LECTURE 2: IMAGING IN CELL BIOLOGY
Anton van Leeuwenhoek (1632-1723)
• Built many simple, single lens microscopes
• First to observe living protazoa and bacteria which he called “animalcules”
• Went on to visualize human red blood cells and sperm
• With great skill at grinding lenses, naturally acute eyesight and lots of patience he was able to achieve a magnification of
• Placed a drop of water, containing cells of interest, on the edge of a pin surrounded by screws
• Screws could move the pin closer to or further away from the magnifying glass
Features of a Modern Compound Microscope
• Light source
• Condenser lens to focus light on specimen
• Objective lens to collect light after it has passed through specimen
• Ocular or eyepiece lens to focus image onto eye
• Typical light microscope magnification is 40 to 1000X
• Only structures with a high refractive index (ability to bend light) are observable
• Compound microscopes, used in conventional bright-field light microscopy, contains several
lenses that magnify the image
• Total magnification is a product of the magnification of the individual lenses: if the objective lens (lens
closest to specimen) magnifies 100X (maximum) and the projection lens/ocular or eyepiece lens
magnifies 10X, the final magnification recorded will be 1000X
• Certain structures inside the cell are able to bend light more – these are observed as darker areas in images (more light
Resolution of Microscopes
• Resolution: the ability to distinguish between two very closely positioned objects as separate entities
• Aconventional microscope can never resolve objects/cellular features that are less than ~0.2 mM apart
• Smaller resolution is better
Resolution = D
• Distance resolved between 2 points
• λ: wavelength of light
• Nsinα: numerical aperture (higher is better)
• N: refractive index of medium between the specimen and the objective lens
• α: ½ angle of light entering objective
• The limit of resolution is 0.2 mm = 200 nm – cannot resolve objects less than 0.2
mm apart or reveal details about 0.2 mm in size
• Optimizing resolution (D) to make it as small as possible is highly favorable:
o Decrease λ
o Increase N; can add oil or water, which have a high refractive index
o Increase α; having a wide cone makes a wide angle
o Gathering more light (increases N and α)
Obtaining contrast in light microscopy by exploiting changes in the phase of light
• Certain parts of the cell (i.e. nucleus) refract light more than other parts
• Cellular constituents with high refractive properties can slow the passage of a light beam by
a quarter wavelength (~1/4λ)
Phase Contrast Microscopy
• Used to examine live “unstained” cells • Generates an image in which the degree of darkness or brightness of a region of the sample
depends on the refractive index of that region
• Small differences in refractive index & thickness within the cell are further exploited and converted
into contrast visible to the eye
• Light that passes through a transparent specimen changes its phase depending on the refractive index and thickness of
cell structures, which are normally not visible to our eyes. Phase-contrast optics, however, converts these differences into
differences in light intensity visible to the eye.
• From technical point of view, the phase-contrast microscope is a bright-field light microscope with the addition of:
o (1) Phase-contrast objectives containing a phase plate (a transparent plate with a phase ring)
o (2)Acondenser annulus (annular stop or diaphragm, a clear ring in an opaque disk).
• The specimen is illuminated with a hollow cone of the light from annulus. Phase plate shifts the phase of the undeviated
light by approximately one-quarter of a wavelength.
• Finally, the refracted and unrefracted light rays are recombined at the image plane to form the contrast image, i.e. phase-
contrast microscopy generate contrast by interference between light scattered by the specimen and a slightly
delayed reference beam of light.
• Inverted phase contrast microscopes are used routinely to monitor live cells in cell culture flasks. This technique is also
very useful for examining the location and movement of large organelles in live cells.
• Suitable for observing single cells or thin cell layers, but not thick tissues
Differential Interference Contrast Microscopy (Normarski Microscopy)
• Used to examine live “unstained” cells
• Small differences in refractive index and thickness within the cell are converted into contrast visible to the eye
• Uses polarized light
• DIC microscope is equipped with polarizers
• Defines the outline of large organelles such as nucleus and vacuole and provides better detail of cell edge
• Similar to phase contrast microscopy, DIC allows for conversion of small differences in refractive index & thickness within
the cell into contrast visible to the eye.
• In comparison with phase contrast microscopy, DIC is based on interference between polarized light and accordingly a DIC
microscope is equipped with polarizers.
