Title: Light Microscopy and Fluorescent Microscopy

Authors: Istvn Csoms, Ferenc Papp and Zoltn Varga

Date: 30. July 2015.

A tananyag elksztst "Az lettudomnyi- klinikai felsoktats gyakorlatorientlt s

hallgatbart korszerstse a vidki kpzhelyek nemzetkzi versenykpessgnek erstsre"
TMOP 4.1.1.C-13/1/KONV-2014-0001 szm projekt tmogatta. A projekt az Eurpai Uni
tmogatsval, az Eurpai Szocilis Alap trsfinanszrozsval valsult meg.

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1.1

Basic principles of microscopy

Introduction

Microscopes are compound optical systems that are used to magnify the image of small objects.
In medical school, tissues, cells and cellular organelles are highly important among small
objects. To describe, identify and document changes at tissue level, optical microscopic
techniques can help us. The resolution of microscopes using visible light is still sufficient to
observe, detect and recognize the intracellular parts of the cell. Later, during your studies at the
medical school, histology, pathology, haematology and other subjects, require the application of
microscopic techniques. Hence, it is reasonable to learn about the various types of microscopes,
their structures and principle of operation even in the first year, at the very beginning of the
medical school.
Microscopes are made of an objective facing the object of observation and an ocular, or eyepiece, facing the observer (Figure 1.). To refresh memories of high school physics, the object has
to be located between the focus (F) and twice the focus (2F) of the objective, and its image,
magnified, real and inverted, is on the other side of the objective and is outside the 2F distance.

1. Figure Image formation of the light microscope

The eye-piece is placed so that this real image is inside the focus of the eye-piece. Looking into
the ocular we see the virtual, upright, magnified image of this first image. In the end, we see the
doubly magnified, inverted, virtual image of the object. If we want to make a picture in the

microscope, we normally take the first real image created by the objective, by projecting it onto
photographic film, or the chip of a digital camera.
The parts of the microscopes can be classified into (1) optical parts serving illumination and
magnification, and (2) mechanical parts that hold and move the former. The optical parts of the
traditional light microscope (see figure 2.) are comprised of the condenser (for illumination), the
objective and the ocular. For looking at the image with both eyes, a binocular tube is employed,
which doubles the first image by means of a prism onto two separate oculars.

ocular

objectives

revolver
stage

condenser lens
condenser
height
adjustment
condenser iris
illumination
intensity

lamp iris

2. Figure Set-up of a conventional microscope

1.2

Magnification, resolution and visibility

The magnification of the microscope is defined by the product of the magnifications of its
optical elements. These are the following: the objective (typically between 10x s 100x), ocular
(typically between 5x and 20x), and sometimes the prism in the binocular tube (generally 1.5x).
The magnification of individual optical elements is the ratio of the image distance to the object
distance.
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The resolution limit, i.e. the smallest distance at the end of which two points are still conceived
as two separate points for points that are light sources themselves is given by

d = 0.61 / NA

where NA= nsin is the numeric aperture of the objective, n is the diffraction index of the
medium between the objective and the sample, and is the angular aperture (1/2 of the angle of
light rays the lens can take in when focussed). Resolving power is defined as the reciprocal of
the resolution limit, R=1/d, but often the d distance is (illogically) called resolving power. When
the object is illuminated from the outside, the NA of illumination is also to taken into
consideration as it limits the resolving power. This is represented in the Abbe formula:

d=/(NAobjective+NAcondenser).
Thus, using green light of 538 nm wavelength, an objective of NA=1.25 and a condenser of
NA=0.9, the resolution limit is ~250 nm.

