Inpektor announces QLF-Vision™

Quantitative Light-induced Fluorescence has become a household word in caries research through the work of Inspektor and the many researchers who have used our systems to explore, develop and validate the tremendous possibilities. As QLF™ gained recognition as the method to detect early lesions and bacterial activity using fluorescence images, fluorescence began to be applied in a variety of ways and the descriptive term QLF does not offer enough discriminating power to distinguish between them.

To identify our technology from other fluorescence-based methods, Inspektor has decided to use the more specific term QLF-Vision™ (first used in 1993) whenever ambiguity may arise. The name was chosen to reflect the fact that Inspektor's use of QLF is based on visualization of the fluorescence of dental surfaces and applies its analysis to the fluorescence images of these dental surfaces. Although completely irrelevant, we also kind of like the visionary association that emanates from the term. In our publications we will from now on use the term QLF-Vision when referring to the use of QLF that we are developing and we will urge everyone who uses this technology to do the same.

QLF-Vision is the best technology that is currently available for quantitative longitudinal monitoring of early lesions and bacterial activity in situ, in vitro and in vivo. The technology is by now even developed so far, that clinical application becomes a definite possibility (see Clinical applications of QLF-Vision and the Focus on: column).

This newsletter tries to inform the reader about the application of QLF-Vision in research as well as in the clinical field and hopes to give some interesting links to information about the technology made available by universities and companies. In this way we hope to share our unwavering belief in the possibilities of the technology in a fair and informative way. In the case the enthusiasm proves to be contagious, we are even willing to help you to get firsthand experience with QLF-Vision: QLF-Vision is available right now!

Roswitha Heinrich-Weltzien and Jan Kühnisch at the ORCA in Graz

Dr. Roswitha Heinrich-Weltzien and Dr. Jan Kühnisch, from the department of Preventive Dentistry, University of Jena in Erfurt, Germany, have published their clinical experiences with QLF-Vision in an article in Die Quintessenz (Quintessenz 53, 2, 131-141 (2002)) titled: Quantitative-Light-induced Fluorescence measurement - a future method for the dentist? (Note that this article was written before the term QLF-Vision was put forward).

The authors provide many clinical results of QLF-Vision. Their conclusion is that QLF-Vision has high potential for the clinical practice. They also stress the high impact of the results of QLF-Vision on patients

The article is in German and an official English version is in preparation for publication. In the meantime, a short description of the article is given below, featuring some of the very beautiful clinical pictures that the article features.

The article gives a lucid explanation of the fundamentals of fluorescence in dental tissue and the history that led to the development of QLF-Vision. They describe the QLF-Vision hard- and software and present the way QLF-Vision was integrated in their clinic. Then they move on to demonstrate the added value that QLF-Vision brings to the clinical assessment of oral health.

Fig. 1a and 1b. The clinical image of the buccal surfaces of permanent upper front teeth and a montage of the same teeth, from separate images made with QLF-Vision. Tooth 12 has a metal porcelain crown that does not fluoresce and is shown in black. Clearly the restoration with a fluorescing composite material can be seen in tooth 21..

One of the nice features of their article is that Jan and Roswitha provide white light images of the same subjects that were looked at with QLF-Vision (fig. 1a and b). This helps to realize how different the QLF-Vision images may be from the normal clinical view. The impact of this approach is heightened by their use of montage of QLF-Vision images to resemble the clinical situation.

The article provides examples of the differences between white light images and their QLF-Vision counterparts for flat surfaces (fig 2), occlusal surfaces and even for special cases such as a hypoplastic occlusal surface (fig. 3) and Amelogenesis Imperfecta Hereditaria (fig. 4).

Fig 2 Clinical image and fluorescence image (montage) of circular initial carious lesions to the milk front teeth. Whilst teeth 52 and 61 have already been restored with fillings, the mesial surface of tooth 51 shows an active initial caries, which is characterized by the red fluorescence of the attached plaque.	Fig 3 Clinical image and fluorescence image of the hypoplastic occlusal surface of a lower premolar. The hypoplasy is related etiologically to frequent use of antibiotics for treating chronic middle ear afflictions between the age of 3 and 5. Although the changes in the dentine structure are not determined by caries, the fluorescence image is similar to that of a carious lesion.
Fig. 4a Clinical situation of a patient with an Amelogenesis imperfecta hereditaria. Whilst the upper middle front teeth have already been treated with transparent composite fillings and the primary molars with steel crowns, the lower front teeth have not been treated yet.	Fig 4b On the basis of the intact dentine structure, the fluorescence of the lower front teeth remains largely complete; simply in the amber-coloured areas the fluorescence has reduced due to increased light absorption.

The article also covers the assessment of sealants (fig. 5) and secondary caries (fig. 6).

