Drug screening is an important step in the development of new drugs. Through appropriate experimental methods and screening models, drugs with specific bioactivity can be transferred from laboratory research to clinical application. Nowadays, traditional efficiency of drug experimentation has been unable to meet the needs of our society. With the rapid development of computer technology, computer-assisted diagnosis and treatment have been gradually accepted and recognized by clinicians and patients. Drug screening process at the cellular level was studied in this paper. We not only compared the advantages and disadvantages of deep learning models and traditional machine learning methods, but also analyzed the performance of different deep learning models. First, Hela cells injected with different anti-stress drugs were divided into groups for experiment. G3BP, TIA-1 and the nucleus were labeled, respectively. The images were obtained using a single-photon microscope. Then, we distinguished the images of Hela cells treated with different drugs through visual observation, traditional machine learning (LBP/Gabor+SVM) and deep learning algorithms (VGGNet, GoogLeNet, ResNext and DenseNet), respectively. Experimental results showed that compared with visual observation, traditional machine learning and deep learning algorithms had better objectivity. Furthermore, deep learning models all had good classification performance. The highest average correct recognition rate was 92.97%, while that of the traditional method was only 80.93%. Therefore, drug screening methods based on deep learning could assist in screening the optimal treatment drugs, which help clinicians choose appropriate therapy.
Singlet oxygen (1O2) is widely considered as the major cytotoxic species generated during Type-II photochemical reaction of photodynamic therapy (PDT), and its production is crucial for the treatment outcome of PDT. However, it is still a challenging task for direct detection of 1O2 by using an optical system as its extremely weak luminescence at 1270 nm. In previous study, a high-sensitivity optical fiber detection system has been developed to measure the time-resolved 1O2 luminescence spectra. In this study, an optical phantom of skin tissue has been built to simulate the skin optical properties for research in photodynamic therapy (PDT). The phantom consists of an absorber (ink) and a scatterer (Intralipid) and phosphate buffer saline (PBS). Rose Bengal (RB) was utilized as the model photosensitizer to generate 1O2. The time-resolved 1O2 luminescence spectroscopy were measured by using a 1O2 luminescence detection system with a fiber prober in tissue phantom. Furthermore, the effect of absorption coefficient (μa) and scattering coefficient (μs) on the photosensitizer triple state lifetime (τT) and 1O2 luminescence lifetime (τD) was investigated as well. The results indicated that the integrated intensity of 1O2 luminescence decrease with the increase of μa and μs. μa has no significant effect on τT and τD. τT increase with the increase of μs, and it finally stabilized around 1.6 μs. Meanwhile, τD decrease firstly and then increase slowly, and it finally stabilized around 14 μs. This result indicates that this system for 1O2 luminescence have a potential for clinical applications in PDT dosimetry.
Singlet oxygen (1O2) is the primary cytotoxic production in type-II photodynamic therapy (PDT). The correlation between 1O2 generation and PDT efficacy during treatment has received considerable attention. The direct detection of 1O2 luminescence is the gold standard for 1O2 identification. However, the intensity of 1O2 luminescence could be influenced by tissue optical properties, location and morphology of lesion, especially for vascular targeting PDT. In this study, the impacts of vascular diameter and depth on 1O2 luminescence imaging have been investigated in tissue simulating phantom. A near-IR sensitive InGaAs camera with adaptive optics and CW laser 532 nm were used for fast imaging of 1O2 luminescence. Rose Bengal (RB) was used as photosensitizer to generate 1O2 during photosensitization. Intralipid was diluted with different concentrations in order to establish the scattering properties of tissues. The capillary tubes, containing solution of RB, with varied diameters from 0.1 to 0.9 mm were used to simulate the vascular with the depth varied from 0 to 5 mm in phantom. The preliminary results indicate that the profile range ratio of 1O2 luminescence images are negatively correlated to the diameter of capillary tube, and the attenuation of intensity of 1O2 luminescence is non-linear with the increase of depth.
KEYWORDS: Luminescence, Optical fibers, Oxygen, Photodynamic therapy, Signal to noise ratio, Signal detection, Sensing systems, Picosecond phenomena, Single photon detectors, Near infrared
Singlet oxygen (1O2) is widely recognized as the primary cytotoxic agent during photodynamic therapy (PDT). The quantitation of 1O2 generation during PDT plays an important role for 1O2 based PDT dosimetry. However, it is still a challenging task for direct detection of 1O2 using an optical system due to its extremely weak luminescence at 1270 nm. In this paper, a highly sensitive optical fiber detection system was developed to measure the time-resolved 1O2 luminescence spectra from two model photosensitizers (Rose Bengal and TMPyP) at various concentrations (1.25, 2.5, 5.0, and 10.0 μM), The 1O2 luminescence signal was excited by a diode-pumped, Q-switched, frequency-doubled 523- nm Nd:YLF laser and collected by an optical fiber probe coupled with a highly sensitive NIR photomultiplier tube (PMT). Experimental results indicate that the 1O2 luminescence intensity shows a linear enhancement both with the increase of concentrations of Rose Bengal and TMPyP. The 1O2 luminescence signal dramatically decreased after the addition of 50 mM sodium azide, a specific 1O2 quencher. Furthermore, the signal-to-noise ratio (SNR), calculated under the condition of 10 mM Rose Bengal solution, is higher than those obtained from optical fiber 1O2 luminescence detection systems based on InGaAs/InP single photon avalanche diode and superconducting nanowire single-photon detector. Our results suggest that the home-built optical fiber system with a high sensitivity for 1O2 luminescence detection will have a great potential for clinical applications in PDT dosimetry.
