Lisheng Lin

Photosensitized singlet oxygen generation and detection: Recent advances and future perspectives in cancer photodynamic therapy

Photodynamic therapy (PDT) uses photosensitizers and visible light in combination with molecular oxygen to produce reactive oxygen species (ROS) that kill malignant cells by apoptosis and/or necrosis, shut down the tumor microvasculature and stimulate the host immune system. The excited singlet state of oxygen (1 O2 ) is recognized to be the main cytotoxic ROS generated during PDT for the majority of photosensitizers used clinically and for many investigational new agents, so that maximizing its production within tumor cells and tissues can improve the therapeutic response, and several emerging and novel approaches for this are summarized. Quantitative techniques for 1 O2 production measurement during photosensitization are also of immense importance of value for both preclinical research and future clinical practice. In this review, emerging strategies for enhanced photosensitized 1 O2 generation are introduced, while recent advances in direct detection and imaging of 1 O2 luminescence are summarized. In addition, the correlation between cumulative 1 O2 luminescence and PDT efficiency will be highlighted. Meanwhile, the validation of 1 O2 luminescence dosimetry for PDT application is also considered. This review concludes with a discussion on future demands of 1 O2 luminescence detection for PDT dosimetry, with particular emphasis on clinical translation. Eye-catching color image for graphical abstract.

Pattern recognition of multiple excitation autofluorescence spectra for colon tissue classification

OBJECTIVES The aim of this study was to explore the usefulness of multiple excitation autofluorescence (AF) and a spectral feature-based pattern recognition in classification of colon tissues. MATERIALS AND METHODS Under four different excitation wavelengths (337, 375, 405 and 460 nm), AF spectra of freshly excised normal and adenocarcinoma colon tissues were measured. Pattern recognition method including features extraction, data reduction using principal component analysis (PCA) and Fishers discriminant analysis (FDA) were performed for classification. RESULTS There was a significantly difference between spectral patterns of normal and adenocarcinoma tissues. Compared with the other three excitation wavelengths, the AF spectra obtained under 337 nm excitation provided more diagnostic information, but also more sensitive to the trivial change resulted from neoplastic transformation. For discriminating normal from adenocarcinoma tissues, the sensitivity, specificity and accuracy using 337 nm excitation in the present study were 88.9%, 80.0% and 83.9%, respectively. Compared these values with those determined from multispectral data analysis, our findings indicate that the latter has higher specificity while maintaining the same sensitivity (sensitivity 88.9% vs. 88.9%, specificity 91.4% vs. 80.0%, and accuracy 90.3% vs. 83.9%). CONCLUSION This study suggests that the pattern recognition of the multiple excitation AF spectra is an effective algorithm for improving the diagnostic accuracy of adenocarcinoma.

SINGLET OXYGEN QUANTUM YIELDS OF PORPHYRIN-BASED PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY

The major cytotoxic agent with most current photosensitizers used in photodynamic therapy (PDT) is widely believed to be singlet oxygen (1O2). Determination of the 1O2 quantum yields for porphyrin-based photosensitizers, including hematoporphyrin derivative (HiPorfin), hematoporphyrin monomethyl ether (HMME) and photocarcinorin (PsD-007) in air-saturated dimethylformamide (DMF) solutions were performed by the direct measurement of their near-infrared luminescence. In addition, 1O2 quencher sodium azide was employed to confirm the 1O2 generation from the investigated photosensitizers. The maximal 1O2 luminescence occurs at about 1280 nm with full width at half maximum of 30 nm. The 1O2 quantum yields were found to be 0.61 ± 0.03, 0.60 ± 0.02 and 0.59 ± 0.03 for HiPorfin, HMME and PsD-007, respectively. These results provide that these porphyrin-based photosensitizers produce 1O2 under irradiation, which is of significance for the study of their photodynamic action in PDT.

Direct imaging of singlet oxygen luminescence generated in blood vessels during photodynamic therapy

Singlet oxygen (1O2) is commonly recognized to be a major phototoxic component for inducing the biological damage during photodynamic therapy (PDT). In this study, a novel configuration of a thermoelectrically-cooled near-infrared sensitive InGaAs camera was developed for imaging of photodynamically-generated 1O2 luminescence. The validation of 1O2 luminescence images for solution samples was performed with the model photosensitizer Rose Bengal (RB). Images of 1O2 luminescence generated in blood vessels in vivo in a well-controlled dorsal skinfold window chamber model were also recorded during PDT. This study demonstrated the capacity of the newly-developed imaging system for imaging of 1O2 luminescence, and the first reported images of 1O2 luminescence in blood vessels in vivo. This system has potential for elucidating the mechanisms of vascular targeted PDT.

Vessel constriction correlated with local singlet oxygen generation during vascular targeted photodynamic therapy

In this study, the vessel constriction was measured as a biological indicator of acute vascular response after vascular targeted photodynamic therapy (V-PDT). During V-PDT treatment, the near-infrared (NIR) singlet oxygen (1O2) luminescence at 1270 nm generated in blood vessels in a dorsal skinfold window chamber model in vivo was directly monitored using a custom built high-sensitive NIR imaging system. In order to compare the acute vascular response, various irradiances with the same light dose were utilized for treatments. The obtained results show that the complete arteriole constriction occurred frequently, while some of the larger veins were constricted partially. For the vessels that have significant constriction after V-PDT, our preliminary data suggest that the vasoconstriction in the selected ROIs are roughly correlated with the local cumulative 1O2 luminescence intensities. This study implies that the 1O2 luminescence dosimetry maybe also effective for evaluating V-PDT efficiency.

