INTRODUCTION TO BIOLOGICAL
INSTRUMENTATION . . . . . . . . . . . . . . . . . . . . . . . . . .1
INTRODUCTION
The light microscope and the spectrophotometer are two instruments that are commonly
used by biologists. The light microscope allows the investigator to examine a level of biological
structure that otherwise would not be visible to the naked eye. The spectrophotometer enables
the biologist to investigate many chemical and physiological processes that characterize living
things. The purpose of the first three exercises is to describe, in simple terms, why and how these
two instruments work and to give instruction on how to operate them. You will be using these
instruments in future lab exercises. Your understanding of how these instruments work and your
ability to use them correctly will directly affect your performance in these future labs. In
addition to these two instruments, you are given instructions for the use of pipettors, pH meters,
and the analytical balance. You will use these instruments for the preparation of many of the
solutions to be used in subsequent exercises
THE LIGHT MICROSCOPE
Significant leaps in our understanding of life have generally come on the advent of a new
instrument or technique. The microscope, first produced in 1590 by Zacharias and Francis
Jensen, was the first powerful tool available to biologists and enabled them to eventually develop
the cell theory. Robert Hooke (1665) used a primitive microscope to describe the texture of cork
as a honeycomb of chambers or cells. The greatly improved microscope of Antony van
Leeuwenhoek (1674) allowed him to observe free cells such as protozoa, bacteria, and
erythrocytes. Finally, based on the information gained from the use of improved microscopes
and preparation techniques, a botanist, Matthias Schleiden (1838), and zoologist, Theodor
Schwann (1839), argued that the cell was the basic unit of life and that each cell was capable of
independent existence, a view that was further expanded upon by Rudolf Virchow (1858), who
stated that every cell arises from a preexisting cell.
Most university students have used a microscope at some point in their education, but
many have not used it properly for maximum advantage. In order to optimize the use of the
microscope, one should know the basic principles of microscopy, how to operate a microscope,
and how to take care of it.
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REFRACTION
When light encounters a change in medium at a boundary surface, some of the light is
absorbed, some reflected, and some is transmitted, depending on the opacity of media
encountered and the angle at which the light strikes. The light that is transmitted abruptly
changes its speed in the new media and changes its direction (unless striking the surface at 90).
The light is “bent” or refracted. The change in direction depends upon the wavelength of light
and the speed of light in the two media. The index of refraction (nr) is defined by the ratio of the
two speeds:
nr = vl / v2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1)
where vl & v2 are the velocities of light in media 1 and 2, respectively. The maximum
value of nr (nor) is when v, is the speed of light in a vacuum (c). Some examples of nor for
various
substances are listed below (at 589 nm wavelength):
flint glass
calcite
crown glass
oil
quartz
water (20C)
air
1.917
1.658
1.517
1.516
1.458
1.333
1.0003
Snell's Law of Refraction is given by the equation:
2n1  sin I = n2  sin r . . . . . . . . . . . . . . . . . . . . . . . . . (2)
where n1 and n2 are the indexes of refraction of two materials; the angle of incidence is I;
and the angle of refraction in medium 2 is r.
It is important to note that different wavelengths of light will be refracted at different
angles. This attribute allows a prism to break up white light into its component wavelengths
(colors) and contributes to the chromatic aberration of lenses. Prisms with the same dimensions
but made up of different material, not only will have differences in nr, but will disperse a
spectrum from white light over a different distance (wide or narrow spectrum). This is called the
dispersion power of the material.
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THIN LENSES
The refractive properties of glass enable this material to be used as a lens in the light
microscope. Refraction at each point on the curved surface of a lens is consistent with Snell's
Law. Most student microscopes utilize convergent lenses. In order to understand how
convergent lenses refract light and form images, one can trace a beam or ray of light through
such a lens (Figure 1.1).
Figure 1.1. The fate of light rays emanating from the focal point (F) through a
convergent lens.
Note that the light rays converge at a given point called the focal point. If an object is
placed beyond the focal point of a convergent lens, a real image is formed that is inverted as
well as magnified (Figure 1.2a). This image would be visible only if a screen were placed at the
point of the image formation. Note that the light ray entering the lens at the center is not diverted
on its exit from the lens. If an object is placed inside of the focal point, a real image is not
formed; but, if the object is viewed through the lens (from the right, Figure 1.2b), a magnified
virtual image is seen that is not inverted. When one uses a simple hand lens for magnifying
objects, one is observing a virtual image. The distance of most distinct vision for the human eye
is 25 cm; the image should be formed 25 cm from the eye. The focal point can be related to the
distance of the object and the image from the lens:
1/p + l/q = l/f . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3)
where f = the focal point, p = the distance of the object from the lens, and q = the distance
of the image from the lens.
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Figure 1.2.
Image determined by means of tracing light rays through a convergent lens.
a. object outside focal point (F), forming real image.
b. object inside F, forming virtual image.
