(A) A schematic representation of PDT where PS is a photoactivatable multifunctional agent, which, upon light activation can serve as both an imaging agent and a therapeutic agent. (B) A schematic representation of the sequence of administration, localization and light activation of the PS for PDT or fluorescence imaging. Typically the PS is delivered systemically and allowed to circulate for an appropriate time interval (the “drug-light interval”), during which the PS accumulates preferentially in the target lesion(s) prior to light activation. In the idealized depiction here the PS is accumulation is shown to be entirely in the target tissue, however, even if this is not the case, light delivery confers a second layer of selectivity so that the cytotoxic effect will be generated only in regions where both drug and light are present. Upon localization of the PS, light activation will result in fluorescence emission which can be implemented for imaging applications, as well as generation cytotoxic species for therapy. In the former case light activation is achieved with a low fluence rate to generate fluorescence emission with little or no cytotoxic effect, while in the latter case a high fluence rate is used to generate a sufficient concentration of cytotoxic species to achieve biological effects.

A timeline of selected milestones in the historical development of PDT.

Imaging platforms for molecular structural and functional imaging across a broad range of size scales. For molecular concentration imaging, (the left side of the figure), optical spectroscopy and magnetic resonance spectroscopy (MRS) are primarily employed, but positron emission tomography (PET) can be used for imaging at these length scales. For structural and molecular imaging at length scales greater than 10’s of nanonmeters (the right side of the figure) a variety of imaging techniques can be employed including various types of microscopy, endoscopy, x-ray computed tomography (CT) and magnetic resonance imaging (MRI) depending on the size and composition of the structure being imaged.

Perrin-Jablonski energy diagram for a photosensitizer (PS) molecule. Various processes during the excited state lifetime of the PS and resulting from its relaxation back to its ground state are highlighted. While in its long lived triplet excited state the PS may undergo excited state reactions to generate cytotoxic species such as singlet molecular oxygen (via energy transfer from the PS to ground state, triplet oxygen). While both the excited singlet state and triplet state are involved in photosensitized cell killing, photodynamic killing comes primarily from the triplet manifold. PS fluorescence and phosphorescence may be used to image PS localization in tissue and time-resolved imaging techniques may be applied to monitor PS interactions with its microenvironment.

A schematic representation of the basic principles of photosensitizer fluorescence detection. The photosensitizer accumulates preferentially in neoplastic tissue (depicted as purple cells), which, upon excitation with light of the appropriate wavelength (blue arrows) emits red fluorescence emission (red arrows). The contrast generated by this fluorescence emission against backscattered illumination (blue arrows with dashed lines) can be used to demarcate the boundaries of neoplastic tissues for sensitive detection of a variety of human cancers and optimization of surgical resection. The backscattered illumination can be useful to observe surrounding non-fluorescent tissue, though in many applications an emission filter is placed before the observer or detector to exclude the backscattered light.

A schematic representation of the heme synthesis pathway which lead to synthesis and accumulation of protoporphyrin IX in vivo. Under normal physiological conditions, synthesis of PpIX is regulated by negative feedback control of free heme on ALA synthase. This feedback is bypassed by addition of exogenous ALA which, due to the relatively low rate of iron insertion by the enzyme ferrochelatase, leads to accumulation of excess PpIX that can be used either therapeutically, for PDT, or to generate fluorescence contrast, for PFD.

Structures of Aminolevulinic acid (ALA, Levulan) and its Methyl and Hexyl Esters (MAL or Metvix^® and HAL or Hexvix^® respectively).

Absorption (A) and fluorescence emission (B) spectrum obtained from PpIX in methanol. Positions of absorption maxima typically (although not exclusively) used for PFD and therapeutic PDT applications are marked with arrows.

Comparison of white light and PpIX fluorescence images of an intermediate grade malignant lesion in the bladder of a human patient, obtained via cystoscopy. In the white light image (A), the lesion is not evident, while in the PpIX fluorescence image obtained under blue illumination (B), the lesion is readily visible as a pink region just above the large air bubble in the lower middle part of the field. (Figure reproduced with permission from ref 62. Copyright 1999 BJU International.)

