LASER EXCITED FLUORESCENCE IMAGING OF LATENT FINGERPRINTS

Estrolita Green B.Sc. (Hons.)

Centre for Forensic Science University of Western Australia

This thesis is presented in partial fulfilment of the requirements for the Master of Forensic Science

2011

1

DECLARATION

I declare that the research presented in this 48 point thesis, as part of the 96 point Master

degree in Forensic Science, at the University of Western Australia, is my own work. The

results of the work have not been submitted for assessment, in full or part, within any other

tertiary institute, except where due acknowledgement has been made in the text.

…………………………………………………

Estrolita Green

2

Abstract

The detection of latent fingerprints is a challenging analytical problem. It involves the

detection of very small quantities of specific chemical compounds in situ on potentially

complex substrates.

Despite the success of the current procedures for photoluminescence detection of

fingerprints, many types of articles remain as difficult substrates because of intense

background fluorescence, which is often spectrally broad and difficult to suppress. Very

few methods are able to detect untreated latent fingerprints. This study investigated whether

(i) confocal laser scanning microscopy (CLSM) equipped with Fluorescence Lifetime

Imaging (FLIM) could suppress background fluorescence sufficiently to reveal good

quality images of such fingerprints and (ii) whether it could further enhance fingerprints

detected with conventional optical techniques. Imaging was performed with a Leica TCS

multi-photon confocal system equipped with FLIM. Methodologies were developed for

untreated fingerprints and fingerprints treated with ninhydrin, cyanoacrylate (CA) and CA

plus fluorescent powder dusting, on various substrates chosen for either their common

place in society or intractability. However, unexpected FLIM results led to the project

focusing on identifying the reasons behind these results. Consequently, selected single

photon lasers were employed to further investigate these findings. Exhaustive and

prolonged testing revealed subtle design errors that were not in accordance with the

expectations of the manufacturer. It was found that excitation light was reaching the

detectors, thereby allowing transmission light to interfere with the emission spectrum of the

3

samples. This amount of light was normally sufficient to obscure any luminescence from

the fingerprint.

None of the methods employed could detect untreated fingerprints on porous substrates,

however, results were more positive for untreated fingerprints on non-porous substrates. It

was found that single photon microscopy produced comparable results to those obtained

with the conventional optical technique for high quality ninhydrin treated fingerprint

deposits. FLIM however was unable to suppress the background fluorescence sufficiently

to reveal discernable fingerprint detail. CLSM techniques offered advantages over the

conventional technique with highly contrasting ridge detail, when dark red glossy card

treated with Greenwop fluorescent powder was examined.

Untreated latent fingerprints on non-porous substrates, glass and foil, showed high contrast

with the CLSM techniques as well as the conventional technique. When these samples were

treated with CA the contrast was further enhanced. The CA plus fluorescent fingerprint

powder method had variable results depending on the substrate the fingerprint was

deposited on. Metal foil samples produced highly contrasted images while the results for

the ziplock bag and juice carton substrates were less successful.

This technology has potential however due to instrumentation limitation in this study the

techniques employed are too immature for application, however with an optimal

instrumentation setup these techniques might be of future forensic use.

4

Acknowledgements I would like to thank Brendan Griffin and Ian Dadour for their supervision and advice. I am

especially grateful to Brendan Griffin for the countless hours spent helping me finish my

thesis. I would like to acknowledge the staff of the Centre for Microscopy, Characterisation

and Analysis for their help with confocal imaging and sample preparation. A special

mention goes to John Murphy who was always accessible and willing to help. I am grateful

to the staff of the Centre for Forensic Science and the Western Australian Police Forensic

Fingerprint Bureau for their assistance throughout this project. Finally, I would like to

thank my family for their patience, love and support over the years.

5

Table of contents

DECLARATION .................................................................................................... 1

ABSTRACT .......................................................................................................... 2

ACKNOWLEDGEMENTS .................................................................................... 4

TABLE OF CONTENTS ....................................................................................... 5

LIST OF FIGURES AND TABLES ....................................................................... 7

CHAPTER 1: INTRODUCTION .......................................................................... 10

1.1 Preface ................................................................................................................................................ 10

1.2 Rationale and aims ................................................................................................................................ 12

CHAPTER 2: LITERATURE REVIEW ................................................................ 13

2.1 Previous fluorescence imaging research .............................................................................................. 13

2.2 The skin .................................................................................................................................................. 152.2.1 The sweat glands ............................................................................................................................. 162.2.2 Latent fingerprint composition ........................................................................................................ 17

2.3 Current detection and enhancement techniques of latent fingerprints ............................................ 18

2.4 Fluorescence microscopy ...................................................................................................................... 192.4.1 Fluorescence .................................................................................................................................... 192.4.2 Fundamentals of Excitation and Emission ...................................................................................... 202.4.3 Confocal laser scanning microscopy ............................................................................................... 232.4.4 Multi photon microscopy ................................................................................................................ 242.4.5 FLIM technique ............................................................................................................................... 25

2.5 Relevant signal enhancement techniques ............................................................................................ 262.5.1 Ninhydrin method ............................................................................................................................ 262.5.2 Cyanoacrylate (Super glue) Method ................................................................................................ 272.5.3 Powder Dusting ............................................................................................................................... 28

CHAPTER 3: MATERIALS AND METHODS ..................................................... 29

3.1 Sample preparation and fingerprint enhancement ............................................................................ 293.1.2 Ninhydrin solution procedure .......................................................................................................... 313.1.3 Cyanoacrylate (Super Glue) Fuming procedure .............................................................................. 31

3.2 Instrumentation ..................................................................................................................................... 32

3.3 Detection ................................................................................................................................................ 33

6

3.3.1 Conventional microscopy ................................................................................................................ 333.3.2 Detection of fingerprints using Leica confocal imaging .................................................................. 333.3.3. Detection of fingerprints using FLIM ............................................................................................. 34

3.4 Processing techniques ........................................................................................................................... 35

CHAPTER 4: RESULTS ..................................................................................... 37

4.1 Porous substrate .................................................................................................................................... 374.1.1 untreated samples ............................................................................................................................ 374.1.2 Ninhydrin treated samples ............................................................................................................... 374.1.3 Fluorescent powder dusting ............................................................................................................. 42

4.2 Non-porous substrates .......................................................................................................................... 434.2.1.Untreated samples ........................................................................................................................... 434.2.2 Cyanoacrylate (superglue) treated samples ..................................................................................... 49

CHAPTER 5: DISCUSSION AND CONCLUSIONS ........................................... 56

5.1 Forensic Application ............................................................................................................................. 59

5.2 Future Study directions ........................................................................................................................ 59

REFERENCES ................................................................................................... 60

7

List of figures and tables Fig 2.1 Image of the Stoke’s Law shift between the excitation and emission spectra

Fig 2.2 Jablonski diagram illustrating single photon and two photon excitation

Fig 2.3 Fluorescence lifetime decay image

Fig 3.1 Fluorescence lifetime decay data of Rhodamine B

Fig 3.2 Image of ninhydrin treated fingerprint deposited on white paper

(a) Contrast and brightness adjusted

(b) FFT bandpass filter applied in addition to contrast and brightness adjustment

Fig 4.1 Ninhydrin treated prints on white paper (conventional technique)

(a) First deposit

(b) Second deposit

(c) Third deposit

Fig 4.2 Ninhydrin treated prints on white paper (single photon CLSM technique)

(a) 488 nm excitation

(b) 561 nm excitation

(c) 594 nm excitation

(d) 561 nm excitation unprocessed

Fig 4.3 Intensity image of fingerprint deposited on white paper and treated with ninhydrin

(a) Intensity image

(b) Histogram of fluorescence lifetime distribution

(c) Fluorescence decay curve of selected point

Fig 4.4 Nynhydrin treated prints on UBD paper obtained with 561nm excitation (single

photon CLSM technique)