• DIC images are sharper than phase contrast images and objects appear to cast a shadow on one side. The “shadow”
primarily represents a difference in refractive index of a specimen rather than its topography (note thatAFM visualizes the
topography of a cell)
• Phase contrast and DIC microscopy can be used in time-lapse microscopy
• Uses a property of certain molecules to fluoresce, i.e. to emit visible light when they absorb light at a specific wavelength
(e.g. invisible UV light)
• The amount of energy emitted as fluorescence emn ) is always less than the amount of energy absorexd (hn ) because
some portion of energy is dissipated due to the interaction with microenvironment
• In terms of wavelengths, the emitted light has a longer wavelength than the absorbed light because of inverse relationship
between n and l (E=hn=hc/l, h – Plank’s constant, c - the velocity of monochromatic light)
• Location of fluorescent dyes or fluorescent protein molecules can be imaged
o Green – Tubulin
o Red – Mitochondria
o Blue – Nuclei
• Can visualize more than one protein or cell structure
• Two major limitations:
o Blurred image caused by superposition of fluorescent images from molecules at many depths in the cell – the
blurring effect makes it difficult to determine the actual molecular arrangements
o To visualize thick specimens, consecutive (serial) images at various depths through the sample must be collected
and then aligned to reconstruct structures in the original thick tissue • Dyes or fluorophores absorb energy kicking electrons (e) into a higher orbital (unstable)
• Instability causes e to drop into its normal orbital releasing
energy as visible light fluorescence
• Fluorescence is a phenomenon in which a material absorbs
light of one color (wavelength) and emits it at a different color
(wavelength).Absorption occurs when an incoming photon (light
particle) causes an electron to move from a stable ground state
to a higher energy, unstable excited state.
• One of the ways for the excited electron to return
to the ground state is to 'jump' back down, emitting a
photon of light. There is always some energy lost to heat in the process, so the emitted photon has less
energy than the original photon. The energy of a photon is
related to its wavelength, which we perceive as color –
higher energy corresponds to shorter wavelengths,
lower energy to longer wavelengths.
How are fluorescence images obtained?
• Fluorescence microscopy is perhaps the most versatile and
powerful technique to characterize cellular organization and to
localize proteins within a cell. If we stain properly cells,
location of fluorescent dyes or fluorescent protein molecules can
be imaged using a fluorescence microscope.
• Fluorescence microscope is a light microscope which
accomplish the following four functions: (1) deliver excitation
light of the appropriate wavelength to the specimen; (2) separate
the excitation light from the emitted fluorescence; (3)
collect as much as the emitted fluorescence from
fluorophores as possible; (4) allow observation of fine
details in the specimen.
• All these functions are available in modern fluorescence microscopes, which are epifluorescence microscopes, i.e.
excitation and observation of the fluorescence are from above (epi–) the specimen. (This is, the excitatory light is passed
from above through the objective lens and then onto the specimen instead of passing it first through the specimen)
• The specific and central part of a fluorescence microscope is a filter cube, which consists of three filters (exicitation filter,
emission filter, and dichroic beam-splitting mirror)
• Excitation filter allows selection of appropriate excitation light which is delivered on the specimen by the objective lens.
• Aspecial mirror, known as a dichroic beam-splitting mirror, allows separation of the excitation light from the emitted
fluorescence. The dichroic mirror has the special property of being able to reflect light below a specific wavelength, yet
allow light above this specific wavelength to pass through the mirror unobstructed.
• In epiillumination, the dichroic mirror reflects the excitation light into the back of aperture of the objective. The
objective acts as condenser and focuses the excitation light onto the specimen.
• Aportion of the emitted fluorescence is collected by the objective and passes through the dichromatic mirror and
emission filter either to eyepiece of detector. Emission filter serves to separate the emitted
fluorescence from the excitation light and any other scattering light before reaching the
The Many Colors of Fluorophores
• Signals are bright on a black background.
• Avariety of fluorophores exist with different excitation and emission wavelengths that
allow labeling of more than one protein or organelle at the same time
• Fluorescent dyes are available to stain cell structures and organelles (e.g. DAPI to stain nuclei blue,
Mitotracker Red to stain mitochondria red)
• Adye can be conjugated with antibodies to localize any molecules of your interest in cells
MonoclonalAntibodies • HAT medium (a selection medium) is toxic for myeloma cells, which have mutation of specific gene.
• Hybrid cells survive in HAT medium because they obtain a missing gene product from spleen cells.
• Hybrid cells are immortal like myeloma cells and produce antibody.
• Primary spleen cells are resistant to HAT medium; they will not grow indefinitely, will eventually die off because they are not
immortal as they have a finite life span
• Producing a monoclonal antibody:
• Step 1 – generate immortal, antibody-producing cells
o Inject synthetic protein into a rat or mouse, mouse’s immune system will recognize it as foreign and an immune
response will be induced, in which B cells proliferate in the spleen
o Immorality is achieved by fusing normal B lymphocytes from an immunized animal with transformed, immortal
lymphocytes called myeloma cells
o Some of the fused cells undergo division, producing hybrid cells
o Fusion of myeloma cell with a normal antibody-producing B cell from spleen yields a hybrid that proliferates into a
clone called a hybridoma
o Hybridoma cells grow rapidly and are immortal
• Step 2 – separate, or select, the hybridoma cells from the unfused parental cells and the self-fused generated by the fusion
o Selection is usually performed by incubating mixture of cells in a special culture medium, called selection medium,
that permits growth of only hybridoma cells
o In i