Characterizing autofluorescence generated from endogenous porphyrins in cancerous tissue of human colon: case studies

The aim of this case study was to explore the relationship between porphyrins and colon adenocarcinoma, and to examine the potential of porphyrin-induced fluorescence for the diagnosis of colon cancer. Further studies were carried on 8 cases ex vivo colon adenocarcinoma samples which exceptionally exhibited 635 nm fluorescence emission under 405 nm excitation. The time-resolved fluorescence spectra at 635 nm emission under 405 nm excitation were also measured and two-exponential decay fitting was performed to determine the fluorescence lifetime at 635 nm emission. Significant difference was observed between the spectra of normal and cancer tissues, which included an emission peak at 635 nm under the excitation wavelengths of 405 nm. There was also a significant difference between the fluorescence lifetimes of 635 nm emission of the normal tissue and cancer tissue (P<0.05). These results demonstrate that the spectroscopic analysis method allows a selective detection of adenocarcinoma tissues. This spectral profile and lifetime of the red fluorescence resemble that of porphyrins, which suggests that porphyrin fluorescence may be a useful biomarker for characterizing colon cancers of certain patient populations.

Extracting autofluorescence spectral features for diagnosis of nasopharyngeal carcinoma

The aim of this study is to investigate the autofluorescence spectral characteristics of normal and cancerous nasopharyngeal tissues and to extract the potential spectral features for diagnosis of nasopharyngeal carcinoma (NPC). The autofluorescence excitation-emission matrix (EEM) of 37 normal and 34 cancerous nasopharyngeal tissues were recorded by a FLS920 spectrofluorimeter system in vitro. Based on the alteration in proportions of collagen and NAD(P)H, the integrated fluorescence intensity of I455 ± 10 nm and I380 ± 10 nm were used to calculated the ratio values by a two-peak ratio algorithm to diagnose NPC tissues at 340 nm excited. Furthermore by applying the receiver operating characteristic curve (ROC), the 340 nm excitation yielded an average sensitivity and specificity of 88.2 and 91.9%, respectively. These results may have practical implications for diagnosis of NPC.

Imaging singlet oxygen luminescence in blood vessels

Photodynamic therapy (PDT) is a minimally invasive treatment for cancer and other diseases that uses light-activated compounds to attack malignant cells and tissues. Using a photosensitizer (an agent that transfers energy from incident light), PDT produces reactive oxygen species—predominantly singlet oxygen (O2)—that are toxic to the targeted cells. Direct imaging of singlet oxygen near-IR (NIR) luminescence at around 1270nm reveals the spatial and temporal heterogeneity of tumors and their response to PDT. However, the imaging process is technically challenging because of the extremely high reactivity of singlet oxygen and its short lifetime in biological microenvironments.1–3 This can limit the chances of luminescence emission. Furthermore, current NIR detectors have very low quantum efficiency (the ratio of incident photons to converted electrons). During the past decade, there have been two main approaches for imaging singlet oxygen luminescence and attempting to correlate it with the biological response during PDT.4 The first method uses an NIR photomultiplier tube or an indium gallium arsenide (InGaAs) photodiode linear array combined with a

Discriminant analysis for classification of colonic tissue autofluorescence spectra

This study evaluates the potential of a discriminant analysis to classify colonic mucosa from autofluorescence spectral characteristics. With 337 nm excitation, the autofluorescence spectra of colonic tissues were measured using a FLS920 spectrofluorimeter. Principal component analysis (PCA) combined with Fishers discriminant analysis was performed for tissue classification. As a result, the sensitivity and specificity of the discriminant analysis is 92.3% and 90.5%, respectively. The results suggest the relative concentrations of collagen and nicotinamide adenine dinucleotide (NADH) are the potential diagnostic biomarkers for colonic tissue classification using autofluorescence spectroscopy, and the discriminant analysis based on PCA is useful to differentiate adenocarcinoma from normal tissue.

Autofluorescence spectroscopic characteristics of nasopharyngeal carcinoma and normal tissue

Light-induced autofluorescence spectra of nasopharyngeal carcinoma and normal tissue in vitro were compared to that of known endogenous fluorophores to explore the possible causes of tissue autofluorescence and to further determine the optimal excitation wavelengths for optical biopsy in vivo. Nasopharyngeal carcinoma and normal tissues were obtained from the suspected patients during pathological biopsy. A FL/FS92O combined TCSPC spectrofluonmeter and a lifetime spectrometer system was used for autofluorescence spectra measurement. Fluorescence excitation wavelengths varying from 260 to 480 nm were used to induce tissue autofluorescence, and the corresponding fluorescence emission spectra were recorded from a range starting 20 nm above the excitation wavelength and extending to 700 nm. The autofluorescence excitation-emission pairs of nasopharyngeal carcinoma and nonnal tissues occur at 300-330, 340-460 and 450-520 nm, and the optimal diagnostic excitation wavelengths for detection of nasopharyngeal carcinoma were 340 and 450 nm. The results abtained in this study could be treated as a reference for the development of optical biopsy system for nasopharyngeal carcinoma.