MAGNIFICATION
Magnification (M) is the ratio of the size of the image to the size of the object and:
M = q/p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4)
where q = size of the image
and p = size of the object.
LENS COMBINATIONS / THE COMPOUND MICROSCOPE
The compound microscope that you will be using consists of a combination of two lenses
(Figure 1.3). The first lens or objective lens produces a magnified real image of the object which
is used by the second lens as a virtual object. This second lens or ocular lens or eyepiece
magnifies the image from the preceding lens and forms a virtual image. The total magnification
of the lens system (Mt) is
Mt = Mo  Me . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .(5)
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where Mo is the magnification of the objective, and Me is the magnification of the
eyepiece.
Figure 1.3.
The image produced with two lenses in combination as found in a microscope. F o =
focal point of objective lens, Fe = focal point for ocular lens (eyepiece), o = object, r =
real image formed by objective, v = virtual image formed by the ocular lens.
RESOLVING POWER
An object is visible if it interferes with light. It cannot interfere with light if it is smaller
than the wavelength of light. The ability of an optical system to distinguish between two
neighboring points is the resolution of the system. Resolution is defined mathematically as the
closest distance (d) that two points can be seen as two separate points. As d decreases, the
resolving power increases. At 10 feet, d for the human eye is 250 m. The resolution imparted
by the magnification of an object by a lens is dependent upon the refractive medium between the
lens and coverslip, the property of the lens and the wavelength of light (). Mathematically, it is
given by Abbe's equation:
d = 0.612
NA
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .(6)
where NA = the numerical aperture of the lens.
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NUMERICAL APERTURE AND RESOLUTION
The numerical aperture of a lens is an index of the light-gathering capability of the lens
and is dependent upon the curvature and the refractive index of the lens:
NA = nor  sin  . . . . . . . . . . . . . . . . . . . . . . . (7)
where nor is the refractive index of the medium between the lens and coverslip, and  is
the aperture angle of the lens.
As the size of the lens diameter increases, or as the curvature of the lens increases, the
value of NA increases (sin  increases). There is a theoretical limit to  (90) and, thus, the
maximum values obtainable for NA:
for a lens in air 1 sin 90 = 1.
for a lens immersed in oil 1.52 sin 90 = 1.52.
Due to the physical limit to size and curvature of lenses that can be ground ( = 85), the
practical limit to NA is 0.95 in air and 1.5 in oil. Using other immersion media can increase the
NA even further; naphthalene NA = 1.6. Spherical aberrations are due to the fact that the lens
equation #3 is an approximation and that, in actuality, different parts of the lens have slightly
different focal points. Spherical aberrations are enhanced when increasing sin .
Spherical and chromatic aberrations can be reduced by the use of objective and ocular
lenses that are properly matched, in that their differences in refractive index and dispersion
power complement. Such lens systems are called compound lenses.
WAVELENGTH AND RESOLUTION
From equation #6, increased resolution can be achieved by decreasing the wavelength of
the light used. For example, using an oil immersion lens and light in the middle of the visible
spectrum ( = 500 nm, green light), d = 0.17 m. Using UV light ( = 365 nm) and a quartz lens
(regular glass absorbs in UV), the best resolution obtainable is 0.10 m. (One does not look
directly at an image from a UV microscope but at a photograph of the image.) To further
increase resolution, the smaller wavelengths of accelerated electrons are utilized with an electron
microscope. (See the exercise on the electron microscope, Exercise #3.)
DEPTH OF FOCUS
An object that you place on a slide and under a coverslip appears 2-dimensional when
viewed under a microscope but is actually 3-dimensional. As the microscope is focused on an
object, it is being focused at a particular plane or depth of the object. Other structures that lie in
planes above or below the present depth of focus will not be visible or will appear out of focus.
In order to see the full detail of the object, you must examine it at several depths of focus.
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ILLUMINATION
The human eye is sensitive within a range of light intensities. The lower limit of detection
is incredibly low, being equivalent to detecting a single candle 15 miles away. At this detection
limit, approximately 320 photons per second are entering the eye or 705 photons /cm2 / sec. Full
sunlight is approximately 1 x 1020 photons / cm2 / sec. Objects are seen because they interfere
(refract, absorb and diffract) with the light. At too low a light intensity, an object will not be
seen. At a very high light intensity, only a small proportion of the total incident light interferes
with the object, and therefore, detail is not seen and the object will be “washed-out”. Therefore,
to optimize the microscope, light of the proper intensity should be used. (This can be
manipulated by adjusting the source intensity or, crudely, by adjusting the iris.)
Ideally, the object should behave as a luminous point, but, since biological specimens are
not luminous, a lens (condenser lens) is used to focus a light source onto the object. For optimal
viewing, the NA of the condenser should be the same as the NA of the objective. Most
compound microscopes have more than one objective lens with different NA’s. By adjusting the
iris, one can approximate the NA of the objective in use.
UNITS OF MEASUREMENT
Name
meter
Abbreviation
m
Equivalent
0.914 meter/yard
decimeter
dm
10-1m
centimeter
cm
10-2m
millimeter
mm
10-3m
micrometer
m
10-6m
nanometer
nm
10-9m
Angstrom

