Ultraviolet-induced fluorescence shedding new light on dental by bgw91912

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 Ultraviolet-induced fluorescence:
 shedding new light on dental
 biofilms and dental caries
 By Professor Laurence J. Walsh and Fardad Shakibaie




                             he ultraviolet (UV) spectrum is traditionally

                      T      divided into three UV bands: UVA (315-400
                             nm, also termed black light, long wave or
                      near UV), UVB (280-315 nm, also termed middle
                      UV - responsible for the sunburn response), and
                      UVC (200-280 nm, also termed short wave UV).1
                      Having the lowest photon energy of the three ultra-
                      violet wavebands, UVA has little effect on
                      microbial pathogens and virtually no effect on
                      human tissue with short-term exposures.
                         UVA is the important band in terms of diagnostic
                      applications in dentistry. The ability of UVA to
                      make biological materials fluoresce is well known         Figure 1. UVA light induces PP9 red fluorescence
                      in medicine and dentistry as well as in industry. For     which can then be imaged for diagnostic purposes.
                      example, UVA sources have application for identi-
                      fying materials such as dyes, inks, minerals,                Luminescence is a general term used to describe
                      chemical and various biological materials (such as        the emission of radiation, which incorporates both
                      blood, when used in forensic science). Light in the       fluorescence (short lived) and phosphorescence
  “The ability        visible violet and UVA wave bands can also initiate       (long lived), as well as other phenomena such as
    of UVA to         chemical reactions which contribute to the photo-         bioluminescence. Many naturally occurring sub-
make biological       polymerization of some dental composite                   stances fluoresce, including minerals, fungi,
                      materials, as well as adhesives, coatings, and resins     bacteria, keratin, collagens and other components
    materials
                      used in fields other than dentistry.                      of body tissue; this is termed primary fluorescence
  fluoresce is                                                                  or autofluorescence.
 well known in        A primer on fluorescence                                     Molecular fluorescence emissions persists only as
 medicine and         The process of fluorescence is of particular interest     long as the stimulating radiation is continued, unlike
dentistry as well     for diagnostic applications in dentistry as well as for   the process of phosphorescence, which persists as an
                      other areas of health care. Upon absorbing UVA            afterglow after the incoming exciting light has been
as in industry...”
                      light, certain molecules (fluorophores) become elec-      turned off. If light emission occurs within one mil-
                      tronically excited to high energy levels, and then        lionth of a second of light exposure, the luminescence
                      decay to lower energy levels by emitting radiation        is fluorescence, whereas if light emission takes longer
                      (emission or luminescence). Fluorescence occurs if        than this, the luminescence is phosphorescence.
                      the transition is between states of the same electron        In molecular fluorescence, the colour of the
                      spin and phosphorescence if the transition occurs         emitted light has a longer wavelength than the colour
                      between states of different spin. At low concentra-       of the exciting light. For example, when a molecule
                      tions, the emission intensity is linearly proportional    absorbs UVA light, the emissions are often in the vis-
                      to the concentration of the molecule present in the       ible spectrum. This relationship is known as Stokes’
                      target tissue. Because of this feature, molecular fluo-   law, named after Sir George Stokes, who published
                      rescence is very useful for quantification.               the first significant paper on fluorescence in 1852.


56 Australasian Dental Practice                                                                       November/December 2007
                                                                                                   the cutting | EDGE

