14.

Compound Microscope: Magnification and

resolution

*Compound optical Microscope: An instrument for magnification of the

angle of sight of small objects invisible to the eye.
Consists of three parts – Mechanical system, Illumination system,
Observation system.

* Mechanical system: Base and arm

Object stage
Tubular barrel
Rotating turret (for objective lens)
Fine and coarse focus adjustment knobs

*Illumination System: Light source

- Collimator, Condenser, diaphragms for shaping the illuminating
light
- Provides the required intensity, diameter, convergence of the
illuminating light beam.

* Observation system: Objective lens and eye piece

Objective lens – Consists of several lens groups. The front lens
determines the magnification, the remaining lenses correct the
aberrations. The objective lens determines the quality of the
microscope image
Eyepiece – Consists of two or more lenses. The upper (eye) lens
determines the magnification of the eyepiece.

* Objective: The object distance is somewhat longer than the focal

length of the objective lens. The Image created by the objective lens
(intermediate image) is at a distance to the eyepiece which is a little
shorter than the focal length. The image is enlarged, inversed and real
* Eyepiece: The intermediate image is the object for the eyepiece. The
image created by the eyepiece is enlarged, upright and virtual.

* Microscope Image: The observer’s eye is located at the back of the

focal point of the eyepiece and views the image created by the
eyepiece at the normal near point of the eye. The microscope image is
enlarged, inversed and virtual.

*Magnification: Transverse magnification of a component of the optical

system is the ratio of a transverse dimension of the image to the
corresponding transverse dimension of the object.

*Objective magnification: Depends on the focal length of the objective

lens fo and the optical length of the tube L (L is the distance between
the adjacent foci of the objective lens and the eyepiece)
Wo = L/fo

*Eyepiece Magnification: Depends on the focal length of the eyepiece fe

and the normal near point distance for the eye ao. We = ao/fe

*Microscope Magnification: Depends on the transverse magnification

of the objective lens Wo and the transverse magnification of the
eyepiece We.
Wm = WoWe = Lao/fofe.

* Infinite optics Microscope:

An additional lens is inserted between the objective and eyepiece
which makes the rays behind the objective parallel to each other.
Adding other optical elements in the parallel light beam has no adverse
effect on image quality. The aberrations are corrected to a higher
degree.
* The Image of a point Object: The Image of a point is a disk surrounded
by diffraction rings. The effect is due to the diffraction of light at the
front lens aperture.

* The image of two points: When the distance between the two points
is small their images are partially overlaid. The two images are
considered separate (distinct) if the distance between the centres is not
less than their radius.

* Resolution: Resolution Limit δ: The smallest distance between two

points of the object at which they are imaged separately. Resolving
power = 1/δ (Depends mainly on the objective)

* Aperture angle γ: The half angle of the maximum cone of light

originating from the object and entering the objective lens. Depends on
the radius and focal length of the objective front lens.

Numerical aperture A: A = n sin γ;

n is the index of refraction of the medium between the object and the
objective lens.

* Resolution Limit: Determined by the numerical aperture of the

objective lens and the wavelength of light δ = λ/A
It can be reduced by using objectives with larger numerical aperture
and light with shorter wavelength.

*Limitations of light Microscope:

The wavelength range of visible light is rather narrow
The theoretical maximum for the aperture angle is 90˚, practical values
reach 70˚.
The index of refraction of air is 1, but liquids (immersions) with higher
values can be used.
* Immersion Objective Lenses:
A liquid transparent medium between the object and objective lens,
the effective aperture angle becomes larger and the resolving power
increases.
Immersions: Water and oils.

* Maximum useful Magnification: Wmax:

The magnification at which
- the object dimensions equal to the resolution of the limit of the
microscope δM
- Imaged equal to the resolution limit of the eye δE
Wmax = δE/δM
- At smaller magnifications the resolving power of the objective lens is
not used efficiently (the eye cannot resolve all the details present in the
image). At larger magnifications the image does not contain additional
details and its quality is worse.

* Selection of the objective and eyepiece lenses:

Objective – Select by numerical aperture to provide the necessary
resolution limit, calculate the maximum useful magnification.
Eyepiece – Select by magnification to provide observation at the
maximum useful magnification.

*Bright Field Microscopy: The image is produced by light transmitted or

reflected by the object.

* Dark Field Microscopy: The image is produced by the light scattered

by the object. It can be used to detect details below the resolution
limit.

* Fluorescence Microscopy: A method for observation of fluorescent

objects by staining with appropriate dyes. It is illuminated by UV light.
Modification of the microscope: Illuminating optics, transparent to UV
light, Filters in the illumination and observation systems

* Multiphoton Fluorescence Microscopy:

- Fluorescence is excited by simultaneous absorption od two or more
infrared photons.
- The illumination is provided by a laser beam focused toa small spot on
the object
- The illuminated spot is scanned over the object to obtain the entire
image
- It can be used to image details under the surface of the object (up to
0.5mm)
- It causes less damage to biological objects than UV light

* Phase contrast Microscopy:

- Used for imaging of low contrast objects
- Light transmitted by such objects is modified by phase but its intensity
is unchanged
- The eye is insensitive to the phase of the light wave
- The method converts changes of phase into changes of intensity,
observable by human eyes.

* Confocal Laser Scanning Microscopy:

- The illumination is provided by a laser beam focused to a small spot
on the object.
- The illumination spot is scanned over the object
- The reflected light at each position is measured and used to construct
the image
- Very high contrast and resolving power can be achieved