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11…New and Developing Treatment Modalities: Two Photon Activation

11…New and Developing Treatment Modalities: Two Photon Activation

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M. K. Kuimova and D. Phillips

Of particular interest for us here is the utilisation of multiphoton processes

requiring high energy pulses. Such multiphoton processes have caused much

excitement in the last decade and offered novel solutions for biological and

medical applications, in particular, multiphoton imaging and two-photon excited

PDT (TPE PDT) [28, 29].

As discussed in earlier chapters, the absorption of a single photon of appropriate

wavelength excites the molecule from the ground state (S0) to the first excited

singlet state (S1), from which it can undergo a series of photochemical processes.

In PDT these photochemical reactions ultimately result in production of ROS and

in ensuing cell death. In simultaneous two photon excitation (TPE), near-infrared

light of twice the wavelength required for the S0-S1 transition can be used to

produce the excited state of the photosensitiser. The sensitiser is then deactivated

in the normal way by either luminescence (which may be utilised in imaging

applications) or by photophysical or photochemical processes to produce cytotoxic

species, which eradicate cells and tissue (as utilised in PDT).

A clear benefit of TPE PDT over conventional one photon PDT is that it

provides the means to excite chromophores in the near-infrared spectral region

(700–900 nm), enabling deeper penetration of useful light, due to minimised tissue

absorption and scattering in the tissue optical window (Fig. 9.2a). However the

major advantage of TPE PDT stems from the fact that biphotonic absorption

depends on the square of the light intensity, so it is confined to the focal volume of

the laser where the intensity is the highest. The latter factor yields considerably

better spatial resolution for TPE imaging, compared to monophotonic confocal

imaging, due to reduced out-of-focus blur. Likewise, TPE PDT has potential

advantages in the treatment of sensitive tissues such as found in the wet form of

age related macular degeneration (wet AMD) in the eye by reducing out-of-focus

damage to adjacent healthy tissue.

Naturally, for TPE PDT and imaging applications to be successful, photosensitisers must be created which combine both desirable biological and photophysical properties with high two-photon absorption cross-sections to enable the

efficient use of biphotonic excitation. In recent years several classes of efficient

TPE PDT sensitisers have been reported [30–33]. For example, it has been demonstrated that TPE PDT using a conjugated porphyrin dimer and 900 nm pulsed

excitation from a Ti–Sapphire laser (150 fs) can efficiently occlude a single blood

vessel in an animal model, avoiding any damage to surrounding blood vessels in a

3D sample [33].

9.11.2 Nanosurgery

Similarly to TPE PDT, intense laser light can be used in two-photon laser ablation

of tissues, termed nanosurgery. The major benefit of using femtosecond laser

pulses for nanosurgery is high peak intensities that reduce the energy threshold for

tissue removal (ablation) and enable laser ablation to proceed with a low-energy

9 Photomedicine


source. With this method, single axons inside the nematode Caenorhabditis elegans (C. elegans) were cut successfully by using near-infrared laser pulses with

relatively low pulse energies of 10–40 nJ at the specimen (200 fs pulses) [34].

In nanosurgery no specific photosensitiser is added to achieve the nanoscale

tissue removal. The minimal energy used is consistent with measured optical

breakdown thresholds in transparent materials. At these low energies, mechanical

effects due to plasma expansion and shock waves are also significantly reduced

with respect to other laser ablation techniques using nanosecond pulsed lasers, that

require much higher energies. Thus nanosurgery using femtosecond pulsed lasers

utilises multiphoton processes to evaporate very small volumes (1 lm3) of a tissue

with no heat accumulation and thermal damage to the environment. This for

example enables the surgeon to cut axons at the nano-scale resolution with minimal damage to the micro-environment and no damage to neighbouring axons [35].

9.12 Conclusions

In this Chapter we aimed to demonstrate that photomedicine is a vibrant and

actively developing branch of medicine that involves the study and practical

applications of light-initiated processes, with respect to health and disease. While

the early medical treatments involving light have been in use since the late

nineteenth century, the new concepts and modalities continue to emerge today.

With a significant amount of research conducted in theoretical and practical

aspects of light-induced medicine and adjoining fields today, from basic chemistry

of photosensitisation and biochemistry of cell death to optimisation of PDT procedures in clinical trials, we believe that the future of photomedicine is bright.

Acknowledgments The current work of MKK in the areas of biological imaging and photomedicine is supported by the UK’s Engineering and Physical Sciences Research Council (EPSRC) in the form of the Career Acceleration Fellowship (EP/E038980/1) and this support is

gratefully acknowledged.