10-10m
A microscope is used to observe objects that otherwise would be too small to see. In order
to determine the size of objects, one is required to use a measurement system. Biologists use the
metric system: A thick human hair has a diameter of 100 m (or 0.1 mm). A sheet of typing
paper is 50 m thick. A red blood cell is 10 m in diameter. A bacterium is approximately 1
m in diameter.
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PROCEDURES AND OBSERVATIONS
1. THE MICROSCOPE
Take a microscope from the locker. Notice that the microscopes are numbered to
correspond to the lockers. Note the manner in which the cord is wrapped around the upper
portion of the microscope. Please replace it in the same fashion at the end of the period. Do not
wind the cord around the substage.
The various parts of the microscope are shown in Figure 1.4.
A.
INITIAL STEPS FOR THE OBSERVATION OF EACH NEW OBJECT
1.
Rotate the scanning or low power lens into viewing position.
2.
Center the slide with the object of study over the opening of the stage.
3.
Center object of study over condenser by moving the entire stage.
4.
Move the stage up to the stop with the coarse focus adjustment knob.
5.
While looking through the ocular lens, move the stage downward with the coarse
adjustment knob, until the object is visible.
6.
Sharpen the focus of the image with the fine focus adjustment knob.
7.
If the entire field is not illuminated, open up the iris and lower the condenser
slightly from its top position.
Some general points of caution:

The low power lens is for general orientation and will often provide the necessary
magnification. Use it! You will find it very helpful.

NEVER change to a higher power without watching the lens from the side. The high
power lens may strike the slide.

NEVER focus the stage up with the coarse adjustment without watching from the
side.
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Figure 1.4. The Compound Light Microscope
B.
PROCEDURE FOR CHANGING FROM LOW POWER TO HIGH POWER
LENS
The CH Olympus binocular microscopes used in this course are conventional
compound microscopes. With the objectives and eyepieces provided, the total
magnifications available are as follows: 40X low power (scanning) objective, 100X
medium power objective, 400X high dry lens and 1000X oil immersion objective.
1.
By moving the entire stage, center the object in the field of view. Since
microscopes of this type are "parfocal," the image should be approximately in
focus when you switch to the higher power lens.
2.
Rotate the objective turret to a higher power objective if necessary. Place one
drop of immersion oil on the slide prior to rotating the turret if you are using the
x100 oil immersion lens. BE CAREFUL TO WATCH THAT THE LENS
DOES NOT HIT THE STAGE. Sharpen the focus using the fine focus knob. It
should require less than ½ turn.
3.
To set the condenser, reduce the iris and move the substage condenser until the
edge of the iris aperture is in focus.
4.
Open up the iris until it is just outside the field of view.
5.
Refocus the image using the fine focus knob.
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NEVER MOVE THE COURSE FOCUSING KNOB WHILE THE HIGH
POWER LENS IS IN VIEWING POSITION.
6.
C.
D.
E.
Before removing a slide from the microscope, return the scanning lens to viewing
position. The slide may then be removed easily, and the microscope is ready for
another preparation.
PROCEDURE FOR PLACING THE MICROSCOPE BACK IN THE CABINET
1.
Remove the slide only when under scanning power.
2.
Move the stage up to the top with the coarse adjustment.
3.
Wind the cord loosely about the upper part of the microscope.
4.
Return the microscope to its correct locker.
5.
When all microscopes are in the cabinet, LOCK the cabinet.
THE ORIENTATION OF THE IMAGE AND MOVEMENT IN RELATIONSHIP
TO THE MOVEMENT OF THE SLIDE
1.
Obtain a clean dry slide and place the small letter “e”, that is available, in the
center of the slide. Answer the questions given below in your lab book.
2.
Position the slide so the “e” is right-side up on the microscope stage. Use the
lowest power objective available on your microscope. How does the letter appear
to you when viewed through the microscope? Inverted? Reversed? Upside down?
3.
Place the letter “e” at the 3 o'clock position (far right) as you view it through the
microscope. Which direction do you have to move the paper in order to move the
image to the LEFT towards the 9 o'clock position as you view it?
4.
Place the letter “e” at the 6 o'clock position (bottom) as you view it through the
microscope. Which direction do you have to move the slide in order to position
the image up to the 12 o'clock position?
MEASUREMENT WITH THE MICROSCOPE
1.
Place a clean, blank slide on the microscope stage; then obtain a small piece of
graph paper, ruled at centimeter and millimeter intervals, and place the paper on
the slide.
2.
Following the steps given earlier in this exercise, focus on the graph paper. The
ruled lines may be used to determine the field diameter provided by the 4X and
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10X objectives (Figure 1.5). The lines are too widely spaced on the graph paper to
use this technique with the 40X and 100X objectives.
Figure 1.5. Relationship of objective power to working distance and diameter of field.
3.
Estimate the field diameters provided by the scanning and low power objectives,
as used with the ocular lens in your optical system.
4.
In the table 1.1, record your estimates of field diameters for the scanning and low
power objectives. Cytologists, and others who work with small objects, find the
millimeter to be a large and cumbersome unit. They consider the micrometer a
more convenient unit of measurement. (To convert millimeters (mm) to
micrometers (m) multiply by 1000.)
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TABLE 1.1
Magnification of Ocular Lens_________________
Objective
Total
Magnification
Field Diameter
(mm)
Field Diameter
(m)
4X
10X
40X
The field diameter provided by the optical system when substituting an objective of higher or
lower magnification may be calculated from the data recorded above, since magnification and
field diameter are directly proportional, but are inversely related. The relationship may be
expressed as:
10X objective magnification =
40X objective magnification
40X objective diameter
10X objective diameter
5.
Calculate the field diameter provided by the 40X objective of your microscope
and record the value in the table above.
6.
When drawings are made, a line indicating the scale dimension should be placed
adjacent to the illustration. The length of this line, which should represent a
convenient number of micrometers, will vary depending on the magnification of
your drawing. However, for each of the objectives the following units should be
represented:
4X objective,
1000 m, approximately one-fourth of the diameter
10X objective,
500 m, approximately one-fourth of the diameter
40X objective
100 m, approximately one-fourth of the diameter
2. OPERATION OF THE BALANCE
A.
TARING THE BALANCE
“Taring” a balance allows the user to automatically compensate for the weight of a
container holding the item that needs to be weighed. The weight of any sample container can be
“tared” at a keystroke and the display set to zero. The taring range encompasses the entire
weighing range of your balance.
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B.

Place the container you wish to tare on the weighing pan.