Fig. 5 Surplus applied sealing material with imperfections at the edge and a central fracture. The red fluorescence at the edge of the cover caused by plaque bacteria is indicative of a poor adhesive bond.	Fig. 6 Amalgam filling with imperfections at the edge on a QLF image.

Each of these examples are provided with a clear and thorough explanation of the observations made and the conclusions drawn, giving a fair impression about the extent of the usefulness of QLF-Vision in clinical practice as well as the limits thereof.

The article concludes with many recommendations to improve the use of QLF-Vision in clinical practice (creating tons of work for our developers as a side effect). Some of these suggestions (such as the automated support for repositioning, the reduction of the influence of ambient light, easier and less subjective analysis, easier and more flexible acquisitions software) have already been addressed today or are being addressed right now (see also QLF-Vision field test in the USA elsewhere in this article). Others, such as the inability to detect approximal caries remain unresolved.

For those who are interested in the Quintessenz article, a provisional English translation was made which is available from Inspektor on request.

For more information on the use of QLF-Vision for clinical purposes, we strongly recommend the have a look at the web-site of Iain Pretty which shows the work of the Liverpool Cariology Group of the University of Liverpool.

All pictures were taken from the article "Die quantitative-lichtinduzierte Fluoreszenzmessung - eine zukünftige Methode für den Zahnartzt" in Die Quintessenz, 53, 2, 131-141 (2002) and are reproduced here with permission of the publisher.

QLF-InVitro demonstration study

	Monique van der Veen

To demonstrate the usability of the QLF-InVitro system, a simple de-remin study was performed by Monique van der Veen (ACTA-CEP, Inspektor Research Systems bv). Eight specimens of human and bovine enamel were subjected to a demineralization challenge and then immersed in a remineralization fluid. QLF-InVitro was used to quantify the levels of de- and remineralization.
Below follows a shortened version of the article, with some of the results and without the references.

The full article including all data and literature references is available from Inspektor on request.

Aim
The aim of the study was to demonstrate and evaluate the use of QLF-InVitro in a relatively straightforward de- and remineralization experiment and to get an estimate of the overhead generated by the acquisition and analysis as performed with QLF-InVitro.

QLF-InVitro

Fig. 1 QLF-InVitro stage (Click on picture)

	Fig. 2 QLF-System box (Click on picture)

The QLF-InVitro system was specifically designed to apply QLF-Vision as a nondestructive method for longitudinal monitoring of fluorescence loss in specimens of dental tissue that are routinely used in in vitro and in situ experiments in caries research.
QLF-InVitro consists of a measurement stage (fig. 1) and a system box (fig. 2) and is connected to a PC that runs special acquisition and analysis software (fig. 3). The stage comes with a dark cover to eliminate the influence of ambient light.

The video signal generated by the video electronics can be connected to the PC through a special video interface card (frame grabber) or, through an analogue-to-digital-video converter, to a IEEE 1394 (firewire or I-link) port. The latter option is especially useful when using laptops that do not support the insertion of PCI boards.
(For a more extensive description of the QLF-InVitro system, the reader is referred to the full article and the May 2001 Newsletter).

Means and methods
Four Ø 4mm bovine enamel discs drilled from the labial surface of incisors and four extracted human premolars were imbedded in acrylic resin after which the enamel surface was ground flat and polished
Before the de- and remin stages, all eight surfaces were partially covered with acid resistant, non-fluorescing, nail varnish (approximately 50% of the surface), leaving the other half exposed to environmental influences.

	Fig. 3 QLF-InVitro analysis screen showing the lesion (blue circle) and the reference areas (magenta rectangle). Click on the picture to enlarge.

All surfaces were measured with the QLF-InVitro system 4 times: at baseline, after the demineralization

challenge and twice during remineralization. Before measurement, all surfaces were rinsed clean, applied varnish was removed with acetone and the specimens were then rehydrated in in de-ionized water for 24 hr prior to the measurement.

During measurement, the samples were kept wet until their repositioning in the QLF-InVitro was sufficiently correct, as determined by the video repositioning software of QLF-InVitro (VidRep). Then, just prior to capturing the fluorescence image, the surfaces were gently wiped with a tissue to dispense of the surplus water on the surface.

When all measurements were made, for each of the surfaces two analysis areas were defined, one in the exposed and one in the protected area, referred to as lesion and Reference areas respectively, see fig. 3. The reference areas are used to correct for the possible differences in lighting conditions between different measurements.

Results
Fig. 4 shows the 4 QLF-InVitro images of two of the eight samples (1 human premolar and 1 bovine enamel specimen). All show clear signs of first de- and then remineralization with respect to baseline as was to be expected.