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Chapter 10

Photochemistry in Medical Diagnostics

Huw D. Summers

Abstract Photochemicals play a leading role in the diagnosis of disease, being

widely used in assays of whole cells, of protein levels and in genetic analysis. This

chapter looks at a number of selected examples of photochemical diagnostics,

aiming to give an overview of the principles of their use through case studies of the

techniques most commonly employed within the clinical setting. These cover

blood based diagnostics, fluorescence assays using immunochemistry and the

photochemical analysis of DNA. The chapter concludes with a look to the future

and consideration of the role of novel nanocrystal photochemicals in the burgeoning field of Nanomedicine.

10.1 Introduction

The application of photochemicals to diagnose disease dates back to the nineteenth

century and the pioneering work of Sir George Stokes on the fluorescent properties

of quinine [1]; interestingly the first practical spectrofluorometer was developed in

the 1950s to monitor quinine in its role as an anti-malarial drug [2]. As the science

of fluorescence spectroscopy was developed in the early twentieth century by

people such as Alexander Jablon´ski, Theodor Förster and Gregorio Weber its

medical application correspondingly increased. Today fluorescence assays are

widely used throughout medicine to diagnose disease, to track the response to

treatment programs and to monitor the general health of patients. This

H. D. Summers (&)

Centre for Nanohealth, Swansea University, Singleton Park,

Swansea SA2 8PP, UK

e-mail: h.d.summers@swansea.ac.uk

R. C. Evans et al. (eds.), Applied Photochemistry,

DOI: 10.1007/978-90-481-3830-2_10,

Ó Springer Science+Business Media Dordrecht 2013



H. D. Summers

pervasiveness of photochemistry in the clinical arena is due to the unrivalled

sensitivity, accuracy and portability of optical sensors; characteristics that have

stemmed from the constant technological advances in the chemical compounds

and the light sources and detectors used in the detection of their fluorescence. In

many cases the performance of photo-techniques has meant that they have

replaced prior, well-established approaches e.g. in the switch from radiochemical

to photochemical assays.

The requirements for photochemical sensors in medicine is the same as in other

biological applications; in addition to the general requirement for high optical

efficiency and wide dynamic range the photo-active compound must be stable in

the biological environment, or behave in a predictable manner, and most importantly be capable of targeting to the tissue or cell of interest. An exhaustive review

of medical photochemistry is beyond the scope of this chapter which rather concentrates on a selection of case studies to provide an introduction by way of

examples. These reflect widely used techniques to be found in daily use in many

hospitals and give a glimpse of photochemistry being used to diagnose disease in

blood, tissue, cells and in chromosomes.

10.2 Blood Diagnostics

The analysis of patient blood is one of the most widely used diagnostics of health

through direct sample measurement. The monitoring of pH and the gas pressure of

O2 and CO2 is routinely used to assess patients during the course of a treatment

regime or following surgery [3, 4]. Blood pH indicates whether the patient is

acidotic or alkalemic, O2 and CO2 levels provide a measure of respiratory

capacity, whilst CO2 also reports on general metabolic processes. Standard procedure involves taking a sample of arterial blood from the patient and then sending

this to a central laboratory for analysis. This is inevitably time-consuming and

does not match the clinical time frame in which the levels of these blood markers

changes over minutes; there is therefore a pressing need for rapid and robust

quantitative measures that can be performed at the bedside or operating theatre [5].

The wide range of assays possible using photo-chemicals and the high sensitivity

of optical measurements make photonic approaches highly appropriate to these


10.2.1 pH and CO2 Measurements

One of the most widely used photo-chemicals for pH measurement is fluorescein

[6], which displays complex optical properties dependent upon its ionic form (See

Chap. 4, compound 4.3). In particular only the two anionic forms present at high

pH are fluorescent. Fluorescein can be used a wavelength ratiometric probe


Photochemistry in Medical Diagnostics


through its pH dependent absorption in the blue-green part of the spectrum

(absorption measurements at 450 and 495 nm are frequently used). One limitation

here is the relatively weak absorption and emission using 450 nm excitation and so

alternatives such as hydroxypyrene trisulfonate (HPTS) (see Fig. 12.1), which

displays strong pH dependent absorption and emission due to ionisation of

hydroxyl groups, have been developed [7].

CO2 is indirectly measured by sensing of the pH of a bicarbonate buffer solution

in equilibrium with the blood CO2, where the Henderson–Hasselbalch relationship

(see Eq. 12.9) allows calculation of CO2 from the pH value [8]. Optical sensing

methods for pH and CO2 are discussed in more detail in Chap. 12.

10.2.2 O2 Sensing

Collisional quenching of fluorescent probes by oxygen molecules is commonly

employed to measure oxygen concentration. This leads to decreases in fluorescence intensity and lifetime (see Chap. 12). To gain high sensitivity and hence

detection of low O2 concentrations (and low cost sensors) long lifetime probes are

required; metal ligand complexes (MLCs) such as ruthenium(II) tris(1,10-phenanthroline) with microsecond lifetimes are therefore commonly used [9, 10].