8

Fig 4.5 Fingerprint deposited on red glossy card and dusted with Green wop

(a) Third deposit

(b) Second deposit

(c) First deposit

(d) Montage image of weak fingerprint deposit

(e) Montage image of strong fingerprint deposit

Fig 4.6 Lifetime decay curve image of a strong deposit print dusted with Greenwop at 815

nm multi-photon excitation

Fig 4.7 Fingerprint deposited on glass (conventional technique)

(a) Third deposit

(b) Second deposit

(c) First deposit

Fig 4.8 Fingerprint deposited on glass (single photon CLSM technique)

(a) Single photon CLSM technique

(b) Multi-photon CLSM technique

(c) Conventional technique

Fig 4.9 Montage image of untreated latent fingerprint on glass excited with

(a) 488 nm excitation

(b) 561 nm excitation

(c) 594 nm excitation

Fig 4.10 Fluorescence lifetime image of an untreated fingerprint deposited on glass

Fig 4.11 Fingerprint deposited on foil

(a) Conventional technique

(b) Single photon CLSM technique (488 nm excitation)

9

(c) Multi-photon CLSM technique (720 nm)

Fig 4.12 Untreated third impression of latent fingerprint on foil (single photn CLSM)

(a) 488 nm excitation

(b) 561 nm excitation

(c) 594 nm excitation

Fig 4.13 Fluorescence lifetime image and its distribution histogram of an untreated

fingerprint deposit on foil at 720 nm excitation

Fig 4.14 Image of a section of a fingerprint deposited on foil and treated with CA

(a) Single photon CLSM technique (488 nm excitation)

(b) Single photon CLSM technique (561 nm excitation)

(c) Single photon CLSM technique (594 nm excitation)

(d) Multi-photon CLSM technique (718 nm excitation)

(e) Conventional technique

Fig 4.15 Montage image of strong finperprint deposit on foil and treted with CA ( single

photon CLSM technique)

(a) 488 nm excitation

(b) 561 nm excitation

(c) 594 nm excitation

10

Chapter 1: Introduction

1.1 Preface The detection and imaging of latent fingerprints plays a significant and important role in the

identification of individuals in law enforcement. A latent fingerprint is the transfer of

material of the friction ridge skin detail to a surface it comes in contact with (1). However

latent fingerprints are almost always invisible (2), hence current imaging techniques are

focused on enhancing the latent print in order to distinguish it from the surface on which it

is found. This study investigates a fluorescence lifetime imaging microscopy (FLIM)

approach to imaging latent fingerprints.

A typical fingerprint is a minuscule deposit of a “complex mixture of natural secretion and

contaminants from the environment” (3). The heterogeneous nature of latent fingerprints as

well as the low average mass of deposited material of approximately 20 micrograms (4)

poses an analytical challenge to fingerprint experts. Approximately 98- 99% of the

fingerprint residue consists of water, leaving a very small amount of material, primarily

mineral salts, organic acids, urea and sugars, to be analysed (5). Additional factors that

would influence the processing of latent fingerprints include “the nature of the surface; the

presence of environmental contaminants; whether or not the surface has been wet or the age

of the fingerprint” (6).

Since the 1980’s a wide range of physical and chemical techniques were developed for the

detection of latent fingerprints (3). Conventional methods vary from dusting with powders

to treating the fingerprint residue chemically to render it visible.

An often serious problem in photo luminescence detection methods is the suppression of

background fluorescence from substrates with an emission wavelength close to that of the

11

fingerprint material. Especially when the background emission is stronger in intensity than

that of the fingerprint emission. Despite the success of treated fingerprints, the possibility

of imaging natural luminescence offers a potential non-destructive technique that leaves

other options available (2). Chemical and physical enhancement methods provide high

sensitivity but they are destructive. Thus the same print cannot be used for further forensic

analyses such as DNA analysis, mass spectrometry, etc. However, in studies to date

imaging inherent luminescence has had limited success (2, 7).

The development of confocal and two-photon laser scanning microscopy have considerably

improved the image quality in fluorescence microscopy (8). This provides a new

opportunity to investigate fluorescence microscopy, coupled with FLIM, as a possible tool

to enhance imaging of latent prints. These advances have been made possible by the use of

more elaborate optical arrangements, more sensitive detectors, faster computers and more

powerful lasers. Confocal microscopy is based on point illumination and point detection

allowing for only light from the focus plane to reach the detector with the out of focus light

blocked by the pinhole, resulting in improved resolution and contrast. Multi photon

confocal microscopy, on the other hand, does not need a pinhole in the optical path to reject

out of focus light because there is no excitation in the out of focus region. Excitation of

fluorophores is limited to the focus region only. This reduces the amount of non-specific

scattered light that reaches the detector. By reducing the detection of scattered light contrast

and detection sensitivity can be improved.

Given the heterogeneous nature of latent fingerprints and substrate background

fluorescence, multi photon microscopy offers multi fluorophore excitation, where near

12

infrared (NIR) radiation at a specific wavelength induces simultaneously the visible

fluorescence of a wide range of fluorophores.

FLIM is a technique that not only characterises the emitted light by the emission intensity

and emission spectrum but also by the decay lifetime. Allowing for fluorophores to be

characterised by spectra as well as their respective fluorescent lifetimes. Applications of

FLIM have been predominantly focused on dynamic biological applications and there is

effectively no published record of previous work other than the few articles that form the

basis for this project.

1.2 Rationale and aims The purpose of this study was to investigate whether (i) confocal fluorescence microscopy

equipped with fluorescence lifetime imaging (FLIM) is capable of imaging inherent latent

fingerprints and (ii) whether it could further enhance prints detected with conventional

optical, chemical and physical enhancement techniques.

13

Chapter 2: Literature Review

2.1 Previous fluorescence imaging research Dalrymple (9) was the first in 1976 to demonstrate that inherent and enhanced

luminescence properties of fingerprint residue can be detected when excited with a

continuous wave argon-ion laser in the visible range. Subsequently many reports have been

published on fingerprint detection using lasers (2, 7, 10-12). Herod and Menzel reported

that latent fingerprints treated with ninhydrin showed fluorescence when excited with

yellow and orange (570-590nm) continuous wave dye lasers (13, 14). A time resolved

photo luminescence imaging technique was first proposed and demonstrated by Menzel in

1979 (15). Time resolved spectroscopy is a useful contrast enhancing method that in

addition to the spatial resolution provides the difference in fluorescence lifetime between

the substrate and the sample. Menzel proposed dusting latent fingerprints with

phosphorescing earth based chemicals prior to illumination, to eliminate substrate

fluorescence. The rationale was that the fingerprint luminescence lifetime can be prolonged

by the application of rare earth based chemicals. Thus the background luminescence will

have decayed and only the fingerprint emission will be recorded.

The basic principle of time resolved luminescence is that the laser is repetitively modulated

(turned on and off). The modulation frequency is adjusted in accordance with the lifetime

of the fluorescence emission to be suppressed. Menzel’s attempt at time resolved

fluorescence imaging made use of a rotating cylinder with two slots, which acted as both

the modulator for the laser and the gate width delay. The main limitations of this method

14

was that sample size was limited and the slow speed of the rotating cylinder allowed the use

of only fingerprint treatments with very long lifetimes (millisecond scale) (15).

Advancement in technology during the 1980’s and 90’s presented new possibilities and

opportunity to build on earlier work done on time resolved luminescence techniques with

improved instrumentation (3, 16). The visualisation of latent fingerprints on white paper

and white card without any treatment was demonstrated by Bramble et al in 1993 (2).

Results from this study show that inherent fluorescence of latent fingerprints are weaker

when excited with light in the visible region of the spectrum, better contrast was observed

in the UV region (2, 16). It has also been reported that fluorescence intensity decreases with

fingerprint age (2, 12) and continuous irradiation (7), but when fingerprints which had

prolonged exposure to laser light were treated with ninhydrin and 1,8-Diazafluoren-9-one

(DFO) no adverse effects were observed. Using the same principles as Menzel (15), Seah et

al in 2004 demonstrated a time resolved imaging technique for the detection of latent

fingerprints with nanosecond resolution with the use of fluorescent powders to enhance

sensitivity (12). This technique was an improvement on the millisecond range experiments

with rare earth based chemicals achieved by Menzel. However the Seah approach fails

when the fingerprint fluorescence is much weaker in intensity than that of the background

fluorescence even if the lifetime decays are different (12). It was also reported that to obtain

the best contrast from a luminescent fingerprint, it is important that the excitation

wavelength is at the wavelength of maximum absorption (9).

