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MICROSCOPY
"micro" (small)
"scopeo" (to watch)
THE RELATIVE SIZES OF MOLECULES,
CELLS AND ORGANISMS
THE RELATIVE SIZES OF MOLECULES,
CELLS AND ORGANISMS
MICROSCOPY
MICROSCOPY
1590
2009
THE LIGHT
Light: electromagnetic radiation of a wavelength that is visible to the human
eye (about 400–700 nm).
Light can exhibit properties of both waves and particles (photons). This
property is referred to as wave–particle duality.
Monochromatic light: light ray possessing one single wavelength
Complex light: mixture of light rays with more different wavelengths.
THE LIGHT
Characteristic parameters
of light waves:
Wavelength: the distance between repeating units of a
propagating wave of a given frequency. (λ, expressed in nm).
Frequency: The number of oscillations within a minute.
Amplitude: distance from the center y position to the peak
COMPONENTS OF VISIBLE LIGHT
PARTS OF A LIGHT MICROSCOPE
MAGNIFICATION
MAGNIFICATION
Total visual magnification of the microscope is derived by multiplying
the magnification values of the objective and the eyepiece.
Ocular: 5-30 x magnification
Objective:
4-100 x magnification
Maximum
magnification:
3000X
Can we apply the maximal magnification?
The resolution
~0.25 mm
Resolution: the shortest distance between two
points on a specimen that can still be distinguished
by the observer or camera system as separate
entities.
The resolution of our optical equipment is better as
closer points can be seen as separate ones.
The resolution of a light microscope
The resolution of the microscope, () :

wavelength

() =
2A numeric aperture
objective
object
A = n·sinα
The letter n is the refraction index of the media between the cover slip
and the objective (air n=1, distilled water n=1.33, cedar oil n=1.51).
α labels the angle closed by the main optical axis and the outermost light
beam (half angle of the objective)
Possibilities of resolution improvement
(i) Reduction of the numerator, i.e. application of light
beam with a shorter wavelength.
With UV light the resolution can be reduced to 0.1 μm, but special quartz lenses and UV-light
detector are needed, therefore the light microscope with UV light source is only a theoretical
possibility.
Possibilities of resolution improvement
(ii) To increase the value of the numeric aperture (A = n·sinα).
Increasing n (refraction index) :
The resolution of the microscope can be enhanced by dropping a solution
with higher refractive index between the front lens and the coverslip.
Those lenses (mainly objectives with 100x magnification) are named as
immersion objectives.
The immersion liquid mentioned above is cedar oil, thus these lenses are objectives with oil immersion (they
are labeled with HI). For the WI labeled objectives distilled water is the liquid which should be used.
Increasing α:
The half angle of a lens can be increased only until 72°, since at larger
angle than this the light beams became totally reflected.
MICROSCOPIC MEASUREMENT
objective micrometer
ocular micrometer
MICROSCOPIC MEASUREMENT
A piece aof
hair
objective micrometer scale
ocular micrometer scale
ocular
micrometer scale
ADVANCED MICROSCOPY
1. Phase contrast microscopy
2. Fluorescence microscopy
3. Laser scanning confocal microscopy
4. Electron microscopy
Phase contrast microscopy
A large spectrum of living biological specimens are virtually transparent when observed in the
optical microscope under brightfield illumination. Phase contrast microscopy provides an
excellent method of improving contrast in unstained biological specimens without significant loss
in resolution, and is widely utilized to examine dynamic events in living cells. Fritz Zernike
received a Nobel prize in 1953 for his discovery of phase contrast. In a phase-contrast
microscope, the annular rings in the objective lens and the condenser separate the light. The light
that passes through the central part of the light path is recombined with the light that travels
around the periphery of the specimen. The interference produced by these two paths produces
images in which the dense structures appear darker than the background.
Phase contrast microscopy
Phase contrast microscopy
Fluorescence microscopy/
Laser scanning confocal microscopy
Fertilization:
Sperm aster, male
and female pronuclei
MT,
DNA,
CS
Transfected tissue culture cells
DNA microtubules F-actin
Microscopy: the past and present
intestine cross section,
haematoxilin/eosin staining
intestine cross section,
multicolor fluorescence image
Microscopy: the past and present
testis cross section,
haematoxilin/eosin staining
testis cross section, multicolor
fluorescence image
Fluorescence
Fluorescence - The process by which a suitable atom or molecule, which is transiently excited by
absorption of external radiation at the proper energy level (usually ultraviolet or visible light), releases the
absorbed energy as a photon having a wavelength longer than the absorbed energy. The fluorescence
excitation and emission processes usually occur in less than a nanosecond.
Fluorescence Microscopy
Fluorescence Microscopy is the most rapidly
expanding microscopy technique employed
today, both in the medical and biological
sciences.