Fig. 3 The series of QLF-InVitro images made for 2 different specimens: a human premolar (top) and bovine enamel (bottom)

The graphs in fig. 5a and 5b show the fluorescence loss calculated by the analysis software each moment in time for the two specimens with and without correction based on the reference areas. In the graph of fig. 5c, the average behavior of the fluorescence intensity over the reference patch is shown (as the normalized difference in fluorescence intensity over the reference area with respect to the baseline image, averaged over all specimens). Interestingly enough, the fluorescence intensity over the reference areas shows the same behavior pattern as the lesion areas.

Fig. 5a Fluorescence loss (ΔF) in specimen 1 (human premolar).	Fig. 5b Fluorescence loss (ΔF) in specimen 7 (bovine enamel).	Fig. 5c Normalized change in fluorescence averaged over all 8 reference areas.
All graphs show ΔF with and without correction. Click on the images to see an enlarged picture of each graph.

Discussion
The fluorescence images and the analysis results all show the expected demineralization followed by remineralization. This is consistent with earlier research that established a good correlation between changes in mineralization and fluorescence loss.

Two explanations were postulated to explain the behavior of the fluorescence intensity of the reference areas:

1. The acid resistant varnish does not completely protect the covered surface and some de- and remineralization takes place even with the varnish in place.
2. Part of the fluorescence intensity measured over the reference area originates in the demineralized part of the tissue. This contribution naturally follows the behavior of the lesion areas.

Further research is needed to establish if these effects play a role and, in the case they do, to what degree they explain the observed effect.

Care must be taken to treat the specimens consistently, especially with respect to the humidity of the specimens as the fluorescence intensity varies significantly when the humidity of the specimens changes. The procedure followed in this demonstration study has proven to be easy to implement and effective in reducing effects of drying on the analysis results.

The average time needed for capturing the images did not exceed 10 minutes for all 8 specimens combined.
The analysis stage took less than half an hour and consisted mainly of the positioning of the two analysis areas to all specimens after they were defined for the first one and then set to default. (Re-)calculating and exporting the analysis data was typically a matter of seconds.

In comparison with other techniques such as Microradiography, Histology, Polarized-light microscopy and Microhardness measurements, QLF-InVitro is nondestructive and much faster and easier.
With respect to the DiagnoDent, another implementation of quantitative-light induced fluorescence that shares the pinpoint nature of microhardness measurements, QLF-Vision provides the advantage of a visual, easily reproducible analysis of the whole surface of the specimens.

Because the analysis is based on images stored on the PC, any area that turns out to become interesting can be defined and analyzed for all moments in time, even if the area was not considered important at earlier stages. This makes the use of QLF-Vision very flexible and increases the amount of data that can be extracted from the measurements.

Conclusions
The lesions created in this simple de/remin model turn out pretty similar as seen with QLF-Vision, which is to be expected.
Work done by Guggenheim and others indicate that the standard deviation in the measurement of ΔF with the QLF-InVitro system is in the order of 1-2%.
The results show clearly that the de- and remineralization process could easily have been monitored with much higher frequency than was actually done, providing much more information about the actual process in time. Considering the very small overhead of the measurement process and the the fact that it is nondestructive, QLF-InVitro enables much closer longitudinal monitoring of de- and remineralization processes without the time-penalty and the need for extra tooth specimens. None of the other systems provide the flexibility of the QLF-InVitro systems where analysis can be done retrospectively on any part of the captured specimen surface.

Compared to other available technologies, QLF-InVitro, provided proper care is taken with the treatment of the specimens, turns out to be easier, faster and more flexible than anything else currently available.

From Jan 6 - 10, 2002, the "International Consensus Workshop on Caries Clinical Trials" was held in Loch Lomond, Scotland. The workshop was the answer to a growing need of industry, government and science to reassess the traditional way in which clinical trials are used to demonstrate anti-caries efficacy of oral care products. The basic attitude of the workshop was reflected in its motto: "Agreeing where the evidence leads us".

As a result of the conference, a final consensus statement was issued on April 19. One of the most interesting conclusions concern the way in which the anti-caries efficacy of oral care products is to be measured. Rather than putting emphasis on the detection of frank caries and cavities, the consensus statement stresses the importance of early de- and remineralization. According to the statement, clinical trials that only record cavitated lesions as outcome measure are to be considered "outmoded".
Instead "New assessment techniques should, in the clinical context, be capable of measuring a wide range of lesion depths and changes in degree of mineralization, either directly or indirectly. Ideally a continuous scale should be used."
De/remin is proposed as the key outcome variable: "Measures of mineral density change are not surrogate outcomes but rather are primary indicators of the cumulative status of the dental caries lesion."

The consensus statement also warns that the we should not throw away existing knowledge:
"While there are many other ways in which the design of CCTs (Caries Clinical Trials) might be improved further, through better diagnostic, design and analytical techniques, it is paramount that the overriding principle behind CCT design validation must be that the results and conclusions from any new design are in line with those shown previously by "conventional CCTs".

