The Compound Light

Microscope
 Concepts
• Magnification - making an object appear bigger

• Resolving power - minimum distance between two

objects at which the objects can just be distinguished
as separate

• Working distance - distance between the objective

lens and the specimen
– The higher the magnification, the smaller the working
distance
 How does a microscope work?
• An incandescent light source emits light
waves traveling in different paths and of
varying wavelengths. Some of the lenses in
a microscope bend these light waves into
parallel paths, magnify and focus the light at
the ocular

• However, the higher the magnification the

closer the lens must be to the specimen.
Since a higher magnification lens bends light
more severely, the specimen is brought into
focus a shorter distance from the lens
 How does a microscope work?
The importance of resolution

• If the points cannot be clearly focused then they are closer together than
the resolution of the microscope and, regardless of the magnification, the
image quality will be poor

Contrast
• A narrow beam provides higher contrast; if all of the light passes through
the cell then no details will be visible
• Staining also provides contrast
 Magnifications
• Ocular – 10x
• Scanner – 4x
• Low power – 10x
• High power – 40x
• Oil immersion – 100x

Total magnification = ocular x objective

e.g. 4x (scanner) x 10x from ocular = 40x
 To add contrast:
• Manipulate light – e.g. iris diaphragm, light
intensity

• Add stains – e.g. crystal violet, safranin, etc.

ELECTRON MICRSOCOPES:

1. Scanning electron microscope (SEM)

-It has high magnification and high resolution. The
specimen is coated in gold and the electrons bounce
off to give you an exterior view of the specimen. The
pictures are in black and white.

2. Transmission electron microscope (TEM)

-Thin slices of specimen are obtained. The electron
beams pass through this. It has high magnification and
high resolution.
 Scanning electron microscope image of pollen.

A TEM image of the polio virus. The polio virus is between 30 and 230 nm in size
Techniques in Optical Microscopy:

1. Bright field microscopy - is the simplest of all

the light microscopy techniques. Sample illumination
is via transmitted white light, i.e. illuminated from
below and observed from above.
-Limitations include low contrast of most biological
samples and low apparent resolution due to the blur
of out of focus material. 
 MICROSCOPY
light microscope
■
light as source of illumination
■
glass lenses
■
limited resolution (loses resolving power at magnifications
above 2000X)
electron microscope
■
beam of electrons directed by magnets into a viewing
screen or photographic plate
■
greater magnification than light microscope
■
greater resolving power than light microscope
 2. Dark field microscopy is a technique for
improving the contrast of unstained,
transparent specimens.
-Dark field illumination uses a carefully
aligned light source to minimize the quantity of
directly-transmitted (unscattered) light
entering the image plane, collecting only the
light scattered by the sample
Dark field microscopy produces an image with a dark background.
3. Flourescence microscopy
-When certain compounds are
illuminated with high energy light,
they then emit light of a different,
lower frequency. This effect is
known as fluorescence.
Yeast cell Lymphocyte

Human Cancer cell