Many MLCs exhibit absorption maxima between 400–500 nm wavelengths and so

can be excited with inexpensive blue LEDs; the microsecond dynamics of their

fluorescence decay also makes it relatively straight forward and inexpensive to

measure decay lifetime using silicon photodetectors, either directly in the timedomain or via phase-based measurement using frequency dependent excitation

(see Chap. 14).

10.3 Immunoassays

Immunoassays are based on the body’s evolved system of antibodies for molecular

recognition. The mammalian immune system can evolve a virtually unlimited

number of antibodies to different antigen molecules; for photochemistry this

provides a readymade targeting technology, through which fluorescent probes can

be selectively attached to diseased cells or specific sub-cellular compartments

within cells. Immunofluorescence assays are therefore widely used in medical



H. D. Summers

10.3.1 Antibodies and Antigens

Antibodies are molecules, produced by the immune system, of very specific form.

They are produced by B cells in response to antigens (antigen : antibody

generator). Each antibody has a unique recognition element (antigen binding site)

that structurally matches its partner antigen, i.e. there is a three-dimensional shape

match between antibody and antigen akin to that between a lock and key [11, 12].

It is this highly selective identification and binding mechanism that makes antibodies ideal for diagnostic assays [13]. Extremely dilute molecules that are known

biomarkers of disease can be located and selected from the myriad background of

similar molecules through the use of their bespoke antibody. The antibodies are

generated by one of two methods:

1. Introduction of the target antigen into an animal system and subsequent harvesting from the blood serum, in this case polyclonal antibodies are generated—a number of different antibodies will be produced that recognise different

binding sites of the antigen.

2. Production of monoclonal antibodies from a single B-cell using cell culture

techniques—a single antibody type is produced with a single antigen binding

format [14]. This avoids the multi-specificity that arises from the use of

polyclonal antibodies.

Identification of recognition events is done through detection of emissions from

radiochemicals or photochemicals tagged to the antibody–antigen pair.

10.3.2 Development of Immunofluorescence

The first steps in the development of fluorescent antibody staining were made by

Coon et al. at the Harvard medical school in the early 1940s [15]. They initially

used anthracence isothiocyanate as a labelling fluorophore, however the pronounced spectral overlap of the anthracence emission (kmax * 400 nm) with

tissue autofluorescence led them to an alternative—fluorescein-isocyanate (FTIC),

which has an emission maximum at 520 nm. Coon was able to demonstrate that

when attached to Pneumococcus antibodies the fluorescein did not prevent antibody binding, that bright fluorescence could be seen in organ tissue sections from

mice infected with the bacterium and that the antibody labeling could differentiate

between different strains of the Pneumococcus. Immunofluorescent staining or

imaging of this sort is now routinely used in the detection and identification of

infectious diseases.

The use of immunolabelling as a quantitative assaying protocol dates back to

the late 1950s and the work of Yalow and Berson on the detection of blood

hormones [16]. Using radiochemical markers they developed a simple and effective assay based on the competitive binding of antibodies. Radioactive insulin was


Photochemistry in Medical Diagnostics


attached to insulin antibodies; these were then mixed with blood samples containing native insulin. As the blood borne insulin binds to antibodies it replaces the

radioactive species and this is monitored as a time dependent decay in total

radioactivity from the sample. This Yalow–Berson method is one of a range of

radioimmunoassay techniques that are extremely sensitive and can detect antigen

concentrations in the picomolar range. The idea that small molecules such as

insulin could stimulate antibody production was extremely controversial at the

time, Yalow and Berson’s original paper was rejected from the journal Science and

only accepted into the Journal of Clinical Investigation after the authors agreed to

substitute the words ‘insulin antibody’ for ‘insulin-binding antibody’ in the title

[17]. It is interesting to note that in 1977 Yalow was awarded the Nobel prize in

Medicine ‘for the development of radioimmunoassays of peptide hormones’.

Today the approach first outlined by Yalow and Berson has developed to

incorporate enzyme-activation of photochemicals allowing easier detection via

optical fluorescence [18–23]. Enzyme-linked immunosorbent assays (ELISA) or

enzyme immunoassays (EIA) use antibody binding to antigens and enzymes on a

substrate to create a surface on which target recognition followed by washing produces bound enzymes. Addition of a suitable enzyme substrate produces a fluorescent signal that is proportional to the amount of antibody–antigen binding. Thus

the antibodies recognise the molecule of interest and the enzymes amplify the readout signal through multiple binding of enzymes onto each antibody–antigen pair.