The advent of tunable lasers made possible the search for an excitation wavelength for

maximum photoluminescence emission (7). Akib et al’s experiments included the

15

investigation of both sebum rich (finger was wiped across the forehead or nose) and eccrine

prints (normal). The author was able to successfully image the sebum rich print, however

no useful images of eccrine fingerprints were obtained. (7).

The application of luminescence techniques requires the use of high intensity light sources

and various types and strengths of lasers are used in fingerprint detection. Argon-ion lasers

(9, 12, 15, 16) and Nd:YAG (2, 7) lasers were more commonly used in the detection of

latent fingerprints. The relatively low success rate in earlier research, can be attributed to

the lack of flexibility in the selection of excitation wavelengths that resulted in non-

optimised fluorescence. In addition laser power was much less than what is available today.

Pulsed lasers were a poor choice then, unable to excite phosphorescers because of their

short pulse width and low repetition. More efficient short pulse lasers are now available (8).

2.2 The skin

The skin covering the palms of the hands and the soles of the feet, is different to the skin on

other parts of the body. It is elevated into minute ridges in patterns of different designs (17).

Here there are no hairs; the epidermis is much thicker and is referred to as thick skin.

Elsewhere the epidermis is thinner and this skin is called thin skin and contains hairs except

in certain locations (18).

The skin consists of two main layers: the epidermis and the dermis.

In the epidermis five layers can be distinguished. The stratum corneum is the surface layer

of the skin. It is comprised of dead cells (keratinised cells) that are continually shed and

16

replaced by cells from the layers beneath, as the skin constantly renews itself. This renewal

process begins in the base of the epidermis (stratum basale), where the cells (keratinocytes)

divide (mitosis) and move toward the surface to replace those, which have been sloughed

off. On their migration to the stratum corneum these cells undergo keratinisation. During

keratinisation the cells loose their nuclei and become surrounded by a tough impermeable

‘envelope’ of various proteins (loricrin, involucrin, filaggrin and keratin). Higher up in the

granular layer complex lipids are also secreted by the keratinocytes and these form into a

semipermeable skin barrier (19).

The dermis, the inner layer of the skin is composed of connective tissue. It contains blood

and lymphatic vessels, nerves, muscles, appendages (e.g. sweat glands, sebaceous glands

and hair follicles) and a variety of immune cells such as mast cells and lymphocytes.

Under the dermis there is another layer, the hypodermis (subcutaneous layer). It is

comprised of subcutaneous fat (18).

2.2.1 The sweat glands

Glands are located in the dermis and the hypodermis. Three types of glands are responsible

for the natural secretions of the skin, the sudoriferous (sweat) eccrine and apocrine glands

and the sebaceous (oil) glands. The physiological function of these secretions are that they

(i) lubricate and keep the skin in good condition and improve traction of ridged skin, (ii)

wash away dead cells, (iii) coating the skin with a water repellent shield, (iv) expel waste

and (v) act as a cooling mechanism.

17

Eccrine sweat glands are found throughout the skin except at the lips and external genitalia.

They are abundant in thick skin, which is devoid of hair. The palms of the hands and the

soles of the feet produce only eccrine gland secretions.

The eccrine sweat gland is a simple coiled tubular gland. The secretory portion of the gland

is located deep in the dermis or in the upper portion of the hypodermis. The glands traverse

the epidermal layer to open at the summits of the papillary ridges to form sudoriferous

(sweat) pores (18).

Sebaceous glands are inactive until puberty. They are responsible for secreting sebum or

grease onto the skin surface (via the hair follicle) and are found in high number on the face

and scalp (19). “Sebum is the most durable matter of the latent print residue and is a

product of the cellular decomposition of keratin” (20) .

Apocrine sweat glands are found in the axillary (armpit), pubic region and scalp and do not

function until puberty. They secrete into the upper parts of the hair follicle and, similar to

sebaceous glands, their secretions reach the surface via the hair follicles. Products of all of

these glands can be found in latent fingerprint deposits (19).

2.2.2 Latent fingerprint composition

The secretion from eccrine glands is present to some degree in latent fingerprints (21). The

chemical composition of eccrine excretion contains about 99 % water but in addition it

contains various inorganic salts, as well as organic matter. These residues might also be

18

contaminated with sebaceous gland secretions. Sebaceous material is often transferred onto

the papillary ridges when fingers are run through the hair and touches the forehead or nose

region (5). Sebaceous components of latent fingerprint residue are predominantly fatty

acids, cholesterol, squalene and triglyceride (22, 23). Amino acids and lipids are latent

fingerprint components that are commonly targeted by chemical detection methods. A

variety of protocols have been employed to determine the composition of latent fingerprints

(21, 22, 24, 25).

The chemical composition of a latent fingerprint residue can include: minerals (sodium,

chloride, potassium, calcium magnesium, iron, iodine, fluorine, bromine); organic

components (lactic acid, glucose, nitrogen, urea, ammonia, thiamine, riboflavin,

pantothenic acid, nicotinic acid, pyridoxine, aminobenzoic acid, inositol, choline, folic

acid) and miscellaneous substance (alcohol, histamine and morphine have also been found

in sweat following their administration to subjects) (21). However this list is not

exhaustive.

2.3 Current detection and enhancement techniques of latent fingerprints

A wide range of techniques are available for the detection and enhancement of latent

fingerprints. The techniques include: optical methods, (diffusion, luminescence, ultra violet

(UV) absorption and reflection) physical methods (powdering, small particle reagent,

vacuum metal deposition), physico-chemical methods, (physical developer, multi-metal

deposition, iodine, cyanoacrylate) and chemical methods (ninhydrin and its analogs, metal

19

complexation after ninhydrin treatment, 1,8-diazafluoren-9-one (DFO), silver nitrate,

osmium- and ruthenium tetroxide and dimethylaminocinnamaldehyde (DMAC).

2.4 Fluorescence microscopy

The first fluorescence microscopes were developed between 1911 and 1913 by German

physicists Otto Heimstädt and Heinrich Lehmann. The fluorescence microscope is based on

the phenomenon that certain material emits energy detectable as visible light when

irradiated with the light of a specific but shorter wavelength. The sample can either be

fluorescing in its natural form, or treated with fluorescing chemicals (26).

2.4.1 Fluorescence

Luminescence is a term describing a family of processes in which susceptible molecules

emit light from electronically excited states created by either a physical (for example,

absorption of light), mechanical (friction), or chemical mechanism. Luminescence through

excitation of a molecule by ultraviolet or visible light photons is a phenomenon termed

photoluminescence. Photo luminescence is formally divided into two categories,

fluorescence and phosphorescence, depending upon the electronic configuration of the

excited state and the emission pathway.

The process of phosphorescence occurs in a manner similar to fluorescence, but with a

much longer excited state lifetime.

20

Molecules capable of undergoing electronic transitions that ultimately result in fluorescence

are known as fluorescent probes or fluorochromes. Fluorochromes that form a larger

macromolecule i.e. lipids, proteins, are called fluorophores. Fluorochromes are described as

intrinsic and extrinsic. Intrinsic fluorochromes, such as naturally occurring amino acids can

be used to form fluorescent proteins. Extrinsic fluorochromes are synthetic dyes or

modified biochemicals that are added to a specimen to produce fluorescence with specific

spectral properties.

2.4.2 Fundamentals of Excitation and Emission

Fluorochromes have unique and characteristic spectra of absorption (usually similar to

excitation) and emission. In order to determine the emission spectrum of a particular

fluorochrome, the wavelength of maximum absorption is determined and the fluorochrome

is excited at that wavelength and intensity of emission of different wavelengths is plotted.

