CHAPTER 4

The Fluorescence Microscope

In the previous chapters we discussed microscopes used in many types of forensic examinations
as well as many different sections of a forensic laboratory. Similar to the polarized light microscope, the uorescence microscope is generally used for specialized examinations in the trace
section. Using high magnication and the additional components found in a uorescence microscope, an analyst can identify many important characteristics for a sample, which can be used for
identication and comparison purposes.
Like other microscopes, the uorescence microscope uses a combination of lenses to produce
a magnied image. In addition to the basic components, several additional parts are added to
enhance the analytical ability of the microscope. A uorescence microscope has the following
basic parts:

visible light source

condenser
sample stage
objective
support and alignment portions
oculars
In addition, some or all of the following components are added:

ultra-violet light source

excitation lters
barrier lters
Basically, the short wavelength light in a uorescence microscope originates from a specialized
illuminator. The light from the illuminator then passes through an excitation lter. The excitation
light is redirected through the objective by a mirror so that it reaches the sample. Part of this
light is absorbed by the sample and re-emitted as uorescence. The re-emitted light then passes
through a barrier lter. This lter allows light to pass that is in the visible range (uorescence
from the sample), permitting it to reach the oculars. Since the barrier lter blocks light emitted by
the excitation lters, the background will appear black, allowing only uorescence from a sample
to be viewed.

Practical Forensic Microscopy: A Laboratory Manual

2008 John Wiley & Sons, Ltd

Barbara P. Wheeler and Lori J. Wilson

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PRACTICAL FORENSIC MICROSCOPY

Fluorescence microscopes generally use oculars and objectives that provide total magnication
within the 40X to 400X range. Because of the higher total magnication, a smaller eld of view
and less depth of eld are obtained. Samples may initially be viewed with transmitted light;
however, reected ltered light is sometimes used to obtain certain microscopic characteristics.
The uorescence microscope is used to identify and characterize samples. The higher level
magnication allows viewing of the initial characteristics of an item or sample, with additional
comparison and microscopic examinations also possible.

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61

Experiment 4A: Familiarization

with the Fluorescence Microscope
Recommended pre-lab reading assignments:
Siegel JI. Fluorescence Microscopy. American Laboratory. 1982; April: 659.

Recommended website:
Abramowitz M, Herman B, Murphy DB, Davidson MW. Anatomy of a Fluorescence Microscope. [Java
Interactive Tutorial]: Olympus American, Inc.; 2007 [updated 2007; cited 2007 December 19]; Available from: http://www.olympusmicro.com.

OBJECTIVE
Upon completion of this practical exercise, the student will have developed a basic understanding of:
1. components of the uorescence microscope
2. uorescence
3. use of the uorescence microscope to observe uorescence

INTRODUCTION
The theory behind a uorescence microscope is fairly simple. Some materials possess the property
that when specic wavelengths of light fall upon them, they absorb this light and in turn re-emit
light of a longer wavelength. This property is called uorescence. The uorescence microscope
allows the forensic scientist to observe this property by selectively irradiating the sample and
viewing the resultant uorescence. Although many samples uoresce, we will study the uorescence properties of bers in this exercise. Fiber evidence is class evidence (i.e., not unique)
because many bers from different sources could be indistinguishable. The discovery of a ber
and its identication as a particular ber type (e.g., acrylic, cotton, nylon, polyester) may not, of
itself, provide much support for a forensic investigation. The probative value of particular bers
found at a crime scene depends on their uniqueness relative to the background of bers normally
encountered. What is often required is information that makes the trace evidence more specic
and discriminating. The uorescence property of a questioned ber may be unique, allowing it to
be easily distinguished from other bers.
In earlier experiments we learned that a microscope is an optical instrument that uses a combination of lenses to produce a magnied image of small objects. Just like a compound or polarized
microscope, the uorescence microscope uses several basic optical components that gather light
and redirect the light path so that a magnied image of the viewed object can be focused within a
short distance. These basic optical components are: light source, condenser, sample stage, objective, support and alignment portions, and oculars. In addition, the uorescence microscope uses
additional parts that enhance the microscopes analytical ability. These additional components are

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excitation and barrier lters and a light source, which extends into the UV region of the spectrum
for excitation. Excitation lters are placed between the illuminator and the sample while the barrier
lters are placed between the sample and the oculars.
The operation of a uorescent microscope is shown in Figure 4A-1. In a uorescence microscope, the short wavelength light originates from a specialized illuminator. Since uorescence is
easily examined with reected light, the illuminator is usually mounted at the top of the microscope. Usually a high-pressure xenon or mercury lamp is used. The light from the lamp is passed
through an excitation lter, which allows only a narrow band of specic wavelengths to pass
through. Excitation lters are normally in the UV range of light. The excitation light is redirected

excitation filter
barrier filter

a)

UV light source

objective

fluorescence excitation
specimen
b)

fluorescence emission

visible light

barrier filter
UV light

objective

specimen

Figure 4A-1 a) Fluorescence excitation occurs when UV light travels from the light source to the
specimen. The wavelengths used to cause excitation vary with each sample so a variety of excitation lters
are used. b) Fluorescence excitation is detected by measuring the light coming off the specimen. UV light
from the light source is blocked by a barrier lter.

