THE LIGHT MICROSCOPE

The light microscope, so called because it employs visible light to detect small
objects, is probably the most well-known and well-used research tool in biology.
A microscope is an instrument designed to make fine details visible. The
microscope must accomplish three tasks: produce a magnified image of the
specimen (magnification), separate the details in the image (resolution), and render
the details visible to the eye, camera, or other imaging device (contrast).
It is used in cellular biology, to observe the cells, the fabrics, in petrography to
recognize the rocks, metallurgy and metallography to examine the structure of a
metal or an alloy. It includes mechanical parts of a very high degree of accuracy and
optical parts, pretty fragile.
The lenses of the optical microscope are used on obtaining an increased
image of the sample to observe. A microscopic system consists of an objective and
an eyepiece, making it possible to solve the problem of the pupillary conjugations
(suppression of the vignettage) and that of the correction of the aberrations of field
(“thick” total system made up of several lenses).
Normally, the object to be observed is placed in front of the lower lens of the
microscope, called “objective”. The objective gives an increased real image of the
object (figure 1). This intermediate image is processed again by the eyepiece “Oc”
which gives of it a virtual image ad infinitum finally observed by the eye. If the object
is beyond the focal distance, one obtains a real image reversed and of different size.
The image is larger than the object if this one is located at a distance lower than the
double of the focal distance from the objective.

Figure 1. Light microscope concept

Characteristic for the microscope is its power, meaning the ratio between the
angle under which the object is seen through the instrument and the length of this
object.
For a comfortable observation, you must preserve a minimal distance between
the ocular lenses and the eye (15 to 20 mm for a naked eye, 20 to 25 mm for an
observation with correction glasses).
The most used illumination technique in microscopy with traditional broad field
is the Köhler illumination, which guarantees optimal image quality.
The microscope parts are (figure 2) :
A. Mechanical parts
a. Static
[1]. Body
[2]. Base
[3]. Stage
b. Mobile
[4]. Coarse focusing knob
[5]. Fine focusing knob
[6]. Stage control knob – front-back
[7]. Stage control knob – left – right (lateral)
[8]. Condenser knob (up-down displacement)
B. Optical parts
[9]. Eyepieces
[10]. Eyepiece support
[11]. Objectives rotative support
[12]. Objectives
[13]. Place for slide (with sample)
[14]. Condenser
[15]. Diaphragm
[16]. Bulb (illuminator)
[17]. Switch (on/off)

Figure 2. Light microscope - components

Light microscope usage:
Place microscope correctly, behind you; you have to stay sit while working,
with the elbows leaning upon the table.
• Check if the smaller objective (x10) is placed in the optical axis and well
fixed.
• Turn on the light by the on/off switch.
• Place the sample (slide) on the stage; place the sample on the slide over the
light. The slide is placed on the stage, between the two metallic supports
that have to embrace it. Generally, the sample should be placed over the
central hole in the stage.
• Coarsely adjust the image by manipulating the coarse focusing knob until
reaching the accurate image, then use the fine focusing knob for the fine
adjustment.
• On the sample, search the most appropriate area for the observation.
• Adapt light intensity by adjusting the diaphragm.
• If required, choose a larger objective for a higher magnification: without
moving the stage by the large focusing knob, switch carefully the objective
spinning support in order to bring the objective of x20 (or x40) in the
optical axis; it should produce a light click when it reaches the correct
position. For image adjustments with the objectives of x20 or x40, use only
the fine focusing knob, otherwise you risk to damage the slide or the glass
slice over the specimen!
For each magnification step, you have to adjust the specimen placement and the
light adjustments. As higher the condenser is placed, the better the specimen will be
illuminated. If you move down the condenser, you will obtain a higher contrast.

Working distances for the objectives:

(optimal values for an 160 mm tube)
OBJECTIVE FOCAL Working distance
10 X 16 mm 6 mm
40 X 4 mm 0.5 mm
100 X 1.6 mm 0.35 mm 0.12 mm
The glass lied over the specimen is already 0.16 - 0.17 mm thick !

Figure 3. Working distances for different objectives

The resolution power (transverse) d for a microscope, meaning the smallest distance
under which two points cannot be seen separately, can be expressed according to λ
(wavelength), n (objective refraction index), and α, the half-angle of the maximum
available light cone angle.

d = λ / 2n sinα = λ / 2NA

where NA means the product n sinα or the objective numerical aperture.