Electrons moving in orbits around the nucleus of an atom are arranged in different energy

levels within their probability distribution functions. In accordance with Quantum

Mechanics, electrons exist only in certain energy levels. Each electronic state is further

subdivided into a number of vibrational and rotational energy levels. Many of the electrons

can absorb additional energy from external sources of electromagnetic radiation, which

results in their promotion to an inherently unstable higher energy level. Fluorescence

occurs in three steps, on time scales separated by several orders of magnitude. First the

excitation of a susceptible molecule by and incoming photon occurs in femtosecond (10E-

15 seconds) scale. Next the vibrational relaxation of excited state electrons to the lowest

21

energy levels takes place. This process is much slower and occurs in the picosecond scale

(10E-12 seconds). Finally, the "excited" electron emits the extra energy as electromagnetic

radiation and returns to its original and stable energy level. This occurs in a time period of

nanoseconds (10E-9). The energy of the emitted radiation equals the energy that was

originally absorbed by the electron minus other small quantities of energy lost through a

number of secondary processes. Thus since the wavelength is inversely proportional to

energy, the wavelength of emission is always longer than the wavelength of excitation

(Stoke’s Law) and the emission peak is always lower than the excitation peak due to none-

radiative energy loss and scattering (Figure. 2.1).

Taken from http://www.olympusmicro.com/primer/lightandcolor/images/excitationemissionfigure2.jp

Fig. 2.1. The Stoke’s Law shift between the excitation and emission spectra.

22

Electromagnetic radiation energy levels can vary to a significant degree depending upon the

energy of source electrons. For example infrared rays, or visible light waves contain far less

energy than ultraviolet light. Higher electromagnetic radiation energies are associated with

shorter wavelengths than similar forms of radiation having lower energy. The relationship

between the energy of an electromagnetic wave and its frequency is expressed by the

equation: E = h = hc/

Where E = energy in kilojoules per mole; h = Planck's constant; =frequency of light; c =

velocity of light; = wavelength of light. Based on this equation, the energy of an

electromagnetic wave is directly proportional to its frequency and inversely proportional to

the wavelength. Thus, as frequency increases (with a corresponding decrease in

wavelength), the electromagnetic wave energy increases, and vice versa.

Göppert-Mayer (27) in 1931 described a nonlinear absorption process where two photons

with a combined energy level equivalent to that of a high energy single photon (linear

process), when arriving almost simultaneously at a molecule of a fluorophore can combine

their energy to promote the molecule to an excited state (Fig. 2.2). For two photon

excitation, usually longer wavelengths in the infrared range are used to excite the

fluorophores.

23

Fig. 2.2. Jablonski diagram illustrating single photon and two photon excitation (after Denk et al.,1990).

2.4.3 Confocal laser scanning microscopy

In conventional optical microscopy focused and out of focus light is detected. All parts of

the sample in the optical path are excited at the same time and the resulting fluorescence is

detected, including a large unfocused background section. In contrast, a confocal

microscope uses point illumination. The confocal microscopy concept is that there is a

point laser beam scanned in the x- and y- direction, at the in focus plane. The fluorescence

light from the sample travels back through the objective lens and scanner and is focused

into a confocal pinhole that is conjugate with the illumination point and the focused point

of the illumination light in the specimen. Light from outside the focal plane is not passed

through the confocal pinhole and so is not included in the image.

A confocal microscope (CLSM) is generally equipped with continuous wave (CW) lasers

of visible range and spectral detectors. CW lasers are used for single photon excitation.

24

CLSM’s can also be operated in multi-photon mode. A pulsed infrared (IR) laser is used as

a laser source.

Advances in CLSM have allowed for the development of new microscopy techniques. One

example of such a technique is fluorescence lifetime imaging (FLIM) (28).

2.4.4 Multi photon microscopy

Multi photon microscopy is based on the simultaneous absorption of two or more photons.

If a molecule of a fluorophore can be excited by the almost simultaneous absorption of two

or three photons that have half or a third of the energy required to elevate the target

molecule to the semi-stable excited state, there is a probability that some of that absorbed

energy may be released as a fluorescence photon. The probability of multi photon

transitions to occur is extremely low and it requires very high light intensities concentrated

in a small focal volume. The photon density is achieved by the use of an ultra short-pulsed

infrared light to excite fluorophores. Excitation is essentially restricted to the point of focus,

a very small volume (approximately 0.1 femtoliter). For this reason the fluorescence

detected will originate only from that volume, allowing extremely efficient detection of all

he signal including scattered light, without any need for confocal pinholes. The combined

energy of two or more low energy photons is equivalent to the energy of one high energy

photon and the resultant fluorescence spectra in both cases (26, 29).

25

2.4.5 FLIM technique

FLIM has been widely applied in biological sciences (30-33). FLIM is a fluorescence

detection mode that allows for the collection of fluorescence lifetime images of objects in

addition to the normally detected intensity images. The imaging of fluorescence lifetimes

provides the opportunity to potentially separate fluorophores with similar spectral

characteristics by their lifetimes if it is sufficiently separated (34). An example of lifetime

measurements in the time domain is shown in Fig. 2.3.

A number of FLIM approaches have been demonstrated (11, 12, 35). FLIM can be

performed in both the frequency domain and the time domain. In the frequency domain

method a continuous wave light source and the fluorescence detector are modulated

periodically and from the phase shift of the fluorescence signal the lifetime can be deduced.

In the time domain approach the fluorescence decay profile is directly measured. A pulsed

(femto second) laser with approximately 80 MHz rate is used for excitation and a point

detector, photomultiplier tube, for fluorescence detection. It is based on the concept of

time-correlated single-photon counting (TCSPC). A clock is started with the arrival of an

excitation pulse at the sample and stops with the detection of an emitted photon at the

detector. A histogram of photon arrival times is built up and fitted to a decay curve to yield

the characteristic fluorescence lifetime (28). TCSPC is a sensitive technique for recording

low light signals with picosecond resolution and high precision.

26

Fig. 2.3. Fluorescence lifetime data.

2.5 Relevant signal enhancement techniques

2.5.1 Ninhydrin method

Ninhydrin is a chemical reagent commonly used for the enhancement of fingerprints on

porous substrates. It is a non-specific amino acid reagent. It reacts with the alpha amino

acid groups of the latent fingerprint residue to form a purple–blue product known as

Ruhemann’s purple resulting in a visible coloured print.

In 1982 Herod and Menzel reported a laser/ninhydrin method that could be applied to both

porous and non- porous substrates. They found that fingerprints that showed discernable

fingerprint ridges under room light after ninhydrin treatment, showed no fluorescence when

Lifetime image

Lifetime distribution histogram

Exponential fit parameters

Lifetime decay curve

Residual curve

Cursor position

27

investigated under argon laser (330 to 360 nm) excitation, however they fluoresced in the

red and near infrared region under yellow and orange dye laser (570 to 590 nm) light (13).

They further reported two modified ninhydrin methods for latent fingerprint enhancement.

In the first modification the fingerprint was treated with trypsin prior to being enhanced by

ninhydrin. In the second modification the prints were treated with a methanol solution

containing trypsin and ninhydrin. The trypsin (a proteolytic enzyme) would cleave proteins

and peptides into smaller amino groups to aid enhancement (14). Samples treated with

trypsin and then with ninhydrin showed better contrast than those treated with a

trypsin/ninhydrin mixture or ninhydrin alone when examined under argon laser light.

However no significant improvement was noticed under dye laser light. In the second

treatment fingerprints developed with ninhydrin were treated with metal salts. The

ninhydrin/zinc chloride complex showed improved fingerprint ridges under argon laser

light (14) and an increased photoluminescence at liquid nitrogen temperatures (36).

