two
sorts of light when the image is
projected results in the specimen
appearing as a dark object.
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Modifications
• Phase contrast condensers
and objective lenses
• To use phase contrast the light
path must be aligned
• Annular ring- is aligned with condenser. This usually involves sliding a
component into the light path or rotating a condenser turret
• Generally, more light is needed for phase contrast than for corresponding
bright field viewing
• since the technique is based on a diminishment of brightness of most objects
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Phase contrast of buccal smear
Wherever there was a change in density - a cell wall, a membrane, or a granule - one could see
different light intensities in the eyepiece. In this way one can see structures within living cells
not otherwise visible
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Cell components show wide variation in refractive index
• Refractive property of structure is directly proportional to bending of light
• The same properties that cause the light to bend also delay the passage of
light by a quarter of a wavelength
• Light from highly refractive structures arrives about a quarter of a wavelength
out of phase as compared to low refractive material
• If the light from an object to the edges of the objective lens is retarded a half
wavelength and the light to the center is not retarded at all, then the light rays
are out of phase by a half wavelength
• They cancel each other when the objective lens brings the image into focus
• A reduction in brightness -object is observed
• The degree of reduction in brightness depends on the refractive index of the
object
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Applications for phase contrast microscopy
• The phase contrast microscope has made it possible to study living cells
• When high magnifications (400x, 1000x) are needed and the specimen is colorless
or the details so fine that color does not show up well
• Cilia and flagella, for example, are nearly invisible in bright field but show up in
sharp contrast in phase contrast
• Amoebae look like vague outlines in bright field, but show a great deal of detail in
phase
• Most living microscopic organisms are more clear in phase contrast
• Phase contrast made it possible to study the cell cycle
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