However, it also recognizes that this may be asking too much. For example, the current gold-standard for CCTs uses de/remin detection that is orders of magnitude less sensitive than new methods. Stookey et al. showed results from a clinical trial where they compared ΔQ as measured with QLF-Vision with the DMFS score. Although QLF-Vision correlated with DMFS very well, for the first three months QLF-Vision showed significant demineralization which the DMFS score did not. Should we now discard QLF-Vision because its results do not match those of the traditional method? Of course not. DMFS is simply not equipped to measure remineralization so there is no way to to use it to evaluate the QLF-Vision result for the first three months. As the QLF-Vision data are consistent with the DMFS scores on the long term and because the standard error of the QLF-Vision data is much lower than those of the DMFS scores, many just take the QLF-Vision results at face value and try to formulate hypotheses that may explain them.

The consensus statement admits the problems that arise when trying to replace an existing gold standard with a new, more sensitive one: "Given the deficiency of current "gold standards", and the challenges of achieving appropriate validation, new reference standards and validation protocols should be developed". Unhappily it does not elaborate.

The consensus statement also states that new diagnostic methods like QLF-Vision should be further developed. Here also, no indication is given how this is to be achieved.

Whatever its limitations, we consider the consensus statement a huge step forward. It confirms the concepts that underlie the development of QLF-Vision with its emphasis on the need for technology that monitors (early) de- and remineralization in dental tissue longitudinally on a continuous scale.

We also think that the enormous effort should be recognized that was exerted by all concerned to arrive at consensus which certainly provides a solid basis for the future. It may well be that this effort and the goodwill that was created by it among the many attendants, even if they did not agree with each other, may well be the most lasting result of this workshop.

	Wolfgang Buchalla and Aine Lennon

Iain Pretty

QLF was again well represented at the ORCA. There was an intriguing study done by Iain Pretty (again, check out his website) using QLF-Vision to study the effects of fluoridated restoration materials. Aine Lennon presented FACE, her brilliant concept that integrates the use of fluorescence with the restoration process which, as she showed, indeed increased the quality of excavations. Wolfgang Buchalla did a neat fundamental job that (among other things) confirmed that the wavelength used by our QLF-Vision systems to excite fluorescence is indeed optimal.

Renske Thomas (left) with Marie-Charlotte Huysmans

Many others (Sue Higham, Monique van der Veen and Renske Thomas among them) tackled the tricky problem of the red fluorescence with varying success. Raymund Hibst showed the results of a very ingenious experiment that aimed to establish the effect of lesion depth on the ability of the DIAGNOdent to detect subsurface caries. His most striking conclusion was that in order to improve the measurement, ideally the total fluorescence over the surface should be taken into account instead of the small surface covered by the tip of the DIAGNOdent.

	Ingo Häberlein

While in the corridors the soft grumbling of many a seasoned hand could be heard about 'the lack of quality' this year (and the last, and the one before that), Ingo Häberlein presented examples of a new (white) impression material that will color dark blue in places where it detects lactic acid. According to Häberlein, the detection is incredibly sensitive and will in his opinion be able to indicate all the sites in the mouth that are 'under attack'. This would mean that, after QLF-Vision has brought the detection of early de- and remineralization to the clinic, the probable cause of this process could now be located in vivo. As a consequence, a whole new range of caries-related detection systems will soon become available to clinicians. We estimate that this will have a profound impact on clinical practice and on the ORCA.

Heinz Duschner

When presenting the German city of Konstanz, host of the first and the 50-th ORCA next year, Heinz Duschner recalled that ORCA was originally founded by clinicians. In those early days, clinicians formed the bulk of the attendees. Now, almost 50 years later, scientists dominate the conference and this is not too surprising as the fundamentals that govern the caries process often need expertise that is not at the reach of every clinician. However, we feel that these years of fundamental 'plodding and prodding' are beginning to bear fruit in the form of tools such as the lactic acid detector and QLF-Vision. These tools will open up many new avenues for in vivo, clinical research on the caries process, involving once again, practicing clinicians. We will therefore not be surprised if the next 50 years of ORCA (correct pronunciation: orrrr-kà, according to Karin Sjögren) will see the pendulum swing slowly back with a rise in the contribution and attendance of clinicians. In our view, this would be another proof of the vitality that ORCA always has aspired to support.

Products that use QLF-Vision™ for detection and monitoring early lesions and bacterial activity are available from Inspektor.
New is the availability of a digital video connection (IEEE 1394) which dispenses with the need of a framegrabber and allows them to be used with notebooks and laptops (if equipped with an IEEE 1394 port).

For fundamental research with in vitro and in situ models, QLF-InVitro™ is the system of choice. For information on prices and the technology click here.

For in vivo research and clinical applications of QLF-Vision we provide QLF™\CLIN. For information about availability, prices, accessories and the technology click here.