Progression free survival data by treatment group for the large multicenter trial reported by Stummer et al in 2006, which compared white light and fluorescence guided resection for treatment of malignant glioma. After 6 months there is a significant enhancement in progression free survival in the 5-ALA group as compared to white light, while the two curves collapse after 15 months. (Figure reproduced with permission from ref 78. Copyright 2006 Elsevier Ltd.)

Intraoperative images of a partially resected brain tumor (A and B) and the surface of the brain (C and D), comparing images obtained by white light (A and C) and mTHPC fluorescence imaging under blue light (B and D). In A, a partially resected tumor is difficult to discern while it is readily apparent in the blue-light mTHPC fluorescence image of the same tissue. Similarly, in C (a white light image of the surface of the brain), there is no apparent tumor, while the mTHPC fluorescence image, which is complemented by spectroscopy (inset) of the same field reveals the presence of malignancy. (Figure reproduced with permission from ref 80. Copyright 2001 American Society for Photobiology.)

Laparoscopic images of peritoneal metastases in a rat model of ovarian cancer obtained by white light imaging (right) and HAL-induced PpIX fluorescence imaging under blue light illumination (left). Image (A) shows a lesion that is only visible in the blue light mode, but not by white light (position marked by a circle) (8mM HAL after 2 h). Image (B) shows three lesions visible by both blue and white light (big circle) and one only detectable by fluorescence (small circle) (8mM HAL after 2 h). (Figure reproduced with permission from ref 94. Copyright 2003 Cancer Research UK.)

Laparoscopic images comparing metastatic ovarian carcinoma lesions in human patients under white and blue light illumination following intraperitoneal instillation of ALA in human patients. As observed in preclinical studies for detection of micrometastatic ovarian cancer using PFD, lesions are visible Tumor was detected on tissue specimens by strong red fluorescence, in lesions measuring <0.5 mm. (Figure reproduced with permission from ref 95. Copyright 2004 American Cancer Society.)

(A) Schematic of the prototype fiber-optic fluorescence microendoscope imaging system. Fluorescence excitation is provided by a light emitting diode (LED) which is directed through the objective via a dicrhoic mirror. Excitation light is directed into the mouse through a flexible sub-millimeter imaging fiber which also collects fluorescence emission. The longer wavelength emission passes the dichroic mirror and is registered on a CCD camera with on chip multiplication gain. (B) Fluorescence image of microscopic tumor nodules (tens of microns in size) detected on the peritoneal wall of an ovarian cancer mouse by the fluorescence microendoscope using BPD as the contrast agent. Scale bar is 100 μm. (C) H & E stain of a 5 μm section from the same region. Arrows indicate tumor nodules. (Reproduced with permission from ref 15. Copyright 2009 Nature Publishing Group.)

Multispectral images (A-D) taken pre-PDT of a superficial BCC on the ankle and the corresponding white-light image (E) taken before ALA application. Real-time image processing displays the difference between the 630 and 600 nm images displayed with a false color scale (F) showing clearly the extent of the lesion and the differential accumulation of PpIX in the SBCC compared with the application site and surrounding healthy tissue. Scale bars for these images were not available. (Figure reproduced with permission from ref 113. Copyright 2001 Wiley.)

MAL induced PpIX fluorescence images of two patients (A-C, and D-F) who received Mohs micrographic surgery (MMS). Images were obtained 3 hours following administration of MAL. Clinical pictures showing the outlines of the first MMS excision (white lines) were conducted without respect to the fluorescence images. The “real margins” of the tumors confirmed by histopathologic analysis are also included (red areas) and were already delineated by the fluorescence seen in panels B and E. The agreement between the fluorescence and histopathology margins suggests that the use of PFD may speed up Mohs surgery by reducing the number of step-wise excisions necessary. (Figure reproduced with permission from ref 110. Copyright 2008 American Medical Association.)

Comparison of white light and ALA induced PpIX fluorescence imaging (excitation wavelengths = 375–440 nm) for identification of oral cancer. (A) White light image of a tumor in the right floor of the mouth. (B) Autofluorescence imaging under excitation with blue-violet light of the same region. (C) The same region imaged again under ordinary white light, 1.5 hours after application of ALA. D. PpIX fluorescence image of the same region. (Figure reproduced with permission from ref 120. Copyright 2000 Wiley.)

