Microscopy

BNN30104
BIOCHEMISTRY
AND
BIOMOLECULAR
TECHNOLOGY
What is a microscope? How does it work?

• 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).
The Human Eye

• Since so many microscope users rely upon direct observation, it is

important to understand the relationship between the microscope and the
eye.
• Our eyes are capable of distinguishing color in the visible portion of the
spectrum:
• from violet to blue to green to yellow to orange to red;
• the eye cannot perceive ultra-violet or infra-red rays.
• The eye also is able to sense differences in brightness or intensity ranging
from black to white and all the grays in-between.
• Thus for an image to be seen by the eye, the image must be presented to
the eye in colors of the visible spectrum and/or varying degrees of light
intensity.
The Human Eye
• The eye receptors of the retina for sensing color are the cone cells;
the cells for distinguishing levels of brightness, not in color, are the
rod cells.
• These cells are located on the retina at the back of the inside of
the eye. The front of the eye, including the iris, the curved cornea,
and the lens are respectively the mechanisms for admitting light
and focusing it on the retina.
• From there, the “message” is sent to the brain via the optic nerve.

Fig 2. Anatomy of the

Human Eye.

Fig 3. Microscopic
Anatomy of the
Retina.
The Human Eye
• For an image to be seen clearly, it must be spread on
the retina at a sufficient visual angle.
• Unless the light falls on non-adjacent rows of retinal
cells (a function of magnification and the spreading of
the image), we are unable to distinguish closely-lying
details as being separate (resolution).
• Further, there must be sufficient contrast between
adjacent details and/or the background to render the
magnified, resolved image visible.
 “SIMPLE” MICROSCOPE
• Because of the limited ability of the eye’s lens to
change its shape, objects brought very close to
the eye cannot have their images brought to focus
on the retina.
• The accepted minimal conventional viewing
distance is 10 inches or 250 millimeters (25
centimeters).
• More than five hundred years ago, simple glass
magnifiers were developed.
• These were convex lenses (thicker in the center
than the periphery).
• The specimen or object could be focused by use
of the magnifier placed between the object and
the eye.
• These “simple microscopes”, along with the
cornea and eye lens, could spread the image on
the retina by magnification through increasing the
visual angle on the retina.
 “SIMPLE” MICROSCOPE

• The “simple microscope” or magnifying glass reached

its highest state of perfection, in the 1600’s, in the
work of Anton von Leeuwenhoek who was able to
see single-celled animals (“animalcules”) and even
some larger bacteria.
• The image produced by such a magnifier, held close
to the observer’s eye, appears as if it were on the
same side of the lens as the object itself.
• Such an image, seen as if it were ten inches from the
eye, is known as a virtual image and cannot be
captured on film.
• These magnifiers had severe limitations in specimen
positioning, illumination, lens aberrations, and
construction.
COMPOUND MICROSCOPE
• Around the beginning of the 1600’s, through work attributed to the
Janssen brothers in the Netherlands and Galileo in Italy, the compound
microscope was developed.
• In its basic form, it consisted of two convex lenses aligned in series:
• an object glass (objective) closer to the object or specimen,
• and an eyepiece (ocular) closer to the observer’s eye—with means
of adjusting the position of the specimen and the microscope lenses.
• The compound microscope achieves a two-stage magnification.
• The objective projects a magnified image into the body tube of the
microscope and the eyepiece further magnifies the image projected by
the objective
• For example, the total visual magnification using a 10X objective and a
15X eyepiece is 150X.
 COMPOUND MICROSCOPE
• When you look into a microscope, you
are not looking at the specimen, you are
looking at an IMAGE of the specimen.
• The image is “floating” in space about
10 millimeters below the top of the
observation tube (at the level of the
fixed diaphragm of the eyepiece) where
the eyepiece is inserted.
• The image you observe is not tangible;
it cannot be grasped.
• It is a “map” or representation of the Fig 7. Compound
specimen in various colors and/or magnifier. In the
shades of gray from black to white. compound
• The expectation is that the image will be microscope, the
an accurate representation of the intermediate image
formed by the
specimen, accurate as to detail, shape objective and tube
and color/intensity. lens is enlarged by
• The implications are that it may well be the eyepiece.
possible (and is) to produce (or even
enhance) highly accurate images.
COMPOUND MICROSCOPE