2.5.2 Cyanoacrylate (Super glue) Method

This method was first devised by the Criminal Identification Division of the Japanese

National Police Agency in the late 1970’s. It is a common forensic analytical tool for the

development of latent fingerprint on non-porous substrates. Many cyanoacrylate methods

have been used with the focus on accelerating the process by the application of heat,

chemicals and the use of vacuum chambers. The technique involves the latent print to be

exposed to cyanoacrylate vapour in an enclosed chamber. The cyanoacrylate, a clear

monomeric liquid when vaporized, “reacts with certain components in the latent fingerprint

28

residue to develop white polymer along the fingerprint ridges”(5). Although the

mechanism of polymerisation is not fully understood, it is believed to be selective and that

certain products in the moisture, eccrine and sebaceous components of the fingerprint

residue act as initiators allowing for polymerisation to take place only on the sites where

the initiators are present (5, 37).

2.5.3 Powder Dusting

The application of powders to enhance latent fingerprints is a simple, common and well

established technique. It is commonly applied to smooth, non absorbent surfaces where the

powders adhere to the greasy and moisture deposit left by the fingerprint ridges. The range

of powders available is extensive and is generally divided into four groups: regular,

luminescent, metallic and thermoplastic (5, 6, 38). Luminescent fingerprint powder

contains natural or synthetic components that fluoresce when exposed to a light source.

Redwop and Greenwop fluorescent fingerprint powders were selected for experimental

purposes. They are very fine grain powders that fluoresce under ultraviolet, argon ion, and

Nd:Yag lasers.

29

Chapter 3: Materials and Methods Methods were developed and optimised for the visualisation of untreated latent fingerprints

and fingerprints processed with ninhydrin, cyanoacrylate and cyanoacrylate plus

fluorescent fingerprint powder.

3.1 Sample preparation and fingerprint enhancement

The substrates examined can be divided into porous and non-porous surfaces. Some sample

substrates (white paper, foil, clear zip lock bag, glass) were selected because of their

common occurrence, while others (purple paper, UBD street directory paper, grease proof

paper) were selected because of their known poor retention of fingerprints using traditional

imaging techniques. A list of the substrates used in this experiment can be found in table

3.1.

A pencil was used to draw fingerprint templates where donors were then asked to place

their fingerprints on the substrates. Donors were randomly selected. Each donor rubbed

their finger on their forehead to obtain sebaceous oil on their fingers (index, middle and

ring finger of both hands). They then laid three consecutive fingerprints without recharging

the finger. This process was repeated for each of the prepared substrates. No limits for

pressure were given to the donors, allowing for different types of contact to be made. These

prints formed two sets. One set was investigated for inherent fluorescence the other was

chemically enhanced. Porous substrates were treated with ninhydrin before examination

and non-porous substrates were processed with cyanoacrylate fuming and dusted with

30

fluorescent powder. Ambient laboratory temperature ranged from 24 - 25C and Relative

Humidity from 52 – 55%.

Table 3.1

Sample Materials and Treatment

Surface Type Sample Reagent

Porous White paper

Purple paper – similar in

colour to ninhydrin

products

UBD street directory paper

Newspaper

GLAD non stick baking

paper

Ninhydrin

Non-porous Glass slide

Clear zip lock bag

Foil

No treatment

Superglue

Superglue + Powder

Reagents

Ninhydrin (C9H6O4), ethyl acetate (CH3OOC2H5), n-Heptane (C7H16), glacial acetic

acid and ethanol were all laboratory grade, obtained from a range of suppliers. Commercial

cyanoacrylate, “Quick Fix” by Selleys, was purchased from a hardware store.

31

3.1.2 Ninhydrin solution procedure

A previous reported heptane formulation of Hewlett, Sears and Suzuki as described in (38)

was used. It consists of:

Ninhydrin 5 g

Acetic acid 3 ml

Ethanol 75 ml

Ethyl acetate 25 ml

Heptane 1 L

The ninhydrin crystals were pulverised using a mortar and pestle. It was then dissolved in a

mixture of the acetic acid, ethanol and ethyl acetate. The mixture was then stirred with a

magnetic stirrer till ninhydrin was completely dissolved before heptane was added to form

a working solution.

The items were briefly (5 seconds) immersed in the ninhydrin solution, removed and air

dried. They were then stored under laboratory conditions in envelopes in a drawer until

examination.

3.1.3 Cyanoacrylate (Super Glue) Fuming procedure

The experiment was performed in a fume cupboard using a closed plastic container.

Samples were suspended from a wire positioned in the upper middle portion of the

32

container. Two to three drops of liquid cyanoacrylate were placed into a small porcelain

dish, and placed onto an electric mat heater positioned at the bottom of the plastic

container.

The items were exposed to the fumes for at least 5 min or until a white coloured fingerprint

appeared (38). Prints were left over night after development to allow the white polymer

formed on the fingerprint ridges to harden. The prints were first examined under laser light

excitation. In order to improve contrast between the polycyanoacrylate on the fingerprint

ridges and the substrate in weaker prints, the same prints were dusted with fluorescent

fingerprint powders.

3.2 Instrumentation

Experiments were performed on a commercial Leica TCS SP2 MP inverted confocal

microscope (2005) equipped with a multiline Ar/ArKr laser, two He-Ne lasers and a

titanium:sapphire (Ti:S) (Mai Tai, Spectra Physics) pulsed femtosecond (fs) laser at the

Centre for Microscopy Characterisation and Analysis. This system is equipped with four

photon multiplier detectors for signal detection in the confocal mode and a FLIM detector

for fluorescence lifetime imaging. The visible light lasers, Ar/ArKr and He-Ne, were used

to obtain single photon scanning images. The Ti:S fs (near infrared tuneable from 710 –

990 nm) laser with a 80 MHz repetition frequency and peak output power > 1.5 W was

used for two photon measurements. Fluorescence lifetime imaging was carried out using

the single photon counting (TCSC) method. The FLIM system is based on Becker &

Hickl’s multi dimensional time correlated single photon counting (TCSPC) technique. The

33

photons detected by the FLIM detector are processed in the TCSPC module, which is

controlled by its SPCM software.

Conventional microscopy was performed on a Zeiss stereo discovery V.8 microscope with

a KL 1500 LD halogen illuminator and charge coupled device (CCD) camera.

3.3 Detection

3.3.1 Conventional microscopy

Conventional optical microscopy uses visible light and a system of lenses for optical

analysis. The halogen illuminator was set to allow for even illumination of the sample.

Images were visualised using a 0.5 X objective and captured with a CCD camera.

3.3.2 Detection of fingerprints using Leica confocal imaging

The performance and optical properties of the microscope were investigated by test

measurements of fluorescent test beads. The Leica confocal was calibrated to standards

with well-characterised 4',6-diamidino-2-phenylindole (DAPI) chromophores.

The technique was first optimized for a set of parameters, excitation and emission

wavelengths for each sample type. The excitation wavelengths and the emission

wavelengths chosen were based on preliminary experimental data and the literature (3, 9).

Solutions of extracts of fingerprint sweat residue have been reported to have absorption and

34

corresponding emission peaks in the UV and the visible region of the spectrum. Single

photon experiments were performed at 488, 561 and 594 nm respectively.

3.3.3. Detection of fingerprints using FLIM

Lifetime analysis performance of the microscope was checked using 100 nM Rhodamine B

solution with a reference lifetime determined at ca 1.6 ns (± 0.2 ns) (Fig. 3.1.).

The multi photon excitation and observation wavelengths were based on preliminary scans.

Samples were scanned over the 710 – 990 nm range of the Mai Tai laser. The path settings

were saved into the acquisition software and used throughout the project. Image acquisition

times ranged from 40 to 200 seconds.

35

Fig. 3.1. Fluorescence lifetime decay image data of Rhodamine B

3.4 Processing techniques

Acquired images were treated with processing functions to optimize visualization of the

prints. Processing tools used included crop, normalization, look-up table function, sharpen,

enhance brightness and contrast, background subtraction and FFT-bandpass filter.

Other techniques applied were to (i) collect a data set of the spectra of the background

without a print and use it to divide the data set with the print and (ii) to subtract the

background data set from the data set with print. These techniques however failed to

improve quality of print.

