Pulsed light imaging for wide-field dosimetry of photodynamic therapy in the skin

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1 Pulsed light imaging for wide-field dosimetry of photodynamic therapy in the skin Scott C. Davis 1, Kristian Sexton 1, Michael Shane Chapman 2, Edward Maytin 3, Tayyaba Hasan 4, and Brian W. Pogue 1 1 Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA scott.c.davis@dartmouth.edu, brian.w.pogue@dartmouth.edu 2 Department of Dermatology, Dartmouth Hitchcock Medical Center, Lebanon, NH, USA 3 Biomedical Engineering, Cleveland Clinic, Cleveland, OH, USA 4 Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA ABSTRACT Photodynamic therapy using aminoluvelinic acid (ALA) is an FDA-approved treatment for actinic keratoses, pre-cancerous skin lesions which pose a significant risk for immunocompromised individuals, such as organ transplant recipients. While PDT is generally effective, response rates vary, largely due to variations in the accumulation of the photosensitizer protoporphyrin IX (PpIX) after ALA application. The ability to quantify PpIX production before treatment could facilitate the use of additional interventions to improve outcomes. While many groups have demonstrated the ability to image PpIX in the clinic, these systems generally require darkening the room lights during imaging, which is unpopular with clinicians. We have developed a novel wide-field imaging system based on pulsed excitation and gated acquisition to image photosensitizer activity in the skin. The tissue is illuminated using four pulsed LED s to excite PpIX, and the remitted light acquired with a synchronized ICCD. This approach facilitates real-time background subtraction of ambient light, precluding the need to darken the exam room. Delivering light in short bursts also allows the use of elevated excitation intensity while remaining under the maximum permissible exposure limits, making the modality more sensitive to photosensitizer fluorescence than standard approaches. Images of tissue phantoms indicate system sensitivity down to 250nM PpIX and images of animals demonstrate detection of PpIX fluorescence in vivo under normal room light conditions. Keywords: Photodynamic therapy, dosimetry, fluorescence imaging, protoporphyrin IX, skin cancer 1. INTRODUCTION Actinic Keratoses (AK) are pre-cancerous skin lesions which are non-life-threatening for the general population but pose a significant risk for organ transplant recipients (OTR) undergoing immunosuppressant therapy. These patients can suffer widespread and recurrent AK leading to aggressive malignant squamous cell carcinoma at alarming rates [1-3]. While there are several treatment modalities available for AK, ALA-based photodynamic therapy (PDT) is particularly attractive for OTR s since it can be applied to wide areas of the skin and repeated frequently; however, response rates vary widely, and a Optical Methods for Tumor Treatment and Detection: Mechanisms and Techniques in Photodynamic Therapy XXIII, edited by David H. Kessel, Tayyaba Hasan, Proc. of SPIE Vol. 8931, 89310W 2014 SPIE CCC code: /14/$18 doi: / Proc. of SPIE Vol W-1

