MICROSCOPY
basic requirement-extra requirement
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.
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
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?
No, because over 1000x- magnification we do not see more details, the points of the image
just get bigger. Therefore the resolution is important, not the magnification.
THE RESOLUTION
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. he
resolution of a good light microscope is ~0.25 m
The resolution of the microscope, () : = /2n·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.
(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.
ADVANCED 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.
FLUORESCENCE MICROSCOPY
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 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 subcellular 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.
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. Epi-illuminators 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.
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.
ORGANELLE PROBES: most cell organelles could be stained with specific fluorochromes.
Mitochondrium: MitoTracker and MitoFluor, Lysosome: LysoTracker and LysoSensor, Golgi apparatus:
BODIPY, Endoplasmic reticulum: DiOC, Blue-White DPX, DNA/nucleus: Acridine orange, Propidium iodide,
DAPI, Hoechst
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).
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)
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. 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 (Z-sectioning).
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.
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.
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. One of the proteins is fused to CFP, the
other is fused to YFP. If there is interaction between the two proteins, we get yellow signal
after exciting CFP, if there is no interaction between the two proteins, we get blue signal after
exciting CFP. In case of interaction, the light emitted by CFP excites YFP.
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
Electron microscopy is taught in detail by physics and anatomy. It uses high energy electron
rays for imaging. Two versions exist: transmission and scanning electron microscopy.