A montage image was formed using Leica software as an entire print could not be

visualised with the low magnification objectives available. The image however had a

36

patchwork appearance (Fig. 3.2 (a)). Fig. 3.2 (b) shows the resulting products of a montage

image of a ninhydrin print deposited on white paper after processing. For the image on the

left the contrast and brightness was adjusted. For the image on the right in addition to the

contrast and brightness adjustment a fast fourier transform (FFT) bandpass filter was

applied to suppress the vertical and horizontal lines formed during the formation of the

montage.

Fig. 3.2. Image of ninhydrin treated fingerprint deposited on white paper: (a) contrast and brightness was adjusted.

(b) The image on the left was further processed by applying a FFT bandpass filter.

b a

37

Chapter 4: Results

4.1 Porous substrate

4.1.1 Untreated samples

Single photon (488, 561 and 594 nm), multi-photon CLSM laser lines, the FLIM technique

and the conventional technique could not detect untreated latent prints on any of the

substrates. The absence of contrast on paper samples was partly due to the fingerprint

residue being absorbed into the porous paper fibers. In addition to their fibrous nature the

highly fluorescent background of the newspaper, purple paper, dark red gloss card and

UBD paper completely masked latent print information. Although the grease fingermarks

were visible on the purple paper under room light, however under laser light illumination

no ridge detail could be discerned. Nothing was visible on the greaseproof paper.

4.1.2 Ninhydrin treated samples

The ninhydrin treatment resulted in the characteristic purple-blue colour reaction products

(Ruheman’s complex) revealing a number of latent prints visible in room light and by laser

illumination. Imaging gave variable results. The variability in the development of the prints

was probably due to a diversified sweat composition and the quantity of fingerprint residue

deposited resulting from varying finger pressures applied.

38

The treated prints slightly fluoresced under laser illumination. Only prints with high

contrast in room light could be visualised under laser illumination (Fig. 4.1).

Fig. 4.1. Ninhydrin treated prints on white paper (conventional technique). (a) First deposit (strong); (b)

Second deposit (medium) and; (c) Third deposit (weak).

Single photon (488, 561nm and 594 nm), multi-photon CLSM laser lines, FLIM and the

conventional technique could detect discernable fingerprint details on white paper (Fig.

4.2). The Multi photon laser line was able to visualise ninhydrin treated prints on white

paper over a range of 795 to 850nm with peak excitation at around 800 nm.

a

c

b

39

Fig. 4.2. Ninhydrin treated prints excited with single photon laser: (a) 488nm excitation; (b) 561nm excitation; (c)

594 nm excitation and (d) unprocessed image obtained by 561 laser excitation. The scale applies to all images.

The FLIM technique proved to be unsuccessful as the highly fluorescent fibrous

background completely masked any fingerprint information. Fig. 4.3 shows the intensity

image calculated from the photons in all time channels of the pixels. The distribution of the

mean lifetime over the entire image is shown in the upper right panel. In the lower panel the

fluorescence decay recorded from a selected spot is shown.

Another drawback was that only a section of a fingerprint could be visualised with the

FLIM technique due to the large size of the samples and lack of a low magnification

objective.

b

d c

a

40

The UBD paper, purple paper and newspaper proved to be difficult substrates, these papers

produced very strong background fluorescence which inturn overwhelms the fingerprint

luminescence. Partial ridge details on UBD paper could be visualised on lighter coloured

areas using the 561 nm laser line and conventional technique (Fig. 4.4). Only background

fluorescence was observed during multi photon excitation. Bold text on the newspaper

samples as well as its fibrous nature obscured luminescence from the fingerprints. A lack of

contrast resulted in no fingerprint image being discernable from the purple paper substrate

as it is similar in colour to the Ruhman’s complex (that forms when ninhydrin reacts with

the amino groups in the fingerprint residue). No fingerprint marks developed on

greaseproof paper or the dark red glossy card when treated with ninhydrin solution.

41

Fig. 4.3. Fluorescence decay curve intensity image of fingerprint deposited on white paper and treated with ninhydrin.

Top left: Intensity image, Top right: Histogram of fluorescence lifetime distribution; Lower panel: Fluorescence decay

curve of selected point.

Fig. 4.4. Ninhydrin treated fingerprint deposited on UBD paper samples: (a) conventional technique, (b) single photon

technique (561 nm laser excitation).

a b

42

4.1.3 Fluorescent powder dusting

Dusting of porous sample with fluorescent powder was unsuccessful with the exception of

the dark red glossy card. No image could be obtained with the conventional technique. The

card samples were excited with the 458 single photon laser and the multi-photon laser.

Fluorescence could be observed over a broad range of excitation, from 722 nm to 840 nm.

Both the single photon and multi-photon techniques yielded good contrasting images for

different levels of fingerprint deposits (Fig. 4.5 and Fig. 4.6). Fingerprints dusted with

Greenwop were found to have a mean lifetime of 1.6 ns.

Fig. 4.5: Fingerprint deposited on red glossy card and dusted with Greenwop: (a - c) Fingerprint deposits of increasing

intensity from left to right for a weak, medium and strong deposit at 458 nm single photon excitation. (d) Montage image

of the weak fingerprint deposit; (e) Montage image of the strong fingerprint deposit.

a c b

d e

43

Fig. 4.6: Lifetime decay curve image of strong image deposit dusted with Green wop at 815 nm multi photon excitation

Top left: Lifetime image, Top right: distribution of the mean lifetime over the entire image, Lower panel: fluorescence decay curve in the selected spot and parameters recovered in the fit.

4.2 Non-porous substrates

4.2.1.Untreated samples

Single photon, multi-photon and conventional microscopy techniques could detect

untreated latent prints on glass. Results obtained with conventional optical microscopy

were of lower contrast than results for images obtained with the single photon and multi-

photon techniques (Fig. 4.7. and Fig. 4.8.). Latent fingerprints on glass produced strong

44

auto fluorescence when excited by single photon and multi photon laser excitation (Fig. 4.8

and Fig. 4.9). Results obtained for the 488nm and 561 nm single photon laser lines were

similar where contrast achieved with 594 nm excitation was less.

Fig. 4.7. Conventional technique image of fingerprint deposited on glass. (a) weak fingerprint deposit; (b) medium

fingerprint deposit; (c) strong fingerprint deposit.

Untreated fingerprints on glass showed two bands of spectra when excited with the multi

photon laser. Images could be extracted from 714nm to 722 nm and 738 nm to753nm

excitation respectively. Within this broad emission, maximum intensity occurred at

excitation wavelengths 718nm and 744 nm. Typically the fluorescence lifetime of

fingerprint residue was found to be in the order of 1.6 to 2.3 ns.

c

a b

45

Fig. 4.8. Fingerprint deposited on glass: (a) single photon excitation, (b) multi-photon excitation and (c) conventional

method.

(Fig. 4.10.) Shows a multi-photon lifetime image of auto fluorescence of a fingerprint

deposited on a glass slide. The histogram of the fluorescence lifetime distribution over the

entire image on the right shows a distribution with a mean at about 1.6 ns calculated from

all values of fluorescent ‘pixels’.

b a

c

46

Fig. 4.9. Montage image of untreated latent print on a glass slide excited with (a), 488 nm, (b) 561nm and (c) 594 laser light

excitation respectively.

Fig. 4.10. Fluorescence lifetime decay image of untreated fingerprint deposited on glass. Top left: Lifetime image, Top

right: The distribution histogram of the mean lifetime, Lower panel: fluorescence decay curve in the selected spot.

a b c

47

Fingerprints could be detected on aluminium foil by all three of the methods applied

(Fig. 4.11). Similar results were observed when foil samples were excited with single

photon laser lights (Fig. 4.12). When samples were imaged using the FLIM technique

fluorescence spectra ranged from 718 – 728 nm with maximum intensity around 719 nm

excitation. Lifetime values calculated were similar (1.6 - 2.35 nm) to those obtained from

prints deposited on glass slides. Fig. 4.13 shows a lifetime image of the auto

fluorescence of fingerprint residue deposited on foil and the distribution of the mean

lifetime over the entire image.