The resolution power can be improved in two ways:

- increasing the refraction index. This can be done by using an immersion objective:
you will plunge the frontal objective lens into a fluid (immersion oil) whose refraction
index is close to 1,5 (glass refraction index).
- reducing the wavelength. However, for the visible light examination, the wavelength
cannot be reduced under 400 nm.

The resolution power for the light microscope is around 0,2 μm.
The electron microscope reaches a limit 100 times smaller.
FLUORESCENCE MICROSCOPY

The term fluorescence describes a light emission induced by various

excitation forms excepting heat (sometimes, fluorescence is called “cold light”).
Fluorescence is the property of some atoms and molecules to absorb light at a
particular wavelength and to subsequently emit light of longer wavelength after a
brief interval, termed the fluorescence lifetime. The fluorescence differs from the
phosphorescence because the light emission undergoes immediately after
excitation. The process of phosphorescence occurs in a manner similar to
fluorescence, but with a much longer excited state lifetime.
Fluorescence is a luminescence process in which susceptible molecules emit
light from electronically excited states created by either a physical (for example,
absorption of light), mechanical (friction), or chemical mechanism. Fluorescent
structures are including in their structure some chemical groups called chromophores
that are responsible for this feature.
The most frequent fluorescent organic molecules include benzene rings.
Under photon impact, the chromophore goes to an excited state, and then it goes
back to the fundamental status (in quantum physics the fundamental status is
generally a status with a lower energy) by light emission.
Fluorescence microscopy is different from other types of light microscopes that
are based on the microscopic sample features as phase gradients, light absorption or
birefringence. Fluorescence microscopy images are formed by the specific
distribution of the molecular species able to emit fluorescence.
By fluorescence microscopy, we can record the particular placement of the
intracellular compounds marked by specific fluorophores and, at the same time, their
diffusion coefficient, transport features, or interactions with other biomolecules.
Moreover, fluorescence changes according to environmental variables allow
exploration of pH, viscosity, refraction index, ionic concentrations, membrane
potential etc.

Fluorescence types
We have two types of fluorescent objects:
- self fluorescent objects (primary fluorescence) – that are natural fluorescent
light emitting (chlorophyll, oil, collagen, elastin, fibrillin, flavones,
indolamines, pigments, amino acids).
- objects that must be combined (physically or chemically) with a fluorescent
substrate in order to generate fluorescence; this phenomenon is called
secondary fluorescence. The fluorescent substrate required to induce
fluorescence to the primary object is called fluorophore (examples: DAPI -
4',6' Di Amidino-2-Phenyl Indole - with is staining DNA in blue
fluorescence.

Fluorescence microscope allows us to directly visualize fluorescent substrates

(table 1).

Table 1. Fluorescent substrates and excitation wave color

Fluorochrome Rhodamine Fluorescein FITC Acridin Orange DAPI
Excitation color GREEN BLUE BLUE BLUE UV
 Some genetic markers as Green Fluorescent Protein or GFP are very useful in
biologic research. Here, GFP is the fluorophore, being a protein synthesized directly
by the cell and does not require other substrate addition. Thus, fluorescence can be
observed directly inside living cells.
Fluorescence microscopy is limited by light diffraction, and thus, resolution
power is only varies around an average of 200 nm.

Fluorescence microscope working principle

The fluorescence microscope includes a special light source, usually a high-
pressure mercury arc lamp (HBO - a burner). The emitted light (UV for this lamp) is
focused by the collector lenses and travels along the interior of an illuminator parallel
to the tabletop and perpendicular to the microscope optical axis. Inside the illuminator
there are some heat filters, diaphragms, and the turret with the fluorescence filters
(including the excitation filter, dichroic mirror and the barrier filter) (figure 4).

Figure 4. Fluorescence microscope structure

The emitted UV beam travels through the excitation filter that keeps only a
narrow part of the spectra. The beam is then directed toward the specimen by a
dichroic mirror; this mirror is special while it exhibits a strong reflection coefficient for
the specific filtered wavelengths and a strong transmission coefficient for the
wavelengths that correspond to the fluorescence emitted by the specimen. The
emitted wavelengths are then filtered by the specific emission filter.
Excitation filter, the dichroic mirror and the emission (barrier) filter are usually
assembled into a single cube and can be exchanged according to a specific range of
fluorophores that we are using in biomedical applications.