A digital micromirror device (DMD) based structural illumination and projection optical system were designed and evaluated for fluorescence imaging and diffuse reflectance imaging of tissue in spatial frequency domain, respectively. Light emitting diodes (LEDs) at discrete wavelengths (532, 620, 656 nm) provided illumination for the diffuse reflectance imaging while a 532 nm laser diode (LD) was used as excitation light source of photosensitizer (PS) fluorescence. Both the LEDs and LD light were collimated, homogenized and converged on a DMD to generate the structural illumination. A projection lens was also designed to project a rectangular structural illumination spot on target tissue. The designed optical system could be applied to provide variable frequency structural illumination for depth sensitive excitation of PS and diffuse reflectance imaging.
Photodynamic therapy (PDT) is an effective therapeutic modality that uses a light source to activate light-sensitive photosensitizers to treat both oncologic and nononcological indications. Photosensitizers are excited to the long-lived triplet state, and they react with biomolecules via type I or II mechanism resulted in cell death and tumor necrosis. Free radicals and radical ions are formed by electron transfer reactions (type I), which rapidly react with oxygen leading to the production of reactive oxygen species (ROS), including superoxide ions, hydroxyl radicals and hydrogen peroxide. Singlet molecular oxygen is produced in a Type II reaction, in which the excited singlet state of the photosensitizer generated upon photon absorption by the ground-state photosensitizer molecule undergoes intersystem crossing to a long-lived triplet state. In this talk, the fundmental mechanisms and detection techniques for ROS generation in PDT will be introduced. In particular, the quantification of singlet oxygen generation for pre-clinical application will be highlighted, which plays an essential role in the establishment of robust singlet oxygen-mediated PDT dosimetry.
Vascular targeted photodynamic therapy (V-PDT) has been widely utilized for the prevention or treatment of
vascular-related diseases, including age-related macular degeneration, port-wine stains and prostate cancer. In order to
quantitative assessment the blood vessel damage during V-PDT, nude mice were implanted with Titanium dorsal skin
window chambers for in vivo V-PDT studies. For treatments, various irradiances including 50, 75, 100 and 200 mW/cm2
provided by a 532 nm semiconductor laser were performed with the same total light dose of 30 J/cm2 after the mice were
intravenously injection of Rose Bengal for 25 mg/Kg body weight. Laser speckle imaging and microscope were used to
monitor blood flow dynamics and vessel constriction during and after V-PDT, respectively. The V-PDT induced vessel
damages between different groups were compared. The results show that significant difference in blood vessel damage was
found between the lower irradiances (50, 75 and 100 mW/cm2) and higher irradiance (200 mW/cm2), and the blood vessel
damage induced by V-PDT is positively correlated with irradiance. This study implies that the optimization of irradiance is
required for enhancing V-PDT therapeutic efficiency.
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.
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.
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.
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 Fisher's 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 spectroscopy is a powerful method to identify the endogenous fluorophores in normal and cancerous
cells. The purpose of this study is to characterize the autofluorescence spectra of human normal and leukemia cells.
Autofluorescence measurements of each cell line are performed over a wide range of cell concentrations. All of the
leukemia cells indicate a statistically significant increase in the tryptophan fluorescence relative to that of the normal
cells, while no statistically significant differences are observed in the reduced nicotinamide adenine dinucleotide
(phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) fluorescence between the normal and leukemia cells.
The results suggest that the differences in autofluorescence spectra for leukemia cells and mononuclear cells may be
attributed in part to differences in endogenous fluorophores of different cells.
Singlet oxygen (1O2) is widely considered to be the predominant cytotoxic agent for photodynamic therapy (PDT), and
the 1O2 quantum yield is a key characteristic for the newly developed photosensitizers. In this study, 1O2 generation from
the HiPorfin is monitored indirectly by using the fluorescence probe Singlet Oxygen Sensor Green (SOSG). The good
linear relationship between the concentration of HiPorfin and the initial reaction rate of SOSG is observed, and the 1O2
quantum yield of HiPorfin is determined to be 0.124 ± 0.001, as compared to the standard reference of Rose Bengal. The
results suggest that SOSG can be useful for 1O2 quantum yield determination for the potential photosensitizers in PDT.
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.
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