In practice, the NA of the condenser lens should be matched to (be roughly equal to) the NA of
the objective, so the above formula is often approximated by
d= / NAobjective.
The resolving power often limits the usefulness of magnification. If, for example, we use a
magnification of 1000x (e.g., objective 100x and ocular 10x), the smallest resolved distance in
the above case, 250 nm, will be magnified to 0.25 mm. Our eye is capable of resolving 10
separate points on a 1 mm line positioned at the distance of clear vision, so in this magnified 0.25
mm line we will be able to distinguish 2 points, and hence will clearly see the two points
separated by the 0.25 mm line. Now suppose we increase our magnification 3-fold by using a
20x ocular and a 1.5x binocular prism. The images of the best-resolved two points will be
separated by 0.75 mm, in which our eye could in principle distinguish 7 individual points, but the
resolving power of the objective only allows for 2 points. Thus we are wasting magnification
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power. The rule of thumb is not using a magnification exceeding 1000x the NA of your
objective.

As it can be deduced from the above, the resolving power can be improved in the following ways
(see the Abbe formula):
- decrease the wavelength
e.g. UV microscope, or, as an extreme example the electron and the X-ray microscope
- increase the NA
e.g. by increasing the angular aperture (i.e. better design of objective, shorter working
distance, larger front lens, or to use 2 or three objectives focussed at the same spot of
observation)
or by increasing the refractive index (use of immersion oil with n=1.55, rather than air
with n=1.0. Immersion oil should be used only with the appropriate objectives marked
with a black ring around their body, or labelled HI. You can use these without oil, but this
will decrease their effective NA to about 2/3 of the nominal value, and significantly
deteriorate the image. Obviously, one can use an immersion objective also as the
condenser, thereby increasing the NA of illumination, which also improves the overall
resolving power of the system.

One should not confuse resolving power with visibility. The latter is not related to the
separability of two points, rather to a single point far away from all others, and states the
minimum conditions of size, brightness and contrast against background under which this point
is still visible. Seeing this object does not mean we know anything about its size or shape.

1.3

Aberrations

Objectives and lenses therein have aberrations that impair image quality. Regarding the
theoretical

background,

we

refer

to

the

Biophysics

lectures

(see

also

http://www.biophys.dote.hu). The main aberrations related to objects on or close to the optical

axes and their countermeasures in microscopy are the following:

1.3.1 Spherical aberration

It occurs because light rays passing through the edge of the lens are refracted more than those
through the centre. Thus the image of a point, even if it is on the axis, will in itself be blurred.
Practically speaking, a single lens with a real physical thickness will not have a focal point, but
rather a focal surface. This aberration is corrected in aspheric lens systems.
1.3.2 Spherical focal surface
It is related to spherical aberration inasmuch it is caused by the physical thickness of the lens.
The overall result is that the image of a larger object is either in focus at the centre or at the edge
of the field, but never both at the same time. In other words, if an object is close to the optical
axis, but not exactly on it, even an aspheric lens will produce this aberration. Correction for this
results in a flat image, at the price of some loss in resolving power. Corrected objectives bear
the forename plan-.
1.3.3 Chromatic aberration
It is caused by the wavelength dependence of the refractive index of any material. Henceforth a
single lens will have different focal lengths for different colours, the shorter wavelengths having
shorter focal lengths. This can be corrected for by making compound lenses from single lenses of
diverse materials that compensate for each others chromatic aberrations. Objectives corrected
for two colours are called achromat, those corrected for three are called apochromat.
Apochromats are further corrected for the fourth colour of the visible spectrum (the four being
blue, green yellow and red) by a special compensating ocular.

1.4

Illumination in the microscope

Once we have a high quality microscope and a good sample, the most common errors that hinder
image quality are problems with adjusting the illumination. The following generic points should
be considered.
1. The intensity of illumination should provide and optimal balance of brightness and contrast.
2. The light ray should be symmetric, centred at the optical axis.
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3. The field of vision should be illuminated evenly, and no light should fall on points outside the
field, as photons scattered from there ruin image quality.
4. The NA of illumination should match that of the objective. Thus, for high NA objectives, the
condenser should be high (close to the specimen) and its diaphragm open, for low NA objectives
vice versa.