Microscopic applications of the fluorescence phenomenon

Fluorescence principles can be applied with different types of microscopes:
 - on a classical light microscope. We can direct the excitation beam to cross
through the objective instead falling onto the specimen. Thus we may define the
epifluorescence microscopy;
- on a confocal laser scanning microscope (CLSM). CLSM is a microscope
used to obtain high-resolution optical images at different depths into the specimen.
Confocal microscope is able to acquire in-focus images from selected depths, a
process known as optical sectioning; final resolution is better than the classical light
microscope. Optical sections can be reconstructed by specific software in order to
obtain a three-dimensional representation of a complex fluorescent object in the
specimen;
- on a total internal reflection fluorescence microscope (TIRFM). This type of
microscope is able to observe just a very thin region of a specimen, usually less than
200 nm. Thus, the field depth is better than the confocal microscope (range - 600
nm), but its availability is reduced to the specimen base, at the interface between the
specimen and the support (glass slide).

Practical applications of fluorescent labeling

Simple labeling is realized by affinity between a fluorochrome and the
molecule to be labeled (ex. DAPI is a DNA label and fluoresces in blue).
An example is represented by the comparison between 3 fluorochromes with
different absorption and emission spectra. Fluorescein (FITC – Fluoresceine Iso-
Thio-Cyanate) absorb blue radiations (max 490 nm) and produces a green
fluorescence (max 520 nm). Rhodamine (TRITC – Tetra-methyl Rhodamine Iso-Thio-
Cyanate) abrorb green radiations (max 541 nm) and produces a red fluoprescence
(max 572 nm). DAPI (Di Aminido Phenyl lndol) absorb violet radiations (max 372 nm)
and produces a blue fluorescence (max 456 nm). The results are varying according
to specific fluorochrome type for different subcellular structures (figure 5).
Immunofluorescence (figure 6) is useful to detect a certain substrate X which
is non-soluble (and fixed) into the cell. First, we use the substrate X as an antigen
which will be injected to another species (ex. rabbit)(6A). The rabbit immune system
is reacting by producing antibodies (immunoglobulins) anti-X (6B). We are separating
the antibodies from the rabbit by affinity chromatography. The antibodies anti-X bind
specifically the X antigens, on cell praparations from the first species (6C). These
antibodies can be directly labeled by fluorescein but it is more useful to bind
fluorescein labeled goat antibodies against rabbit immunoglobulin (6D). When
exposed to ultraviolet light, the fluorescein emits green fluorescence and allow the
localization of the X substrate (6E).
Other techniques for fluorescent labeling:
- FISH technique (Fluorescent In Situ Hybridization) is used to label
nucleotid sequences by fluorescent labeled probes and used on tissue
slices. FISH is a cytogenetic technique that allows us to explore elements
inside a cell.
- FRAP (Fluorescence Recovery After Photobleaching – fluorescence
redistribution following photobleaching) is a technique in which we apply a
laser flash in a small specimen area. In this region, the molecules are
losing their fluorescence. However, if the molecules are mobile in the
environment the fluorescent populations are being redistributed until
homogenization. These techniques allow the investigations on lateral
diffusion for labeled molecules, depending on the molecular displacement
 speed in the environment (at higher diffusion speed the fluorescence if
quickly recovered).
- FRET (Forster Resonance Energy Transfer) uses two fluorochromes, a
donor which transmits its energy to another acceptor fluorophore. This
technique allows examination of intermolecular interactions.
- BRET (Bioluminescence Resonance Energy Transfer), is similar to FRET
but the donor is bioluminescent (luciferase).

FITC for actin labeling in cell

cytoskeleton

TRITC for actin labeling in

cell cytoskeleton

DAPI labeling for cell nucleus

Figure 5. Simple labeling for different cell parts by different fluorophores

Figure 6. Immunofluorescence principle
PHASE CONTRAST MICROSCOPY
When light travels through a substrate, it interacts with it and undergoes
changes in amplitude and phase according to medium properties. Amplitude changes
give rise to light absorption, a wavelength dependent process that gives rise to
colors. Phase changes are not easily observed by free eye, even if these changes
bring large amount of information. Phase contrast technique is used to transform
differences in refraction index into contrast differences. It was developed in the '30s,
by the Dutch physicist Frederik Zernike (Nobel Prize winner in 1953).
Phase contrast microscopy speculates the slowing down of the light while
crossing transparent areas of a specimen, according to non-influenced light. This
phase difference is not visible for the human eye. Phase changes can be improved
with half-wavelength by a phase transparent plate and can produce light intensity
differences. This makes the transparent object to become more shiny then the
environment.
Phase contrast microscopy is used especially to observe living cells, non-fixed
and not stained. The method is actually used together with reflected fluorescence
method in order to observe specimen areas that are not-fluorescent. Phase contrast
microscopy is also useful to observe thin specimens or dispersed in the examination
field.