2 sizeable percentage of lesions have no response. This is predominantly due to lack of photosensitizer accumulation in the lesions, which mitigates the effect of the light dose; however, there are currently no clinical tools to determine drug uptake before light delivery to facilitate patient-specific treatment parameters. Point-probe measurements provide exceptional sensitivity to photosensitizer concentration and tissue optical properties; however, are inherently limited in the tissue volume sampled. Because of this, and of the spatial variability in PpIX production, even within lesions, we and others have observed relatively poor reproducibility metrics using probe systems for AK assessment. Wide-field imaging approaches, while often less sensitive, reveal the heterogeneity in the spatial distribution of fluorescence. Imaging systems with varying levels of sophistication [4-10] have been used in many studies of PDT of the skin; however, one of the primary challenges in imaging fluorescence in the clinic is addressing the contaminating signal from ambient light. Fluorescence imaging systems which are currently being translated into the clinic, or are in later research phases, generally handle ambient light in one of the following ways: (1) Using fluorophores that fluoresce near 800 nm, which has relatively low background light, (2) selective filtering of the ambient room lights, which can be disruptive, or (3) darkening the room when imaging in fluorescence mode. The latter is the most common approach, and often the only approach used when imaging fluorescence in the visible waveband, such as PpIX. Darkening the room is disruptive to the clinical workflow and generally unpopular with clinicians. Therefore, a more flexible technology which facilitates fluorescence imaging without adjusting the ambient lighting would be a significant step to facilitate translation of fluorescence imaging into the clinic. Herein, we report on a pulsed imaging system which is capable of imaging visible PpIX fluorescence under normal room light conditions. The system uses very short, high intensity excitation light pulses and gated camera detection to minimize the background signal collected and to allow real-time background subtraction of the ambient light. We demonstrate the ability of the system to track PpIX fluorescence in normal mouse skin after topical application of ALA. 2. METHODS A diagram and photographs of the pulsed light imaging system are shown in Fig. 1. Details on the system components have been describe elsewhere [11]. Briefly, the system is composed of an array of eight SpecBright pulsed LED area lights (ProPhotonix, Salem, New Hampshire), four of which were chosen for red light excitation (630 nm) of protoporphyrin IX. Remitted light is collected with a 70 mm macro camera lens and passes through a motorized filter wheel to a gated intensified CCD (PI-MAX 3, Princeton Instruments, Acton, Massachusetts). For image acquisition, the four LED s and camera are synchronized to illuminate/acquire in 500 μs bursts. Using short pulses allows the LED s to be overdriven safely at 10 times the current allowed in CW mode. This significantly increases the ratio of excitation light intensity to ambient background light intensity while acquiring fluorescence images. The camera is also triggered to acquire images when the LED s are not illuminating the tissue for real-time background light correction, as shown in Fig. 1(a). Proc. of SPIE Vol W-2

3 (a) ICCD (Pi-Max3) Filter wheel Camera Lens Trigger/controller LED array (b) (c) (d) Figure 1. (a) A diagram showing the major components of the system and illustrations of the pulse sequence routine. Photographs of the imaging system provide views of (b) the PI-MAX camera, (c) the camera lens surrounded by the pulsed LED s, and (d) the control software interface and the imaging head mounted on an articulating arm. Using liquid tissue phantoms, we previously compared the performance of the pulsed system with a state of the art clinical fluorescence imaging system (Zeiss Pentero surgical microscope) and demonstrated that the pulsed system is more sensitive to PpIX. Minimum detectable concentrations were 1 μm for the commercial system operating in a dark room and 250 nm for the pulsed system operating in a fully lit laboratory [11]. Here, we examine the ability of the system to track PpIX accumulation in the skin of athymic mice after topical application of ALA. Over one week prior to imaging, mice were put on a specialized non-chlorophyll diet to reduce the normal skin fluorescence, which is much higher than the fluorescence in normal human skin. ALA was applied to the back of mice and images acquired every 45 minutes to an hour for 3 hrs. RGB filters were used to acquire co-registered color images at each time point. 3. RESULTS AND DISCUSSION Color and fluorescence images under pulsed red light excitation approximately one hour after ALA administration of three mice are shown in Fig. 2. Black marker dots mark the region in which ALA was applied topically. The fluorescence images were acquired under normal ambient lighting conditions (fluorescent lights in a laboratory) and are presented as grayscale images after background subtraction. These images indicate robust PpIX fluorescence in each animal, yet the spatial distribution of PpIX varied dramatically between animals. Proc. of SPIE Vol W-3

4 Mouse 1 Mouse 2 Mouse 3 Figure 2. Color images (left) and corresponding grayscale fluorescence images (right) for three mice one hour after topical application of ALA. The pro-drug was applied between the black marker dots. Mouse 1 Fluorescence images (false color green) overlaid on color images for each measurement during the 3 hours after ALA application are shown in Fig. 3. Quantitative analysis of the images (not shown) reveals an expected increase in PpIX accumulation over the time course. Qualitative inspection also suggests that the spatial distribution of PpIX for each animal was generally maintained throughout the imaging session. These preliminary data are the first to show topical ALA-induced PpIX fluorescence images acquired under room light conditions using pulsed-red light excitation. PpIX fluorescence after topical ALA application was readily detectable, and spatial heterogeneities in the fluorescence distribution were clearly discernible. 50 min 90 min 105 min 150 min 180 min Pre-ALA 70 min 100 min 130 min 170 min 200 min Pre-ALA 85 min 115 min 140 min 190 min 210 min Mouse 3 Mouse 2 Pre-ALA Figure 3. PpIX fluorescence image overlays (green false color) on RGB images for each mouse over the 3 hour time period. These images were acquired with red light excitation under normal room lighting conditions. Proc. of SPIE Vol W-4