    Fluorophores are excited by a range of
wavelengths, and also emit over a broad
range. Thus, for any fluorophore there will
be some overlap between its absorption
(excitation) spectrum and its emission
spectrum. The difference between the
absorption maximum and the emission
maximum is known as the Stokes’ shift.
    Light sources used for fluorescence must
produce light within the absorption region
of the fluorophore of interest, at sufficient
intensity. Until recently, the types of UVA
light sources used included nitrogen lasers,
                                                  Figure 2. Demonstration of calculus (left) and mature plaque deposits (right) because
helium cadmium lasers, high pressure mer-
                                                  of their PP9 fluorescence.
cury lamps, high pressure xenon lamps, and
metal-halide arc lamps. These light sources
are large, expensive, and have limited lifes-
pans. With advances in LED technology,
UVA emitting LEDs have now been devel-
oped. Common UVA wavelengths in
commercially available LEDs are: 375,
385, 395 and 405 nm. UVA LEDs are
small, long lasting and very reliable, by
comparison with other light sources. Exam-
ples of dual wavelength dental curing lights
with visible blue LEDs and UVA LEDs are
the Ultradent UltraLume 5™ and the GC G-
Light™. UVA LEDs have also been
incorporated into dental imaging systems
and examples of this include the Durr
Vistaproof™ and the Morita Penscope™. The
LEDs used in such devices are spectrally
broad and also produce some faint light in
the visible violet range (400-440 nm).            Figure 3. Visible spectrum (400-700 nm) showing the short wavelengths on the left and long
    To observe fluorescence, optical filters      wavelengths on the right. A wavelength of 405 nm is useful for exciting PP9 fluorescence.
are used which pass the fluorescence wave-
lengths but not the excitation wavelengths.       dental plaque and dental calculus respec-      coccus mutans, Enterococcus faecalis and
These filters can range from coloured glass       tively, when using UVA light sources. The      various lactobacilli are weaker or negative
or polymers (such as coloured spectacles)         fluorescence arises because of the pres-       for porphyrin fluorescence in the red
through to the more expensive interference        ence of porphyrin compound, particularly       spectral region.
filters made by depositing layer upon layer       protoporphyrin-IX (PP9), in bacteria. PP9         Thus, the maturity of dental plaque,
of dielectric materials onto a glass surface,     and similar porphyrin molecules are            rather than the presence of cariogenic
each of which has different refractive            derivatives of haemoglobin and are             streptococci, is the basis for the red fluo-
indices. Constructive and destructive inter-      involved in the biosynthetic pathway for       rescence which occurs with UVA light.
ference occurs with different wavelengths of      heme. Because PP9 is found in high             The emission of the red light from dental
light, causing some to be transmitted             amounts in Gram negative oral bacteria,        plaque corresponds with known emission
through and others reflected back (rejected).     and the levels of Gram negative bacteria       peaks of UVA-excited PP9 (Figure 1). As
For instruments which measure fluores-            increase as the dental plaque biofilm          well, the rapid decay of fluorescence once
cence, interference filters are typically used.   becomes more mature, red fluorescence is       the incoming UVA light is ceased con-
                                                  associated with mature dental plaque on        firms that the process is PP9 fluorescence
Plaque and calculus detection                     teeth as well as on appliances such as den-    rather than phosphorescence. PP9 is also
UVA light emits visible red fluorescence          tures. Laboratory studies have shown that      excited by visible red light (655 nm)
from deposits of mature dental plaque on          Actinomyces odontolyticus (found in den-       giving near-infrared emissions. This
the surface of teeth, restorations, or dental     tine carious lesions), Bacteroides             longer wavelength is used in the KaVo
appliances. This has been studied since           intermedius, Corynebacterium spp. and          DIAGNOdent™ system, which measures
the seminal work of Bommer and Bene-              Candida albicans all emit at 620-635 nm        fluorescence quantitatively.
dict in the mid-1920’s, who first reported        and 700 nm when excited by 407 nm UVA             In the mid-1920’s, Bommer detected an
the characteristic red fluorescence from          light, while the Gram positive Strepto-        orange and red fluorescence of dental


November/December 2007                                                                             Australasian Dental Practice 57
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                                                                       Figure 5. Clinical examples of the software in use. Top left, cari-
                                                                       ous molar; Top right; the same area with superimposed fluores-
Figure 4. Dürr Vistaproof system, showing camera (centre), shrouds     cence scores. Note the intensely stained area on the right hand
for the imaging head (upper left) and software (lower right).          side. Lower left, UVA induced green fluorescence of normal
                                                                       tooth structure, with red PP9 fluorescence from fissures; Lower
                                                                       right, software analysis of the same image.