1.4.1 Setting the correct illumination in a conventional light microscope:

1. Centering the illumination light. Focus on the specimen with the lowest magnification
objective. Minimize the iris aperture of the lamp and of the condenser. Center the small light spot
in the field of view using the adjustment screws of the lamp and of the condenser.
2. Adjusting the brightness and angle of illumination.. Focus on the specimen with the required
objective. Open the iris aperture of the condenser and by changing the position of the condenser
focus the image of the lamp diaphragm. Then open the lamp diaphragm until the field of view is
illuminated evenly, but no light rays fall outside the field of view. Remove (one of) the ocular(s)
and adjust the size of the condenser diaphragm so that it is just a little bit smaller than the
diameter of the back lens of the objective.

1.5

Some important types of microscopes

1.5.1 Bright field:

This microscopy applies white light to illuminate the object that is required to be studied. Both
unstained and stained cells can be visualized with this instrument. In the case of the former ones,
the different parts of cells are distinguished from protoplasm as darkened objects as the result of
the light absorption and the scattering away from the objective lens. The latter ones are stained
with various dyes (red, blue, green etc.) which bind to a certain organelle or to molecules in the
cell (e.g. the nucleus). These dyes absorb all wavelengths except that region of the spectrum that
corresponds to their colour. (Some examples for such staining can be found in the Cell Biology
lab manual to be used during the second semester, and also in your Histology course.) Reducing
the intensity of light illuminating the specimen by lowering the condenser and narrowing its
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diaphragm can improve contrast and depth of field. However, this will result in the decrease of
effective numerical aperture and resolving power.

Koehler illumination
To see sharp images, the microscope must be equipped with Koehler illumination. In the
case of microscopy using an external light source, this provides more even illumination with
adjustable intensity without decreasing the resolution. It is achieved by focussing the light source
onto the sub-stage (condenser) iris diaphragm which is at the same time in the rear focal plane of
the condenser lens. From every point of the image of the light source formed on the iris
diaphragm, a parallel ray is projected towards the sample. This results in a nearly uniform bundle
of parallel rays impinging on the specimen, and hence a more precise and detailed image with
less artefacts.

1.5.2 Dark field

Dark field illumination involves the technique of blocking out the direct light completely and
viewing an object by means of diffracted light. A special condenser prevents the direct light from
reaching the objective but light from rays that are scattered on objects will still enter the
objective. This technique enables us to study sudden changes in refractive properties at the edges
or discontinuities in an object, the appearance and movement of small light scattering objects in
the field of view, as well as the actual colour of objects. This method can also reveal the presence
of objects that are sized below the resolving power of the lens system used (even molecules
hence the name ultra-microscope), because of the sensitivity of human eye to intensity
differences (dark background - bright object).

1.5.3 Phase contrast

It was developed in 1932 by F. Zernike (Nobel Prize in 1953). The phase contrast microscope
enhances contrast also of unfixed and unstained cells. Most cellular components are transparent
to light and have a high refractive index. Light transmitted through these structures is withheld a
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bit, resulting ~ wavelength phase delay relative to the light passing through the clear medium.
The phase contrast microscope transforms this phase difference to difference in brightness. The
diffracted and non-diffracted light beams are separated, and one of them is retarded by an
additional wavelength. Afterwards the two beams are recombined to form the final image. The
interference of in-phase (diffracted/scattered) and the out of phase (non-diffracted, or direct)
beams produces exaggerated contrast of objects with various indices of refraction. Separation of
diffracted and direct beams is achieved by a transparent annulus (ring) normally built into the
condenser as well as a phase plate in the objective. The transparent annulus in the condenser
creates a ring of light that illuminates the sample. This ring of light narrows as it reaches the
sample because of the focusing effect of the condenser lens, and at the plane of the object (where
it is focussed at) it creates a spot of even illumination. Then the cone of light broadens again
shines a ring on the objective. This image of the annulus is adjusted to coincide with a
corresponding ring (the phase ring) in the phase plate, which is in, or on one of the lenses of the
objective. Photons that impinge on the object in this cone of light and go through directly without
being scattered must therefore also pass through the phase ring in the objective. Most diffracted
(scattered) photons go off track and therefore arrive ate areas of the phase plate that is inside or
outside the phase ring. The few that go through the phase plate are negligible as regards image
formation. There are two strategies of phase contrast microscopy:
Positive (dark) phase:

