Histology Lesson I:
The Light Microscope and Special Microscopes
Objective: Students will investigate the light microscope and other special
microscopes in order to explore the various types of microscopes used.
Warm Up: Which types of microscopes/ forms of microscopy do you already know? Create a list below:
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The Compound Light Microscope:
The compound microscope uses lenses and
light to enlarge the image and is also called
an optical or light microscope.
The compound light microscope has two
systems of lenses for greater magnification:
1) The Ocular/ Eyepiece Lenses, or the
lens you look through.
2) The Objective Lenses, or the lens
closest to the object.
Total Magnification:
- The Total Magnification of a microscope is a product of multiplication of the magnification of the Ocular and
Objective lenses. Ocular lenses typically have a 10x magnification. What would be the total magnification if
you were using a 40x Objective Lens?
Resolving Power is described by Abbes Formula:
- The most important property of a microscope is the resolving power: the
minimum distance between two points which can be sensed as separate.
d=resolution λ= wavelength of light n= refraction index α =aperature angle of lens
Other Parts of the Microscope:
Body Tube/ Column: Connects the eyepiece to the objective lenses
Arm: Supports the tube and connects it to the base
Base: The bottom of the microscope, used for support
Illuminator: A steady light source (110 volts) used in place of a mirror.
Stage: The flat platform where you place your slides. A mechanical stage allows the slide to move
around by turning two knobs. One moves it left and right, the other moves it up and down
Revolving Nosepiece: Part that holds two or more objective lenses, rotate to change magnification.
Objective Lenses: Usually you will find 3 or 4 objective lenses on a microscope. They almost always
consist of 4X, 10X, 40X and 100X powers
Diaphragm: a rotating disk under the stage used to vary the intensity of light that is projected
Special Microscopes:
1. Stereo Microscopy:
 Includes two complete light microscopes with two separate lens
systems and two separate oculars.
 Low magnification objective lenses used to study relatively large
objects such as smaller anatomical parts, ie. the papillae of the
tongue
2. Brightfield Microscopy
 Utilizes confocal light microscopy
 Tissue sample are prepared using very tissue specific stains.
3. Dark Field Microscopy
 Has a special condenser which produces a dark background in the
microscope
 Condenser filters the centrally located beam and lets only the
peripheral ones through. These beams meet the object, change
direction, and the scattered light is detected only.
 The studied cells appear as illuminated objects against a black
background
 Useful to identify bacteria which cannot be stained properly with
stains.
4. Phase Contrast Microscopy
 Technique converts phase shifts in light passing through a
transparent specimen to brightness changes in the image.
 Phase shifts themselves are invisible, but become visible when
shown as brightness variations.
 Used when studying living, unfixed, unstained specimen
5. Polarization Microscopy
 Microscope has two Polaroid filters, the polarizer and the analyzer
placed between the objective and ocular lenses
 Useful for studying biological membranes and diagnosis of certain
diseases such as silicosis, oxalate nephrosis, etc.
6. Fluorescence Microscopy
 The light source is a powerful UV lamp. Light passes through two sets of filters
 The specimen is illuminated with light of a specific wavelength (or wavelengths) which is
absorbed by the fluorophores, causing them to emit light of longer wavelengths (i.e., of a
different color than the absorbed light).
 Various cellular structures can be labeled using immunocytochemistry which can absorb
different wavelengths of light, labeling cell structures different colors
7. Confocal Scanning Microscopy
 Most recently developed method of microscopy, uses a laser beam as its light source.
 Laser beam rapidly moves from one spot to another and images from this is recorded by a
videocamera and stored in a computer, which is able to compose a 3-D image.
 Used for studies of thicker samples
Practice Problems / Homework
1. Determine the total magnification for the following objective lenses
a.
10 x ocular & 4 x objective =
b.
10 x ocular
& 10 x objective
=
c.
10 x ocular
& 40 x objective
=
2. What type of microscope or microscopy would be the most useful clinical tool?
a. Following tissue staining, you investigate stained cells:
b. Assessing pond water sample for single cellular protist life
c. Analyzing urine sample for oxalate crystals:
d. Completing a delicate surgery where low magnification could assist:
3. Research how the following historical scientists contributed to modern day microscopy?
a. Galileo
b. Malpighi
c. Faber of Bamberg
d. Frits Zernike