One of the most commonly employed enzyme-substrate pairs used in ELISA is

horseradish peroxidase and diaminobenzidine; oxidation of this benzidine derivative in the presence of hydrogen peroxide converts it to a fluorescent form [24].

10.3.3 Immunofluorescence Assay Types

Immunofluorescent assays can be implemented in a number of different ways and

are categorised by their structure of the protocol used. The main classifications are

depicted in Fig. 10.1.

In direct or primary assays the fluorescently labelled antigen binds directly to the

target antibody, providing one photochemical reporter per antibody. In the competitive or substitution form of this assay both labelled and unlabelled antigens

compete to bind to the antibodies; this is the basis of the Yalow–Berson method,

where antigen concentration is measured by its displacement of fluorescent reporter

antigens. In indirect or secondary immunoassays two antibodies are used; the primary antibody binds to the antigen to be detected, and a secondary, fluorescently

labelled antibody is then used to bind to a different molecular motif of the antigen.

This indirect technique brings two main advantages: (i) multiple fluorophores can be

attached to the secondary antibody to provide signal amplification and (ii) many

different primary antibodies can be developed whilst maintaining the same fluorescently labelled secondary antibody. This minimises the requirement for development of potentially expensive fluorescently labelled antibodies.


H. D. Summers

Direct or primary assay:

labelled antigen directly attaches

to primary antibody

Competitive or substitution assay:

unlabelled antigen replaces labelled

antigen (Yalow-Berson method)

Indirect or secondary assay:

antigen attaches to primary

antibody, secondary antibody with multiple reporter

labelling then attaches to

same antigen

Fig. 10.1 Immunofluorescent assay types

10.4 Gene Level Diagnostics

The use of stains to enhance image contrast in biological tissues and cells has been

common since the earliest days of microscopy when Antoni van Leeuwenhoek

(1632–1723) used saffron to dye muscle fibres. The first applications sought to

identify structures which are otherwise invisible as the bulk of cellular material is

transparent in the optical regime. The use of stains in medical diagnosis began in

the nineteenth century with Carl Weigert’s work on the identification of blood

disorders through dye absorption, Paul Ehrlich’s discovery of trypan red which

kills the protozoa responsible for sleeping sickness and the work of Hans Gram

whose bacterial staining techniques led to the classification of gram positive and

gram negative cells. Technological development has been a constant theme ever

since the first synthetic dye—mauveine, an aminobenzene with an intense purple

colour, was discovered by William Perkin in 1856. One strand of this development

has been the unravelling of the cell’s structure through the use of photochemical

compounds with first whole cells, then sub-cellular organelles, and finally chromosomes and individual genes being identified. In modern medicine diagnosis is

now commonly implemented at the genetic level through the staining of DNA

sequences within whole cells using fluorophores. This method attaches fluorophores to specific target sequences of DNA within chromosomes allowing identification of numerical and structural abnormalities, monitoring of the effects of

therapy, recognition of tumour cells and a host of pre-birth diagnoses from foetal

DNA, e.g. Down’s syndrome.


Photochemistry in Medical Diagnostics


10.4.1 Karyotyping

The complete set of human chromosomes in their condensed form, just prior to cell

division, is called the human karyotype. This can be visualised using staining

procedures developed over the past 30 years that use selective binding of fluorescent dyes to DNA that is rich in either adenine–thymine (A–T) or cytosine–

guanine (C–G) nucleotide pairs [25, 26]. The presence of the dye produces distinctive banding of the chromosomes which can be visualised under a microscope.

A–T areas are known as G-bands as they stain with Giemsa stain and G–C bonding

areas are correspondingly known as R-bands because they form the reverse of the

G-band pattern (see Fig. 10.2). Examination of these chromosome banding patterns provides a ready measure of chromosome number and fidelity and can be

used in diagnosis of diseases such as cancer, where polyploidy cells are common

(cells containing multiple chromosome copies), or genetic disorders where, the

banding patterns are altered due to DNA miscoding.

10.4.2 Fluorescence In Situ Hybridisation (FISH)

Whilst karyotyping provides a means to genetic disease diagnosis it is non-specific

and subjective, relying on expert assessment by someone ‘trained in the art’. To

obtain greater precision and reliability in situ DNA hybridisation techniques have

been developed that bind specific, fluorescently-labelled RNA sequences (riboprobes) to matching base sequences on chromosomes within cells [27–30]. A wide

variety of probes, matching specific sequences of DNA, are available, having been

developed as part of the Human Genome Project. Individual DNA sequences have

Fig. 10.2 Karyotype of a human male (reproduced with permission from the National Human

Genome Research Institute: http://www.genome.gov/)

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