Briefly press the <<O/T>> key to start the taring process.

Taring runs automatically. If you tare the balance when it is unstable, the
taring operation will be shown in the display by horizontal segments.

On completion of taring, the zero display appears and your balance is
ready for weighing.

By pressing the <<O/T>> key again in the unstable (not yet tared)
condition, you can abort taring.
WEIGHING SAMPLE
Once the weight of the container has been tared, you may directly read the weight of the
item added to the container.
3. THE USE OF THE pH METER
The pH meter provides a means of determining the concentration of H+ and OH - in any
aqueous solution in the acidity range between 1.0 M H+ and 1.0 M OH -. The term pH is defined
as
pH = log10 1 = -log10[H+]
[H+]
In a precisely neutral solution at 25C
[H+] = [OH-] = 1.0 x 10-7 M
The pH of this solution is
pH = log
1
1 x 10-7
= 7.0
A solution that differs from another by 1 pH unit implies that one solution has 10X the
hydrogen ion concentration of the other.
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Measurement of pH is one of the most common and useful analytical procedures in cell
and molecular biology and biochemistry since the pH of intra- and extracellular fluids governs
important aspects of the charge, structure, and therefore functional activity of many biological
macromolecules.
The pH of a solution is measured using a glass electrode. In order to obtain accurate
measurements, the electrode must first be calibrated using standards of known pH.
CALIBRATION AND USE OF THE pH METER.
1.
See instructions for pH meter as posted above the instrument. Your instructor
will demonstrate how to use it.
2.
Determine the pH of your unknown solution provided. Record the solution name,
its concentration, and the determined pH here.___________________________.
4. PIPETTOR OPERATION
1.
You will see a number of pipettors capable of dispensing variable amounts of
liquid. The maximum dispensing capacity is indicated on the plunger and is in l
increments (10, 100, 1000 l). Hold the pipetter in one hand. With the other
hand turn the volume adjustment knob 1/3 revolution above the desired setting
then slowly down until the required volume shows on the digital indicator. This
prevents mechanical backlash from affecting accuracy. If you pass the desired
setting, turn the dial to 1/3 revolution above the desired setting and reset the
volume. The friction ring prevents the setting from being changed unintentionally.
ALWAYS DIAL DOWN to the desired volume.
2.
Attach a new disposable tip to the pipette shaft. Press firmly to ensure a positive
airtight seal.
3.
Depress the plunger to the first stop. This part of the stroke is the calibrated
volume displayed on the digital volume indicator.
4.
Holding the pipettor vertically, immerse the disposable tip into the sample liquid.
5.
Allow the push-button to return slowly to the UP position. NEVER LET IT
SNAP UP!
6.
Wait a moment to ensure that the full volume of sample is drawn into the tip.
7.
Withdraw the tip from the sample liquid. Should any liquid remain on the outside
of the tip, wipe it carefully with a lint-free tissue, taking care not to touch the tip
orifice.
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8.
To dispense sample, touch the tip end against the side wall of the receiving vessel
and depress the plunger slowly to the FIRST STOP. Wait about 2 seconds (longer
for viscous solutions), then press the plunger to the SECOND STOP (bottom of
stroke), expelling any residual liquid in the tip.
9.
With the plunger fully depressed, withdraw the pipettor from the vessel carefully,
with the tip sliding along the wall of the vessel.
10.
Allow the plunger to return to the “UP” position.
11.
Discard the tip by depressing the tip ejector button. A fresh tip should be used for
each sample to prevent sample carryover.
TEST OF PIPETTING ACCURACY
Your instructor will assign one of the following volumes to pipette: 1000 l, 500 l , or
50 l. Choose the appropriate pipettor. Pipette a water sample on a tared weigh boat. Record
the weight. Repeat two more times by re-taring the weigh boat before each sample is transferred.
(It is important that you weigh directly after pipetting due to inaccuracies caused by
evaporation.) Calculate the % error as follows: First take the average of the weighed samples.
Calculate the mean error percent (E%; as directed by the manufacturer Rainin)--
E% =  - vo x 100
vo
Where  = mean weights of samples in mg
vo = volume shown on pipette in l
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