Fig. 4.11. Fingerprint deposits on foil. (a) conventional technique, (b) single photon excitation (488 nm) and (c) multi-photon

excitation (720 nm).

b a

c

48

Fig. 4.12. Untreated third impression (weak) of latent fingerprint on foil: (a) 488 nm excitation, (b) 561 nm excitation,

(c) 594 nm excitation.

Figure. 4.13. Untreated foil lifetime image and its distribution histogram image at 720 nm excitation.

b a

c

49

Single photon, multi-photon CLSM and the conventional techniques could not image

untreated fingerprint deposits on plastic ziplock bag and juice carton samples. The

fingerprint deposits could not be distinguished from the light clear plastic background

while the intense background fluorescence of the juice carton overwhelms any auto

fluorescence present from the fingerprint.

4.2.2 Cyanoacrylate (superglue) treated samples

Fingerprints deposited on foil when treated with CA revealed good contrasting images

when observed under single photon, multi-photon laser excitation and the conventional

method (Fig. 4.14). Images showed improved contrast when compared with those of

untreated fingerprints. Intensity images could be obtained using 718 – 728 nm excitation

with optimum excitation at 725 nm, however no fluorescence lifetime values could be

measured using the FLIM technique.

50

Fig. 4.14 Image of partial fingerprint on foil treated with CA: (a) 488 nm excitation, (b) 561 nm excitation, (c)

594 nm excitation, (d) multi photon 71 8nm excitation and (e) conventional technique.

b a

c d

e

51

Fig. 4.15. Montage images of a fingerprint deposited on foil and treated with CA: (a) 488 nm excitation, (b) 561 nm

excitation, (c) 594 nm excitation and (d) conventional technique image.

Foil fingerprint samples treated with CA and dusted with Redwop fluorescent powder

produced highly contrasting images with the conventional as well as the single photon and

multi photon CLSM techniques (Fig. 4.16).

FLIM produced highly contrasted images. The excitation range of Redwop ranged from

726 to 900 nm with emission above 800 nm. It was found that the fluorescent lifetimes of

CA fingerprints dusted with Redwop powder has a mean lifetime of 2.5 nm (Fig. 4.17).

b a

c d

52

Fig. 4.16: Images of CA treated foil samples dusted with Redwop fluorescent powder: (a) conventional technique (b)

montage image of single photon excitation (488 nm), (c) lifetime image with mean lifetime encoded by colour (green

line through image is an artefact).

b a

c

53

Fig. 4.17 FLIM image of a weak fingerprint deposit on foil, treated with CA and dusted with Redwop fluorescent

powder. Top left: Intensity image, Top right: The distribution histogram of the mean lifetime over the entire image,

Lower panel: fluorescence decay curve in the selected spot and parameters recovered in the fit.

Plastic ziplock bags treated with CA showed high contrast under 488, 561 nm and multi

photon laser illumination, when contrast was occasionally observed under 594 nm

illumination, detail was generally poor (Fig. 4.18).

54

Fig. 4.18. Montage of fingerprint deposited on ziplock bag treated with cyanoacrylate excited with (a) 488 nm and (b) 561 nm

respectively

Dusting CA treated prints on zip lock bags was ineffective with random distribution of

powder over the entire substrate.

CA treated fingerprints on juice carton could not be imaged using the conventional

technique however showed faint luminescence under 488 nm and 561 nm laser

investigation. The quality of the images was generally poor under 594 nm excitation. When

CA samples were dusted with Redwop fluorescent powder the reverse was the case.

Maximum contrast was achieved under 594 nm excitation (Fig. 4.19). FLIM images

showed no defined fingerprint detail. Although a higher concentration of the Redwop

fluorescent powder is visible on perceived ridges, the powder settling on the background

makes it difficult to discern clear ridge details (Fig. 4.19).

b a

55

Fig.4.19: Fingerprints on juice carton: (a) Image of fluorescent juice carton background; (b) Image of a section of a

fingerprint treated with CA; (c) CA treated fingerprint dusted with Redwop fluorescent fingerprint powder and (d)

Lifetime image of the CA treated fingerprint dusted with Redwop fluorescent fingerprint powder.

b a

c d

56

Chapter 5: Discussion and Conclusions

The methodology was developed on the basis of literature and the defined capabilities of

the Leica TCS SP2 confocal and Leica FLIM (Becker & Hickl SPC-730/830). In practice a

number of experimental challenges were faced through the project. The challenges resulted

in a large proportion of the project being spent identifying the reasons behind a number of

unexpected results. The project was conceived in good faith however after exhaustive and

prolonged testing it was found that subtle instrument design errors, that are not in

accordance with the expectation of the manufacturers defined specifications were the

source of these unexpected results.

Challenges encountered using the FLIM system centred around the filter block not

completely blocking excitation light, thereby allowing excitation light to interfere with the

emission spectrum of the samples. The tuneable multi-photon laser also offered its own

challenges. The workable range for this laser was decreased as the inefficient filter block

would allow excitation light through at the lower end of the laser wavelength range and it

would completely drop off after 965 nm rather than 990nm. The laser would also stop

pulsing during data acquisition for wavelengths under 820 nm. The FLIM set up only

allows for a section of the fingerprint to be imaged due to the lack of a suitable low

magnification objective. For single photon and multi-photon imaging this was easily

overcome in principle by performing a sequential tile scan that allows the software to

construct a montage image. However uneven illumination resulted in a loss of detail where

the separate image sections are joined. When experiments were performed using the lower

1.6 X magnification objective in the FLIM mode the detector performance is also

significantly affected, with the SYNC, CFD, TAC and ADC signals all decreased. The

57

decay curve data also included a cyclic interference for experiments performed at

wavelengths lower than 730 nm. This limitation disappeared when imaging was performed

using the 5X objective, suggesting that the 1.6 X objective was unsuitable for imaging in

the FLIM mode.

The core forensic questions addressed by the present study were whether (i) confocal

fluorescence microscopy equipped with FLIM is capable of imaging inherent latent

fingerprints and (ii) whether it could further enhance prints detected with conventional

optical, chemical and physical enhancement techniques.

For fingerprints deposited on all porous substrates without any chemical enhancement none

of the techniques used could detect any fluorescence. Auto fluorescence of inherent prints

is generally weak (2, 7). It was found that single photon microscopy produced comparable

image results (Fig. 4.1 and Fig. 4.3) to the conventional optical technique for high quality

ninhydrin treated fingerprint deposits on white paper and UBD paper. However for weaker

prints the best results were obtained with the conventional technique (Fig. 4.1). FLIM was

unable to suppress the background fluorescence sufficiently to reveal any fingerprint

luminescence.

Single photon and multi-photon microscopy showed highly contrasting images of

fingerprints deposited on dark red glossy card and treated with Greenwop fluorescent

powder where no defined ridges were visible with the conventional technique. The FLIM

technique could in addition to the spatial resolution, measure lifetime values for prints

treated with fluorescent powder. These results are consistent with results reported by Seah

et al (12) where they used magnetic fingerprint powders blitz-red and blitz-green to obtain

lifetime data.

58

For fingerprints deposited on non porous substrates both the single and multi-photon

CLSM and the conventional technique could detect auto fluorescence of fingerprint residue

on glass and foil substrates but not on the juice carton. The untreated glass samples showed

comparable results for single and multi-photon excitation however results obtained by

using the conventional technique were of lower contrast than those of the CLSM techniques

(Fig. 4.8). The opposite was true for fingerprints deposited on foil where the best results

were obtained by the conventional technique. FLIM was also unable to measure lifetime

data for untreated fingerprints on foil.

Foil samples treated with CA showed comparable results across all the techniques used

(Fig. 4.11). CA treated fingerprints on juice carton samples were not detected using the

conventional technique, however, they fluoresced slightly under single photon excitation.

When the CA samples were dusted with Redwop fluorescent powder the CLSM techniques

showed better results than the results obtained by using the conventional technique (Fig.

4.16).