Principle of the phase contrast microscope

Not stained specimens that are not absorbing the light are called phase
contrast objects while they are slightly changing the phase of the light diffracted by
the specimen; usually this wavelengths are slowed down by approximately 1/4 of a
wavelength according to the direct light, which is not deviated, while it passes
unaffected near the specimen. Our eyes, like photographic film, are unable to detect
such small phase differences. Human eye is sensible only to visible colors (light
waves frequency variations) or to the differences into light intensity (wave amplitude
variations).
In phase specimens, direct ordinary light crosses without being deviated
through or near the specimen. Meanwhile, the light that is diffracted by the specimen
is not reduced in amplitude (as it should be for an absorptive object) but it is delayed
due to refractive index or to the thickness of the specimen or both. This refracted light
remains behind with about 1/4 wavelength and reaches the image plan being out of
phase, compared to the non-deviated light, but with no diminished intensity. This
result in a slight contrast on the ocular lenses and the details are merely visible. The
phase contrast microscope is accelerating the direct transmitted light by 1/4
wavelength; thus, the difference between direct and diffracted light becomes 1/2
wavelength. Thus, direct and diffracted light arrive at the ocular lenses are able to
produce destructive interference. This procedure gives darker details on a brighter
background. This represents the positive or dark phase contrast microscopy. If the
direct light is delayed by 1/4 wavelength then the diffracted light and the direct light
arrive at the same time onto the ocular lenses. The interference is then constructive
and the image is bright on a dark background. The phase contrast is bright, or
negative.
 Figure 7. Phase contrast microscope
principle

Phase contrast microscopy applications

Phase contrast microscopy is an excellent method used in order to amplify the
contrast for fine sliced or transparent specimens without any resolution loss; it is also
used to observe dynamic phenomena in the living cells, not fixed ant not stained.
We may observe:
- living cells,
- not stained, not fixed tissue slices
- transparent microorganims under brightfield illumination
- intracellular structures,
- membranes,
- nuclei,
- mitochondria,
- mitotic spindle and chromosomes,
- Golgi system
- cytoplasmic granules in animal or vegetal cells
Phase contrast microscopy is also used to observe tumor cells and cell
development, dynamics of cultured cells. Cultured cells require an inverted
microscope for direct observation.
POLARIZED LIGHT MICROSCOPE

Polarized light is a contrast-enhancing technique that improves the quality of

the image obtained with birefringent materials. Polarized light is a contrast-enhancing
technique that improves the quality of the image obtained with birefringent materials.
It is particularly used in mineralogy but also in biology and biomedical research.

Principle of the polarized light microscope (figure 9)

Isotropic materials, which include a variety of gases, liquids, unstressed
glasses and cubic crystals, demonstrate the same optical properties when probed in
all directions. These materials have only one refractive index and no restriction on the
vibration direction of light passing through them. In contrast, anisotropic materials,
which include 90 percent of all solid substances, have optical properties that vary
with the orientation of incident light with the crystallographic axes. They demonstrate
a range of refractive indices depending both on the propagation direction of light
through the substance and on the vibrational plane coordinates. More importantly,
anisotropic materials act as beamsplitters and divide light rays into two orthogonal
components.
The two orthogonal components of light (ordinary and extraordinary waves)
travel at different speeds through the specimen and experience different refractive
indices, a phenomena known as birefringence. A quantitative measurement of
birefringence is the numerical difference between the wavefront refractive indices.
The faster beam emerges first from the specimen with an optical path difference
(OPD), which may be regarded as a "winning margin" over the slower one. The
analyzer recombines only components of the two beams traveling in the same
direction and vibrating in the same plane. The polarizer ensures that the two beams
have the same amplitude at the time of recombination for maximum contrast.
Birefringence, or double refraction, is the decomposition of a ray of light into
two rays when it passes through certain anisotropic materials, such as crystals of
calcite or boron nitride. The effect was first described by the Danish scientist Rasmus
Bartholin in 1669, who saw it in calcite. [1] The effect is now known to also occur in
certain plastics, magnetic materials, various noncrystalline materials, and liquid
crystals

Examples de birefringence (double refraction)

Polarized light microscopy applications

Polarized light microscopy is utilized in a broad range of disciplines, including
medicine, biology, geology, materials science, and the food industry. The specimens
that are readily examined between crossed polarizers originate from a variety of
natural and synthetic sources and include gout crystals, amyloid, muscle tissue,
teeth, minerals, solid crystals, liquid crystals, fibers, fats, glasses, ceramics, metals,
alloys, among others.