5 There are four main advantages to using pulsed light illumination and gated detection for clinical fluorescence imaging in a well-lit room: (1) The approach minimizes the image exposure time, which reduces the total background light detected. For example, a 500 μs exposure collects 1/60 th the background light compared with a 30 ms exposure time typically used for video-rate imaging. (2) Pulsing LED s below 1 ms allows the lights to be overdriven, producing significantly more light than when run in CW mode, thus facilitating more efficient packaging. (3) Rapid acquisition of interleaved light-on and -off images facilitates real-time background subtraction to isolate the fluorescence component of the images. (4) The maximum permissible limits per pulse are much higher for shorter pulses, per the ANSI guidelines, thus allowing higher excitation intensities. These characteristics increase the signal to background ratio significantly. 4. SUMMARY Dosimetric imaging of photosensitizer fluorescence may enable the tailoring of treatment parameters for individual patients to improve response rates of PDT. Improving response is critical for the subpopulation of patients for which skin lesions represent a high risk of morbidity and mortality, such as OTR s. Technologies which provide high fidelity information while minimizing the impact in the clinical theater, such as the system presented here, can accelerate translation of PDT dosimetry into the clinic. Currently, the pulsed imaging system is being deployed in an observational clinical study for PDT of AK to further assess the impact on the clinical workflow and the potential to use the system to predict response. Acknowledgements This work was funded by the National Institutes of Health grants R01 CA and P01 CA084203, and Department of Defense award W81XWH REFERENCES [1] M. M. Hartevelt, J. N. B. Bavinck, A. M. M. Kootte et al., Incidence of Skin Cancer After Renal Transplantation in the Netherlands, Transplantation, 49(3), (1990). [2] C. Ulrich, J. Kanitakis, E. Stockfleth et al., Skin Cancer in Organ Transplant Recipients Where Do We Stand Today?, American Journal of Transplantation, 8(11), (2008). [3] D. Berg, and C. C. Otley, Skin cancer in organ transplant recipients: Epidemiology, pathogenesis, and management, Journal of the American Academy of Dermatology, 47(1), 1-20 (2002). [4] R. B. Saager, D. J. Cuccia, S. Saggese et al., Quantitative fluorescence imaging of protoporphyrin IX through determination of tissue optical properties in the spatial frequency domain, J Biomed Opt, 16(12), (2011). [5] F. Piffaretti, M. Zellweger, B. Kasraee et al., Correlation between protoporphyrin IX fluorescence intensity, photobleaching, pain and clinical outcome of actinic keratosis treated by photodynamic therapy, Dermatology, 227(3), (2013). Proc. of SPIE Vol W-5

6 [6] C. Fritsch, P. Lehmann, W. Stahl et al., Optimum porphyrin accumulation in epithelial skin tumours and psoriatic lesions after topical application of deltaaminolaevulinic acid, Br J Cancer, 79(9-10), (1999). [7] J. Hewett, V. Nadeau, J. Ferguson et al., The application of a compact multispectral imaging system with integrated excitation source to in vivo monitoring of fluorescence during topical photodynamic therapy of superficial skin cancers, Photochemistry and Photobiology, 73(3), (2001). [8] M. Stefanidou, A. Tosca, G. Themelis et al., In vivo fluorescence kinetics and photodynamic therapy efficacy of delta-aminolevulinic acid-induced porphyrins in basal cell carcinomas and actinic keratoses; implications for optimization of photodynamic therapy, European Journal of Dermatology, 10(5), (2000). [9] T. Smits, C. A. Robles, P. E. J. van Erp et al., Correlation between macroscopic fluorescence and protoporphyrin IX content in psoriasis and actinic keratosis following application of aminolevulinic acid, Journal of Investigative Dermatology, 125(4), (2005). [10] M. M. Kleinpenning, E. W. Wolberink, T. Smits et al., Fluorescence diagnosis in actinic keratosis and squamous cell carcinoma, Photodermatology Photoimmunology & Photomedicine, 26(6), (2010). [11] K. Sexton, S. C. Davis, D. McClatchy et al., Pulsed-light imaging for fluorescence guided surgery under normal room lighting, Optics Letters, 38(17), (2013). Proc. of SPIE Vol W-6