For the red dark glossy card samples and juice carton samples when treated with

fluorescent powder and CA respectively, confocal microscopy equipped with FLIM

showed an advantage over the conventional technique that was unable to detect any

fingerprint details. However when comparing the costs of the inexpensive conventional

optical light microscope with that of the expensive CLSM system the above mentioned

advantages seems insignificant

59

5.1 Forensic Application Single photon and multi-photon CLSM techniques used in this project are too immature for

forensic application due to the various instrumentation limitations noted in the discussion.

Although the CLSM system used in this project gave higher contrasting images than the

conventional digital optical microscope it has no significant advantage for this forensic

application. The lack of suitable objectives for low magnification imaging with both the

single photon and multi-photon FLIM techniques results in partial image collection that has

to be processed to form an entire fingerprint image. The stitching artefact that occurs when

a montage is obtained could also be partly removed with processing however important

detail might be lost.

Latent fingerprint matching is already a challenging process for fingerprint experts. Partial

fingerprint matching even poses a greater challenge. “They do not know how likely it is

that two partial prints from different people might match and are expected to testify only to

absolute certainty”(1). Partial fingerprint matching is also highly dependent on image

quality and size (39). With these all being additional shortcomings of the techniques

evaluated here it is fair to say that they are premature for application in a forensic set-up.

5.2 Future Study directions With proper low magnification objectives a stable multi-photon laser and effective filter

block these techniques might be of future forensic use. Also with recent work performed on

orientation modelling of partial fingerprint identifications (39), partial imaging could be

useful in casework.

60

References 1. Thornton J. The General Assumption and Rationale of Forensic Identification. In:

Faigman D, Kaye D, Saks M, Sanders J, editors. Modern Scientific Evidence: The Law and

Science of Expert Testimony. St Paul: West Publishing Co; 1997.

2. Bramble SK, Creer KE, GuiQiang W, Sheard B. Ultraviolet Luminescence from

latent fingerprints. Forensic Science International. 1993;59:3-14.

3. Menzel ER. Recent advances in photoluminescence detection of fingerprints. The

Scientific world. 2001;1:498-509.

4. Wilkinson D. A one-step fluorescent detection method for lipid fingerprints;

Eu(TTA)3 2TOPO. Forensic Science International. 1999;99:5-23.

5. Lennard C, Patterson T. Latent fingerprints. [10 June 2007]; Available from:

http://www.policense.com/info/fingerprints.

6. Lennard C, editor. The detection and enhancement of latent fingerprints. 13th

Interpol Forensic Science Symposium; 2001.

7. Akiba N, Saitoh N, Kuroki K. Fluorescence spectra and images of latent

fingerprints excited with a tunable laser in ultraviolet region. Journal of Forensic Science.

2007;52(5):1103-6.

8. Raarup MK, Nyengaard JR. Quantative confocal laset scanning microscopy. Image

Anal Stereol. 2006;25:111-20.

9. Dalrymple BE, Duff JM, Menzel ER. Inherent fingerprint luminescence-Detection

by laser. Journal of Forensic Science. 1976;22:106-15.

10. Burt JA, Menzel ER. Laser detection of latent fingerprints: Difficult surfaces.

Forensic Science International. 1985;13(2):364-70.

61

11. Dinish US, Chao ZX, Seah LK, Singh A, Murukeshan VM. Formulation and

implementation of a phase-resolved fluorescence technique for latent-fingerprint imaging:

theoretical and experimental analysis. Journal of Applied Optics. 2005;44(3):297-304.

12. Seah LK, Dinish US, Ong SK, Chao Z, X, Murukeshan VM. Time-resolved

imaging of latent fingerprints with nanosecond resolution. Optics and Laser Technology.

2004;36:371-6.

13. Herod DW, Menzel ER. Laser Detection of latent Fingerprints: Ninhydrin. Journal

of Forensic Sciences. 1982;27(1):200-4.

14. Herod DW, Menzel ER. Laser detection of latent fingerprints: Ninhydrin followed

by zinc chloride. Journal of Forensic Sciences. 1982;27(3):513-8.

15. Menzel ER. Laser detection of latent fingerprints: treatment with phosphorescers.

Journal of Forensic Sciences. 1979;24:582-5.

16. Murdock RH, Menzel ER. A computer interfaced time resolved lumenscence

imaging system. Journal of Forensic Science. 1993;38:521-9.

17. O'neill EM. Fingerprints in criminal investigation. Journal of Criminal Law and

Criminology. 1940;30(6):929-40.

18. Ross MH, Reith EJ. Histology a text and atlas. New York: Harper & Row; 1985.

19. Kumaar P, Clark M. Clinical Medicine. Fourth Edition ed. London: W.B. Saunders;

1998.

20. Latent fingerprint composition [Training module]: Victoria Police; 2002. Available

from: http://www.nifs.com.au/F_S_A/Latent fingerprint composition.pdf.

21. Robinson S, Robinson A. Chemical composition of sweat. Physiological Reviews.

1954;34:2002-20.

62

22. Bramble S. Separation of latent fingermark residue by thin-layer chromatography.

Journal of forensic Sciences. 1995;40:969-75.

23. Nakkari T, Schreibman PH, Ahrens EH. In vivo studies of sterol and squalene

secretion by human skin. Journal of Lipid Research. 1974;15:563-72.

24. Collins K. Composition of palmar and forearm sweat. Journal of Applied

Physiology. 1961;17:99-102.

25. Croxton R, Baron M, Butler D, Kent T, Sears V. Development of a GC_MS

method for simultaneous analysis of latent fingerprint components. Journal of Forensic

Sciences. 2006;51:1329-33.

26. Denk W, Strickler JH, Webb WW. Two-photon laser scanning fluorescence

miroscopy. Science. 1990;248:73-6.

27. Goppert-Mayer M. Uber elementarakte mit zwei quantensprungern. Annalen der

physik. 1931;401(3).

28. Wolfgang B. The bh TCSPC Handbook. 2nd Edition ed. Germany2006.

29. Helmchen F, Denk W. Deep tissue two-photon microscopy. National Methods.

2005;2:932-40.

30. Konig K. Multiphoton microscopy in life science. Journal of Microscopy.

2000;200:83-104.

31. Konig K. High-resoluton multiphoton tomography of human skin with subcellular

spatial resolution and picosecond time resolution. Journal of Biomedical Optics.

2003;8:432-9.

32. Breusegem S, Levi M, Barry N. fluorescence correlation spectroscopy and

fluorescence lifetime imaging microscopy. Nephron Experimental Nephrology.

2006;103:41-9.

63

33. Chen J, Zhuo S, Luo T, Jiang X, Zhao F. Spectral characteristics of

autofluorescence and secon harmonic generation from ex vivo human skin induced by

femtosecond laser and visible lasers. Scanning. 2006;28:319-26.

34. Weigart R, Sramkova M, Parente L, Amornphimoltham P, Masedunskas A.

Intravital microscopy: a novel tool to study cell biology in living animals. Histochemistry

and Cell Biology. 2010;133:481-91.

35. Krishnan RV, Masuda A, Centonze VE, Herman B. Quantitative imaging of

protein-protein interactions by multiphoton fluorescence lifetime imaging microscopy using

a streak camera. Journal of Biomedical Optics. 2003;8:362-7.

36. Kobus HJ, Stoilovic M, Warrener RN. A simple luminescent post-ninhydrin

treatment for the improved visualisation of fingerprints on documents in cases where

ninhydrin alone gives poor results. Forensic Science International. 1983;22:161-70.

37. Wargacki S, Lewis L, Dadmun M. Understanding the chemistry of the development

of latent fingerprints by superglue fuming. Journal Forensic Sciences. 2007;52(5):1057-62.

38. Lee H, Gaensslen R, editors. Advances in Fingerprint Technology. Second edition

ed. London: CRC Press; 2001.

39. Wang Y, Hu J. Global ridge orientation modeling for partial fingerprint

identification. IEEE transactions on pattern analysis and machine intelligence.

2011;33(1):72-87.