When coupled to the optical microscope,
fluorescence enables investigators to study a
wide spectrum of phenomena in cellular
biology. Foremost is the analysis of
intracellular distribution of specific
macromolecules in sub-cellular assemblies,
such as the nucleus, membranes, cytoskeletal
filaments, mitochondria, Golgi apparatus, and
endoplasmic reticulum. In addition to steady
state observations of cellular anatomy,
fluorescence is also useful to probe
intracellular dynamics and the interactions
between various macromolecules, including
diffusion, binding constants, enzymatic
reaction rates, and a variety of reaction
mechanisms, in time-resolved measurements.
For example, fluorescent probes have been
employed to monitor intracellular pH and the
localized concentration of important ions.
Fluorescence Microscope Structure
Fluorescence Filter Cube Structure
Exciter filter: permits only selected wavelengths from the illuminator to pass
through on the way toward the specimen.
Barrier filter: blocks the excitation wavelengths and permit only selected
emission wavelengths to pass toward the eye or other detector.
Dichromatic beamsplitter (dichroic mirror): reflects excitation
wavelengths and passes emission wavelengths.
Fluorescence Microscope Structure
Microscopes with an inverted-style frame are designed primarily for tissue culture applications and are
capable of producing fluorescence illumination through an episcopic and optical pathway. Epiilluminators usually consist of a mercury or xenon lamphouse (or laser system) stationed in a port at the
rear of the microscope frame. Fluorescence illumination from the arc lamp passes through a collector lens
and into a cube that contains a set of interference filters, including a dichroic mirror, barrier filter, and
excitation filter. Light reflected from the dichroic mirror is restricted in wavelength by the excitation filter
and enters the objective (now acting as a condenser) to bathe the specimen with a cone of illumination
whose size and shape is determined by the objective numerical aperture. Secondary fluorescence, emitted
by the specimen, returns through the objective, dichroic mirror and barrier filter before being routed
through the microscope optical train. The microscope presented above contains a trinocular observation
tube that is equipped with a port and extension tube for mounting a traditional or CCD camera system (a
Peltier-cooled CCD camera is illustrated). Another port, located near the base at the front of the
microscope, can also serve as an attachment point for a camera system (a traditional 35-millimeter camera
is shown in the figure). In the figure presented above, wide-spectrum fluorescence illumination is filtered
to produce a narrow bandwidth of green excitation wavelengths, which are capable of exciting specific
fluorophores in the specimen. Secondary fluorescence (red light) passes back through the objective and is
distributed throughout the microscope optical system.
Transmitted illumination is provided by a tungsten-halogen lamphouse that is positioned on the
microscope pillar, above the stage. Light from the lamphouse passes through a collector lens, a series of
filters, and the field diaphragm before entering the condenser front aperture. After being focused by the
condenser lens elements, transmitted illumination is projected onto the specimen, which is placed on the
stage. The light that is diffracted, refracted, and not absorbed by the specimen continues through the
objective and into the microscope optical train where it can be directed to the eyepieces or to a camera
system.
Fluorochromes
Fluorochrome - A natural or synthetic dye or molecule that is
capable of exhibiting fluorescence. Fluorochromes (also termed
fluorescent molecules, probes, or fluorescent dyes) are usually
polynuclear heterocyclic molecules containing nitrogen, sulfur,
and/or oxygen with delocalized electron systems and reactive
moieties that enable the compounds to be attached to a biological
species.
Fluorochromes
Fluorochromes
Bovine pulmonary artery endothelial cells visualized using components of the SelectFX Nuclear Labeling
Kit and Alexa Fluor phalloidin conjugates. Nuclei and F-actin were stained, respectively, with (top left)
DAPI and Alexa Fluor 680 phalloidin, (top right) SYTOX Green dye and Alexa Fluor 568 phalloidin,
(bottom left) 7-AAD and Alexa Fluor 488 phalloidin, and (bottom right) TO-PRO-3 dye and Alexa Fluor
350 phalloidin.
Organelle/DNA/Ca2+ probes
Organelle probes:
a fluorochrome nucleus attached to a target-specific
moiety that assists in localizing the fluorophore
Mitochondrium: MitoTracker and MitoFluor
Lysosome: LysoTracker and LysoSensor
Golgi apparatus: BODIPY
Endoplasmic reticulum: DiOC, Blue-White DPX
DNA: Acridine orange, Propidium iodide, DAPI,
Hoechst
Ca2+: fura-2 and indo-1, fluo-3, fura red
Bovine pulmonary artery endothelial cell labeled with
probes to visualize mitochondria, peroxisomes, and the
nucleus. Mitochondria were stained with the MitoTracker
Red CMXRos reagent. Peroxisomal labeling was
achieved with a primary antibody directed against
PMP70, visualized using green-fluorescent Alexa Fluor
488 goat anti–rabbit IgG. The nucleus was stained with
blue-fluorescent DAPI.