plaque in patients using a UVA light           other dyes such as the red-maroon com-          Dental caries
source (Wood’s lamp). Today, this process      pounds rhodamine B and rhodamine                In white spot lesions, PP9 is trapped in
is fully understood at the molecular level.    123,9-10 giving visible yellow emissions.       small surface porosities and can be
In short, when dental plaque or calculus is                                                    detected because of its fluorescence prop-
present, there is an increase in the absorp-   Mineral loss                                    erties. A recent large scale clinical trial of
tion in the UVA spectral region at 350-420     and dysmineralization                           this using the DIAGNOdent (which has a
nm, with the appearance of a fluorescence      Prior to the first World War, Stubel in         detection limit of 1 picomole,12 showed
signal in the visible red spectral region at   1911 investigated the fluorescent charac-       detectable fluorescence in buccal white
590-650 nm.2-3 Using a polarizing element      teristics of various biological tissues         spot lesions on deciduous teeth.13
(to reduce reflection and scatter) and a       when irradiated by ultraviolet light and           Similarly, once the overlying plaque is
long pass orange-red filter, these emis-       found that teeth brilliantly fluoresce an       removed, dyes applied to the tooth surface
sions can be seen with the naked eye, and      intense blue-green colour. Later work           will be retained in areas of porous enamel
can also be recorded with CCD or CMOS          showed that the organic (protein) compo-        affected by early caries, assisting the iden-
sensors in imaging systems.                    nents of tooth structure were responsible       tification of these areas by fluorescence,
   Following professional prophylaxis,         for this, rather than the mineral compo-        e.g. using sodium fluorescein.
residual deposits of plaque and calculus       nents, with the amino acid tryptophan              UVA-induced fluorescence can detect
appear as red fluorescing areas.4 As well      attracting attention as a natural fluo-         more demineralized pre-cavitated enamel
as the obvious diagnostic benefits to clin-    rophore of sound dentine.                       areas (white spot lesions) than a conven-
ical operators, UVA-induced fluorescence          With excitation at 375 nm, emission          tional visual examination.14-16 A significant
can be used to educate patients and to         peaks for human enamel occur at 460             decrease in the intensity of the fluores-
assist in oral hygiene instruction, since it   (blue) and 560 nm (green). Because of           cence signal occurs in both demineralized
is not necessary to use disclosing dyes        this, areas of mineral loss are readily         teeth and in teeth with dentine carious
(Figures 2 and 3).                             apparent because of their reduced positive      lesions.17-19 For wavelengths from 400 to
   If one wishes to use a dye, UVA is a        signal. Thus, under ultraviolet light,          420 nm, carious lesions with cavitations
powerful inducer of bright yellow fluores-     enamel with white spot lesions is darker        in dentine containing bacteria showed
cence in sodium fluorescein or fluorescein     compared to the adjacent luminescent            emissions at 600-700 nm typical for por-
diacetate.5-7 These dyes can be applied        sound enamel. The same method will              phyrin compounds.19-20
topically in the mouth or to dental appli-     identify dysmineralization defects which
ances either intra- or extra-orally, and       occur during tooth formation, as well as        Clinical applications
following rinsing the location of the          dental fluorosis, which can have a texture      The Durr Vistaproof system uses six LEDs
retained dye ,can be used to assist in oral    and colour similar to those of initial caries   emitting at 405 nm and is the ideal wave-
hygiene education.5-8 UVA can excite           lesions but another shape and location.11       length in the UVA waveband for revealing


58 Australasian Dental Practice                                                                             November/December 2007
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 Table 1. UVA excitation and emission interactions                                                                     Table 2. Some dental
                                                                                                                       applications of UVA light
                                       Excitation            Emission                 Colour of emission
 Sound tooth structure                 337 nm                430-450 nm               Light blue                       • Detect mineral loss (white spot
                                                             480-500 nm               Aqua blue                          caries, dental erosion);
                                       375 nm                460 nm                   Light blue                       • Detect dysmineralization (develop-
                                                             560 nm                   Light green                        mental lesions, fluorosis);

 Bacteria (mature plaque, 320-380 nm                         590-650 nm               Red                              • Detect carious lesions which involve
 dental caries)           407 nm                             635 nm                   Red                                the DEJ or dentine;
                          360-580 nm                         600-700 nm               Red                              • Check caries removal during
                          655 nm                             720-800 nm               (Infrared)                         excavation;

                                                                                                                       • Demonstrate plaque and calculus
 Supra and                             420 nm                595 nm                   Red
                                                                                                                         during patient education;
 subgingival calculus                                        635 nm                   Red
                                                             650 nm                   Red                              • Visualize plaque and calculus
                                                             695 nm                   Deep Red                           remaining after debridement; and