FLUORESCENCE MICROSCOPE

63

through the objective by a mirror so that it reaches the sample. Part of this light is absorbed by
the sample and re-emitted as uorescence. The re-emitted light then passes through a barrier lter,
which is used to separate the light used to excite the sample from the uorescence. It does this by
only allowing light to pass that is in the visible range (uorescence from the sample), permitting
it to reach the oculars. Since the barrier lter blocks light emitted by the excitation lters, the
background will appear black, allowing any uorescence from a sample to be easily viewed.
Samples may initially be viewed with transmitted light; however, reected ltered light is used to
obtain their uorescent microscopic characteristics. A series of four excitation lters ultraviolet,
violet, blue, and green are usually recommended for forensic use. It is important to note that glass
slides and most mounting mediums can absorb UV radiation or uoresce in and of themselves.
A strongly uorescing sample may still be visible with a glass cover slip, but weak uorescence
might be missed. In order to minimize interfering uorescence, it is best to use quartz slides
and cover slips. Since most of the common mounting media also uoresce, it is important to use
mounting mediums such as methanol, xylene, Norland 65, and XAM, which do not uoresce.

EQUIPMENT AND SUPPLIES

Fluorescence microscope with objectives of various magnications (e.g., 4X, 10X, 20X, 40X) and
various excitation and barrier lters
Micro kit
Fibers with known uorescence
Quartz microscope slides
Quartz cover slips
Mounting medium such as methanol, xylene, Norland 65, or XAM

SAFETY
Use standard laboratory safety procedures as described in guidelines set by your instructor. Mercury
or xenon lamps generate extreme heat and may explode. Do not look at the lamp directly to avoid
eye damage. Use the safety shield if it is provided. Be cautious of microscope light levels to
avoid eye damage. Know the hazards associated with the mounting mediums, and use them with
appropriate precautions as set by your instructor. Dispose of glass in an appropriate container.

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PRACTICAL FORENSIC MICROSCOPY

PART I: PARTS OF A FLUORESCENCE MICROSCOPE

Label the parts of the uorescence microscope in Figure 4A-2 by writing the name next to the
appropriate number. A copy of this worksheet can be obtained from http://www.wileyeurope.
com/college/wheeler.

1)

2)

4)
11)
5)

10)

6)
8)
3)

9)
7)

Figure 4A-2 A typical uorescence microscope with additional lter cubes.

In the space below write a single sentence explaining the function of each part. Attach additional
pages if necessary.

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65

PART II: OPERATION OF THE FLUORESCENCE MICROSCOPE

Procedure
1. Familiarize yourself with the uorescence microscope. Locate each part of the microscope.
Place a prepared microscope slide on the stage. After turning on the transmitted light source,
manipulate the oculars of the microscope to adjust the interpupillary distance so that when
viewing an object, the right and left images merge as one.
2. Adjust the focus up and down. Using the non-adjustable ocular, focus on an item to obtain a
clear image of an item.
3. Focus the second ocular if necessary.
4. Set up Kohler illumination and have your instructor sign the Kohler Check Sheet.
5. Determine the location of the specialized illuminator.
6. Determine the location of the excitation and barrier lters.

PART III: OBSERVING SAMPLE FLUORESCENCE

Procedure
1. Turn on the power supply for the specialized illuminator. Slide lamp diaphragm into the closed
position while the lamp warms up.
2. Prepare the sample using non-uorescent slides, cover slips, and mounting medium.
3. Turn on the power supply for the base light illuminator. Adjust the microscope to obtain Kohler
illumination.
4. Using the lowest magnication, place the prepared sample on the microscope stage and bring
it into focus. Lower the intensity of the base light and room lighting. (Steps 3 and 4 are only
necessary if the base light will be used to aid in the visualization of samples.)
5. Open the lamp diaphragm on the specialized illuminator. Refocusing of the sample may be
necessary.
6. Observe the sample and note the color of any light being emitted. Change excitation and barrier
lters and repeat your observations. In your notes fully describe the uorescence characteristics
of your sample.
7. Repeat this examination with three more samples.

REPORT REQUIREMENTS
Include all drawings, calculations, or other information obtained during the laboratory procedure.
Notes and/or drawings should include the sample identication, magnication, and a complete
description.

REPORT QUESTIONS
1. Explain how a uorescence microscope is used in forensic science. Give the purpose and
value of the examination.
2. What is uorescence? How is uorescence initiated?

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3. What are the advantages and disadvantages of this optical property for forensic samples?
4. Explain the optics used in a uorescence microscope.
5. If a forensic scientist has a polarizing microscope such as a Leica DMEP, which they want to
convert to a uorescence microscope, could they use the same light bulb? Why or why not?
6. What is the purpose of the excitation and barrier lters?
7. How are excitation and barrier lters paired?
8. Does the color of light emitted from a uorescent sample have any signicance in distinguishing and identifying the sample?
9. Why were quartz slides and cover slips used?
10. Name two types of evidence that could be examined with a uorescence microscope. Of what
would the examination consist?
11. What types of evidence would not be examined with the uorescence microscope? Why?

RECOMMENDED AND FURTHER READING

Abramowitz M, Herman B, Murphy DB, Davidson MW. Anatomy of a Fluorescence Microscope. [Java
Interactive Tutorial]: Olympus American, Inc.; 2007 [updated 2007; cited 2007 December 19]; Available
from: http://www.olympusmicro.com.
Bell S. Forensic Chemistry. Upper Saddle River, NJ: Pearson Education, 2006.
Fong W. Analytical Methods for Developing Fibers as Forensic-Science Proof a Review with Comments.
Journal of Forensic Sciences. 1989; 34(2): 295311.
Herman B. Fluorescence Microscopy. 2nd ed. Oxford, UK: Bios Scientic Publishers; Springer in Association
with the Royal Microscopical Society, 1998.
Siegel JI. Fluorescence Microscopy. American Laboratory. 1982; April: 659.
Wang XF, Herman B. Fluorescence Imaging Spectroscopy and Microscopy. New York: John Wiley & Sons,
Inc., 1996.
Wiggins K, Holness JA. A Further Study of Dye Batch Variation in Textile and Carpet Fibres. Science &
Justice. 2005; 45(2): 936.