• Conversely, it may be (and often is) all too easy to degrade an image
through improper technique or poor equipment.
• Essentially, this is how a microscope functions.
• Light from a lamp passes through a substage condenser and then through
a transparent specimen placed over an opening in the stage.
• Light is then gathered by the objective.
• The objective, together with the built-in tube lens, focuses the image of the
specimen at the level of the fixed diaphragm of the eyepiece.
• The image is then seen by the observer as if it were at a distance of
approximately 10 inches (250 millimeters) from the eye.
1665 – English
physicist, Robert
Hooke looked at a
sliver of cork
through a
microscope lens and
noticed some
"pores" or "cells" in
it.
•Compound/Light Microscope
•Dissection/Stereo Microscope
•Scanning Electron Microscope (SEM)
•Transmission Electron Microscope (TEM)
Important optical component of the
microscope: (1) OBJECTIVE
• Its basic function is to gather the light passing through the specimen and then to project an
accurate, real, inverted IMAGE of the specimen up into the body of the microscope.
• Other related functions of the objective are to house special devices such as
• an iris for darkfield microscopy,
• or a phase plate for phase contrast microscopy.
• The objective must have the capacity to reconstitute the various points of the specimen into the
various corresponding points in the image, sometimes called the “anti-points”.
• The objective must be constructed so that it will be focused close enough to the specimen so that it
will project a magnified, real image up into the microscope.
Important optical component of the
microscope: (2) EYEPIECE
• Its basic function is to “look at” the focused, magnified real
image projected by the objective and magnify that image a
second time as a virtual image seen as if 10 inches from
the eye.
• In recording, a Photo eyepiece “picks up” the real image
projected by the objective a second time as a real image
able to be captured by a camera.
• The eyepiece houses a fixed diaphragm.
• It is at the plane of that fixed diaphragm that the image
projected by the objective will be “seen”.
• On the shelf of the fixed diaphragm, the eyepiece can be
fitted with scales or markers or pointers or crosshairs that
will be in simultaneous focus with the focused image.
 Stereo microscope
• an optical microscope variant designed
for low magnification observation of a
Sample
• using light reflected from the surface of an object rather than
transmitted through it.
• uses two separate optical paths with
two objectives and eyepieces
• provide slightly different viewing angles
to the left and right eyes - produces a 3D visualization
LIGHT microscope

• to view magnified images of small objects on a glass slide.

• higher levels of magnification than stereo or other low power
microscopes and reduce chromatic aberration.
• through the use of two or more lenses in the objective and the
eyepiece.
LIGHT microscope

• The objective lens located on the nosepiece have a short focal length
and are close to the target object where it collects light and focuses
the image of the object into the microscope.
• The second lens, in the eyepiece,
has a longer focal length
and further enlarges the image
Scanning electron microscope (SEM)

• electron microscope that produces images of a sample

by scanning the surface with a focused beam of electrons.
• The electrons interact with atoms in the sample, producing various
signals that contain information about the surface topography and
composition of the sample.
Scanning electron microscope (SEM)
Transmission electron microscope

• a form of electron microscope in which an image is derived from

electrons which have passed through the specimen, in particular one
in which the whole image is formed at once rather than by scanning.
• Always carry with 2 hands
• Never touch the lenses with your fingers.
• Only use lens paper for cleaning
• Keep objects clear of desk and cords
• When you are finished with your "scope", rotate
the nosepiece so that it's on the low power
objective, roll the stage down to lowest level,
rubber band the cord, then replace the dust cover.
 Ocular lens
Body Tube

Revolving Nosepiece
Arm
Objective Lens

Stage
Stage
Clips Coarse adjustment knob
Diaphragm
Fine adjustment knob
Light

Base
Ocular lens

magnifies; where you

look through to see the
image of your specimen.
They are usually 10X or
15X power. Our
microscopes have an ocular
lens power of 10x.
 supports the tube and
connects it to the
arm base
 the flat platform
where you place
your slides
stage
 moves stage (or body
tube) up and down

coarse adjustment knob

 small, round knob on
the side of the
microscope used to
fine-tune the focus of
your specimen
fine adjustment knob
after using the coarse
adjustment knob
 the bottom of the
microscope, used for
support