Immunofluorescence Microscopy
A targeted molecular species (protein, nucleic acid, membrane, etc.) in a specimen is labeled with
a highly specific fluorescent antibody. After the labeled antibodies have been excited by a selected
region of wavelengths, secondary fluorescence emission is gathered by the objective to form an
image of the specimen. Antibodies are labeled either by coupling directly with a fluorochrome
(fluorescent dye; termed direct immunofluorescence), or with a second fluorescent antibody that
recognizes epitopes on the primary antibody (indirect immunofluorescence).
Immunofluorescence Microscopy
Immunofluorescence Microscopy
MT
DNS
CS
Fluorescent Proteins
Green Fluorescent Protein (GFP) - A naturally occurring protein fluorescent probe derived from the
jellyfish Aequorea victoria, which is commonly employed to determine the location, concentration,
interactions, and dynamics of a target protein in living cells and tissues. The excitation and emission
spectra of enhanced GFP (a genetic derivative) have maxima at 489 nanometers and 508 nanometers,
respectively. In order to incorporate the GFP (or any of its genetic derivatives) into a cell, the DNA
sequence for the gene is ligated to the DNA encoding the protein of interest. After cultured cells have been
transfected with the modified DNA, they are able to express chimeric fluorescent proteins for observation
in the microscope. There are genetically modified variants of GFP such as blue fluorescent protein (BFP),
cyan fluorescent protein (CFP), yellow fluorescent protein (YFP)
DsRed fluorescent protein
Fluorescent Proteins
Tubulin-GFP
GFP-FUSION PROTEINS
gene of interest
plasmid with GFP gene
transfection
fusion protein
emits green light
upon excitation
by blue light,
intracellular
localization
determined
GFP
transcription,
translation
Confocal Laser Scanning Microscopy
Confocal Laser Scanning
Microscopy - A popular mode of
optical microscopy in which a
focused laser beam is scanned
laterally along the x and y axes of
a specimen in a raster pattern. The
emitted fluorescence (reflected
light signal) is sensed by a
photomultiplier tube and
displayed in pixels on a computer
monitor. The pixel display
dimensions are determined by the
sampling rate of the electronics
and the dimensions of the raster.
Signal photons that are emitted
away from the focal plane are
blocked by a pinhole aperture
located in a plane confocal with
the specimen. This technique
enables the specimen to be
optically sectioned along the z
axis.
Confocal Laser Scanning Microscope
270000 EURO
Light sources for fluorescence/confocal microscopy
As opposed to traditional arc-discharge lamps used with the shortest range (10-20 nanometers) bandpass
interference filters in widefield fluorescence microscopy, the laser systems used for fluorophore
excitation in scanning confocal microscopy restrict excitation to specific laser spectral lines that
encompass only a few nanometers.
Z-sectioning
Z-sectioning
Drosophila Egg Chamber
Z-sectioning
Drosophila Egg Chambers
3D Reconstruction
After Z-sectioning, the computer attached to the confocal
microscope can generate virtual images of the specimen what
can be viewed from any desired angles.
3D Reconstruction
Drosophila
Egg
Chambers
4D imaging
The confocal microscope can be programmed to take
pictures of the living sample at any desired time
intervals. The resulting pictures are put together as
movie files.
4D imaging
Tubulin-GFP
C. Elegans
embryonic
divisions
4D imaging
Tubulin-GFP, Histon-RFP
Drosophila
embryonic
divisions
FRET
Fluorescence Resonance Energy Transfer (FRET) - An adaptation of
the resonance energy transfer phenomenon to fluorescence microscopy
in order to obtain quantitative temporal and spatial information about the
binding and interaction of proteins, lipids, enzymes, and nucleic acids in
living cells.
FRET: fluorescence resonance energy transfer
CFP
CFP
YFP
YFP
FISH
Fluorescence in situ Hybridization
(FISH) - The fluorescence FISH
technique is based on hybridization
between target sequences of
chromosomal DNA with fluorescently
labeled single-stranded
complementary sequences (termed
cDNA) to ascertain the location of
specific genetic sequences. In medical
practice, the most important
application of FISH is the prenatal
diagnosis of chromosome number
abnormalities (and other chromosomal
mutations).
ELECTRON MICROSCOPY
Elecron ray source (Electron gun)
Filament
Heat
-
-
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Wehnelt cap
(negative potential)
-
-
Space charge
e-
e-
-
ee-
+++++++++++
+++++++++++
Anode (positive potential)
Transmission electron microscope (TEM)
Electron ray source
Condenser lenses
Condenser aperture
Sample
OBJECTIVE
Objective apertures
Intermediate lenses
PROJECTOR
Detector
Transmission electron microscope (TEM)
Scanning Electron Microscope (SEM)
Electron ray source
1st condenser lens
Condenser aperture
2nd condenser lens
Objective aperture
Scan coils
Amplifier/Detector
OBJECTIVE
Backscattered
electrons
Sample
2nd electrons
Scanning Electron Microscope (SEM)