It is applied when objects of higher refractive index are desired to

look darker than their surroundings. The direct (non-diffracted) light is retarded by an additional
wavelength on the phase ring, e.g. by using a thin magnesium fluoride or aluminium layer on
the phase plate, but only on the phase ring, where the image of the illuminating (condenser)
annulus is formed and the direct photons go through. This light beam will thus be delayed ~
phase in the denser parts of the sample and further phase on the phase plate, which totals a
phase relative to the reference beam that gets diffracted on the border of denser objects and thus
goes without phase delay through the environment of these objects, and through the non-delaying
part of the phase plate (inside or outside the phase ring). The interference of the direct, and the
refracted reference beams will result in dark spots at the eyepiece corresponding to highly
refractive areas in the specimen. On the other hand, spots in the specimen that are of lesser
refractive index than their environment will cause the phase of photons directly passing through
them to be O ahead of the diffracted reference beam. However, this phase shift will be cut
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back by the phase ring, so direct and reference beams arrive in-phase at the ocular and thus
constructive interference makes these spots look brighter than their surroundings.
Negative (bright) phase: In this approach, the diffracted light is retarded by a layer at the
phase plate inside and outside the phase ring, and as a result, the diffracted and the direct beam,
both being retarded, come into phase again for highly refractive spots, yielding a brighter image
of these spots relative to the darker background.
To enhance the contrast effect, the direct beam must be attenuated to the level of the
diffracted beam by means of an absorbing layer at the phase plate. The transmission through this
layer is 5-25%, therefore only a little fraction of the original intensity can be observed. This can
be compensated for by stronger illumination, or, in photomicrography, by longer exposure. A
practical problem in phase microscopy is the wavelength-dependence of refractive index: to
avoid this, narrow band filters are used to select a range of wavelengths. Most frequently yellowgreen filters are used with maximum transmission around 550 nm, since the human eye is very
sensitive to this region of spectrum.

1.5.4 Fluorescence microscopy

In contrast to the previously mentioned microscopic techniques where absorption or scattering in
the specimen is exploited, fluorescence microscopy makes use of luminescence from (properly
excited) submicroscopic objects. In this type of microscopy, samples (cells, tissues) are stained
(e.g. when studying viability using FDA) or some of their molecules (e.g. various membrane
antigens) are labelled specifically with fluorescent dyes and are illuminated with a wavelength of
light that corresponds to the excitation (absorption) maximum of the dye. For light sources,
mercury or xenon lamps are used to ensure a wide spectrum of excitation. With application of
proper filters, the desired wavelength of light can be selected. The emitted fluorescence light,
which is of longer wavelengths (owed to the Stokes shift) is detected. To observe only the
emitted flourescent light, filters and dichroic mirrors are placed between the objective and the
eyepiece. Dichroic mirrors are transparent to a certain wavelength range (i.e. that emitted by the
dye observed) and the remainder of the spectrum is reflected (including that used for excitation).
Emission filters are designed to absorb the scattered photons of exciting light that with a low
probability get through the dichroic mirror towards the observer, so optimally only the emitted
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photons reach the ocular. Fluorescent objects appear as bright spots (with color corresponding to
their emission spectrum) in the dark background. It is thus easier to study fluorescent samples in
a darkened room. By applying different types of dyes, many parameters of the cell can be
studied: viability, membrane potential, ligand-receptor bindings, conformational changes of
proteins etc. For details please see the Cell Biology lab manual.
Figure 3. below shows the light path/optical system of a conventional upright fluorescence
microscope (from Wikipedia):

3. Figure Optical system of a conventional fluorescence microscope

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Tasks for students

1) To calibrate the ocular scale with the calibration scale.
2) To measure the long diameters of the frog red blood cells (RBC).
3) To determine the following:
a. The average, the corrected standard deviation (SD) and the standard error of the mean
(SEM) of RBC long diameters!
b. The average of RBC long diameters in m unit!
4) To determine the resolution of the fluorescence microscope based on the given data.
5) To examine fluorescent bead populations.

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