base
 body tube

connects the eyepiece

to the objective
lenses
the part that holds two
or more objective lenses
revolving nosepiece
and can be rotated to
easily change power
 Adds to the magnification
Usually you will find 3 or
4 objective lenses on a
microscope. They almost
objective lens
always consist of 4X, 10X,
40X and 100X
powers. When coupled
with a 10X (most common)
eyepiece lens, we get total
magnifications of 40X (4X
times 10X), 100X , 400X
and 1000X.
The shortest objective lenses
lens is the lowest power, the
longest one is the lens with
the greatest power. Lenses
are color coded.
The high power objective
lenses are retractable (i.e.
40XR). This means that if
they hit a slide, the end of
the lens will objective lenses
push in (spring loaded)
thereby protecting the lens
and the slide.
Stage clips hold the slides in
place. If your microscope
has a mechanical stage, you
will be able to move the
slide around by turning two
knobs. One stage clips
moves it left and right, the
other moves it up and down.
 controls the amount of light
going through the specimen
Many microscopes have a
rotating disk under the
stage. This diaphragm has
different sized holes and is
diaphragm
used to vary the intensity
and size of the cone of light
that is projected upward into
the slide. There is no set rule
regarding which setting to use
for a particular power. Rather,
the setting is a function of the
transparency of the specimen,
the degree of contrast diaphragm
you desire and the particular
objective lens in use.
makes the specimen
easier to see

light
The proper way to focus a microscope is
to start with the lowest power objective
lens first and while looking from the
side, crank the lens down as close to the
specimen as possible without touching
it. Now, look through the eyepiece lens
and focus upward only until the image
is sharp. If you can't get it in focus,
repeat the process again.
Once the image is sharp with the low
power lens, you should be able to
simply click in the next power lens and
do minor adjustments with the focus
knob. If your microscope has a fine
focus adjustment, turning it a bit should
be all that's necessary. Continue with
subsequent objective lenses and fine
focus each time.
Rotate to 40x objective, locate desired
portion of specimen in the center of the
field. Refocus very carefully so that the
specimen is focused as sharply as
possible. (Do not
alter focus for the
Following steps )
Partially rotate so that 40x and 100x
objectives straddle the specimen.
Place a small drop of oil on the slide in
the center of the lighted area. (Take care
not to dribble on the stage.)
Put the small drop
of oil directly over
the area of the
specimen to be
Examined.
Rotate so that the 100x oil immersion
objective touches the oil and clicks into
place.
Focus only with fine focus. Hopefully,
the specimen will come into focus easily.
Do not change focus dramatically.
Clean up!: When you have finished for
the day, wipe the 100x oil immersion
objective carefully with lens paper to
remove all oil. Wipe oil from the slide
thoroughly with a Kimwipe. Cleanse
stage should any oil have spilled on it.
Recap the immersion oil container
securely, replace in drawer.
 Note: this table is intended as a simple

Types of Microscopy guide. Actual performance and usage may

be different in certain applications.

Type Probe Technique Best Resolution Penetration Uses and Constraints

Optical Microscopy Visible Light Detect reflected light (opaque ~200 nm Surface or volume
samples) or transmitted light (can probe through
(transparent samples). transparent
Light focused using lenses. materials)
Near-Field Optical Visible Light Detect reflected light (opaque ~10 nm Surface or volume Biological samples.
Microscopy (NSOM) samples) or transmitted light (can probe through
(transparent samples). transparent
Uses an aperture very close to the materials)
sample surface.
X-Ray Microscopy (TXM, X-Rays Image derived from x-ray scattering or ~20 nm Surface or volume Can be tuned to specific frequencies
SXM, STXM) interference patterns. (x-rays can to provide element identification and
X-rays focused using a “zone plate” penetrate some mapping.
(Fresnel lens). materials)
Scanning Electron Electrons Detect electrons back-scattered by ~1 nm Surface Sample must be in a vacuum.
Microscopy (SEM) the sample.
Electrons focused using
electromagnets.
Transmission Electron Electrons Detect electrons scattered as they ~0.05 nm Volume Samples must be <100 nm thick.
Microscopy (TEM, STEM) move through the sample.
Electrons focused using
electromagnets.
Focused Ion Beam (FIB) Ions Detect ions back-scattered by the ~10 nm Surface Due to the large masses of the ions,
sample. this probe can be destructive to the
Ions focused using electromagnets. surface of the sample. Therefore, it
can also be used to etch the
sample.
Scanning Tunneling Cantilever Tip Detect the quantum tunneling current ~0.1 nm Surface Sample must be conductive material
Microscopy (STM) of electrons from the sample to the and must be in a vacuum.
probe tip. Can be used to manipulate atoms
on the sample surface.

Atomic Force Microscopy Cantilever Tip Detect the electrostatic force between ~0.1 nm Surface Can be used to manipulate atoms
(AFM) the sample and the probe tip. on the sample surface.

Magnetic Force Cantilever Tip Detect the magnetic force between ~10 nm Surface Sample must be ferromagnetic or
Microscopy (MFM) the sample and the probe tip. paramagnetic.