Disease-targeted constructs for PFD and PDT, showing the targeting moeity, imaging agent, and biological application. (A) Ovarian cancer cells incubated for 15h with 140nM equivalent BPD-C225 construct. A confocal laser scanning fluorescence microscope is used to monitor the subcellular localization of the PS with high spatial resolution, fluorescence from the mitochondrial markers is shown in false color as green and BPD is shown in false color as red; 139 (B) confocal microscopy shows that TPC conjugated to a membrane-penetrating arginine oligopeptide (R7) enters MDA-MB-468 (human breast carcinoma) cells efficiently where red represents fluorescence signal from TPC; 142 (C) monitoring fluorescence signal distribution after intravenous injection of 100 nmol of Pyro-GDEVDGSGK-Folate conjugate to double tumor-bearing mice (with a FR positive tumor on the right side and negative one on the left side), indicates preferential accumulation of construct in the receptor positive tumor, establishing NIR imaging ability of the targeted PS construct; 143 (D) fluorescence from aortic segments 24 hr post injection of Ce6-maleylated albumin conjugate indicates the constructs ability to detect and/or photodynamically treat inflamed plaques. Red represents Ce6 and yellow is tissue autofluorescence from the elastic fibers; 146 (E) post-PDT changes in VEGF expression are monitored with the molecular imaging strategy, where an Avastin-Alexa Fluor construct was imaged in PDT-treated subcutaneous PC-3 (prostate cancer) tumors, 6 h following laser irradiation and fluorescence image of tumor labeling is pseudocolored in gold; 147 and (F) T2-weighted magnetic resonance images at day 8 after PDT treatment from F3-targeted Photofrin-containing nanoparticles in a 9L brain tumor showing imaging and monitoring of therapeutic efficacy post treatment.22 (Figures reproduced with permission from: (A) ref 139. Copyright 2006, The International Society for Optical Engineering (SPIE); (B) ref 142. Copyright 2006, Wiley; (C) ref 143. Copyright 2007, American Chemical Society; (D) ref 146. Copyright 2008, The Royal Society of Chemistry and Owner Societies; (E) ref 147. Copyright 2008, American Association for Cancer Research; and (F) ref 22. Copyright 2006, American Association for Cancer Research.)

Concept design and application of PS fluorescence in site-activated constructs. (A) Release of Ce6 for PFD and PDT following cleavage of a cathepsin-B-specific construct in a subcutaneous murine model for human fibrosarcoma. Three-dimensional fluorescence-mediated tomography was used to image the HT1080 fibrosarcomas following 24 h incubation with a poly-L-lysine-Ce6 construct (0.125 mg Ce6 eq./kg).152 (B) Proof-of-concept study demonstrating the cleavage of a peptide linker by MMP-7 for PFD and PDT in a subcutaneous murine model for human epidermoid cancer. The PP_MMP7B construct (drug) was injected intravenously (80nmol) in a single mouse bearing two KB tumors on each hind leg. Only one tumor, left leg, was treated and this mouse was monitored by white light and fluorescence imaging before treatment (left image, 3 h after drug injection) and 1 h after PDT (right image, 5 h after drug injection) on one hind flank. Right Image: Treated tumor on the left leg became edematous one hour post PDT while no fluorescence change is observed in untreated right tumor.154 (C) Mechanism for the cleavage of an enzyme activated prodrug where the blue balls represent the inactive PSs in the uncleaved construct, and the red balls represent the photoactive PSs. The image shows PS fluorescence in cellular co-cultures of S. aureus with human foreskin fibroblasts (HFF) where significantly greater fluorescence intensity is observed in bacterial cells, than in the neighboring fibroblasts indicating cleavage of construct only at the site of infection.13 (Figures reproduced with permission from: (A) ref 152; Copyright 2006, American Association for Cancer Research. (B) ref 154; Copyright 2007, The National Academy of Sciences of the USA. (C) ref 13; Copyright 2009, Wiley.)

A schematic diagram depicting the intersecting roles of imaging in key steps of pre-treatment planning, therapy monitoring, and outcome assessment.