                                                                                                                       • Reveal bound or trapped marker
 Protoporphyrin IX                     407 nm                590 nm                   Red
                                                                                                                         dyes (porosity/leakage).
                                                             620 nm                   Red
                                                             635 nm                   Red
                                                                                                                      14. Hafstrom-Bjorkman U, Sundstrom F, ten Bosch JJ.
                                                                                                                      Fluorescence in dissolved fractions of human enamel.
 Sodium fluorescein                    400-465 nm            520-530 nm               Green                           Acta Odontol Scand. 1991;49(3):133-8.
                                                                                                                      15. Angmar-Mansson B, al-Khateeb S, Tranaeus S.
                                                                                                                      Monitoring the caries process. Optical methods for
PP9 fluorescence. The image sensor is a                   4. Coulthwaite L, Pretty IA, Smith PW, Higham SM,
                                                                                                                      clinical diagnosis and quantification of enamel caries.
6mm CCD, and the camera optics are                        Verran J. The microbiological origin of fluorescence
                                                                                                                      Eur J Oral Sci. 1996;104 (4 ( Pt 2)):480-5.
                                                          observed in plaque on dentures during QLF analysis.
shrouded to excluded ambient light when                   Caries Res. 2006;40(2):112-6.
                                                                                                                      16. Stookey GK.Quantitative light fluorescence: a
examining a tooth, thus increasing the                                                                                technology for early monitoring of the caries process.
                                                          5. Lang NP, Ostergaard E, Loe H. A fluorescent plaque
                                                                                                                      Dent Clin North Am. 2005 Oct;49(4):753-70.
signal to noise ratio. The shroud also acts               disclosing agent. J Periodontal Res. 1972;7(1):59-67.
                                                                                                                      17. Sundstrom F, Fredriksson K, Montan S, Hafstrom-
as a spacer, giving a constant distance                   6. Gillings BR. Recent developments in dental plaque
                                                                                                                      Bjorkman U, Strom J. Laser-induced fluorescence
                                                          disclosants. Aust Dent J. 1977; 22(4):260-6.
(Figure 4). Captured images from the                      7. Gelskey S, Brecx M, Netuschil L, MacDonald L,
                                                                                                                      from sound and carious tooth substance: spectroscopic
camera unit are ported back to a computer                 Brownstone E, Stoddart M. Vital fluorescence: a new
                                                                                                                      studies. Swed Dent J. 1985;9(2):71-80.
via a USB 2.0 interface for analysis                                                                                  18. Borisova EG, Uzunov TT, Avramov LA. Early dif-
                                                          measure of periodontal treatment effect. J Can Dent
                                                                                                                      ferentiation between caries and tooth demineralization
(Figure 5). The camera is powered from its                Assoc. 1993; 59(7):615-8.
                                                                                                                      using laser-induced autofluorescence spectroscopy.
own source, not via the USB interface,                            CPD POINTS AVAILABLE                                Lasers Surg Med. 2004;34(3):249-53.
thereby making it suitable for use with                       Continuing Education credits are                        19. Subhash N, Thomas SS, Mallia RJ, Jose M. Tooth
laptop computers as well as desktops. The                                                                             caries detection by curve fitting of laser-induced fluo-
                                                           available on this article for subscribers                  rescence emission: a comparative evaluation with
DBSWIN analysis software highlights the                      by answering the questionnaire at                        reflectance spectroscopy Lasers Surg Med.
intensity of the red signal from PP9, pro-                       www.dentalpractice.com.au                            2005;37(4):320-8.
viding a numerical score that can assist                  8. Sagel PA, Lapujade PG, Miller JM, Sunberg RJ.
                                                                                                                      20. Buchalla W. Comparative fluorescence spec-
treatment decisions, in much the same way                 Objective quantification of plaque using digital image
                                                                                                                      troscopy shows differences in non-cavitated enamel
as has been done with QLF (Inspektor™)                                                                                lesions. Caries Res. 2005;39(2):150-6.
                                                          analysis. Monogr Oral Sci. 2000;17:130-43.
and DIAGNOdent. Toggling between view                     9. Hart SJ, JiJi RD. Light emitting diode excitation
and analysis modes, and freezing images is                emission matrix fluorescence spectroscopy. Analyst.         About the author
                                                          2002; 127(12):1693-9.                                       Professor Laurence J. Walsh is the tech-
a simple task using a footswitch.                         10. Kuo JS, Kuyper CL, Allen PB, Fiorini GS, Chiu
                                                          DT. High-power blue/UV light-emitting diodes as
                                                                                                                      nology editor of Australasian Dental
                                                          excitation sources for sensitive detection. Elec-           Practice magazine. He is also a noted
References                                                trophoresis. 2004; 25(21-22):3796-804.                      commentator on and user of new technolo-
1. Murphy GF, Walsh LJ, Kaidbey K, Lavker RM.             11. Angmar-Mansson B, de Josselin de Jong E, Sund-          gies and is the Head of The University of
Mechanism(s) of dermal endothelial activation             strom F, ten Bosch JJ. Strategies for improving the
                                                                                                                      Queensland School of Dentistry.
elicited by specific regions of the electromagnetic       assessment of dental fluorosis: focus on optical tech-
spectrum. Clin Res. 1991; 39:195.                         niques. Adv Dent Res. 1994;8(1):75-9.
2. Borisova E, Uzunov T, Avramov L. Laser-induced         12. Hibst R, Paulus R, Lussi A. Detection of occlusal       Dr Shakibaie is a PhD student in the UQ
autofluorescence study of caries model in vitro. Lasers   caries by laser fluorescence: Basic and clinical investi-   School of Dentistry working in the field of
Med Sci. 2006;21(1):34-41.                                gations. Med Laser Appl. 2001; 16(3):205-13.                biophotonics. A dental graduate of the Uni-
3. Kühnisch J, Heinrich-Weltzien R, Tranæus S,            13. Walsh LJ, Groeneveld G, Hoppe V, Keles F, van
Angmar-Månsson B, Stößer L. Confounding factors in        Uum W, Clifford H. Longitudinal assessment of
                                                                                                                      versity of Queensland, he completed his
clinical studies using QLF. Int Poster J Dent Oral Med.   changes in enamel mineral in vivo using laser fluores-      MPhil in 2006 working on laser-induced
2003, 5(2): 177.                                          cence. Aust Dent J. 2006; 51(4): S26.                       fluorescence for diagnostic applications


60 Australasian Dental Practice                                                                                                      November/December 2007

								
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