Imaging biological samples with two-photon fluorescence (2PF) microscopy has the unique advantage of resulting high contrast 3D resolution subcellular image that can reach up to several millimeters depth. 2PF probes that absorb and emit at near IR region need to be developed. Two-photon excitation (2PE) wavelengths are less concerned as 2PE uses wavelengths doubles the absorption wavelength of the probe, which means 2PE wavelengths for probes even with absorption at visible wavelength will fall into NIR region. Therefore, probes that fluoresce at near IR region with high quantum yields are needed. A series of dyes based on 5-thienyl-2, 1, 3-benzothiadiazole and 5-thienyl-2, 1, 3-benzoselenadiazole core were synthesized as near infrared two-photon fluorophores. Fluorescence maxima wavelengths as long as 714 nm and fluorescence quantum yields as high as 0.67 were achieved. The fluorescence quantum yields of the dyes were nearly constant, regardless of solvents polarity. These diazoles exhibited large Stokes shift (<114nm), high two-photon absorption cross sections (up to 2,800 GM), and high two-photon fluorescence figure of merit (FM , 1.04×10-2 GM). Cells incubated on a 3D scaffold with one of the new probes (encapsulated in Pluronic micelles) exhibited bright fluorescence, enabling 3D two-photon fluorescence imaging to a depth of 100 µm.
{"title":"Fluorenyl benzothiadiazole and benzoselenadiazole near-IR fluorescent probes for two-photon fluorescence imaging (Conference Presentation)","authors":"K. Belfield, S. Yao, Bosung Kim, Xiling Yue","doi":"10.1117/12.2211199","DOIUrl":"https://doi.org/10.1117/12.2211199","url":null,"abstract":"Imaging biological samples with two-photon fluorescence (2PF) microscopy has the unique advantage of resulting high contrast 3D resolution subcellular image that can reach up to several millimeters depth. 2PF probes that absorb and emit at near IR region need to be developed. Two-photon excitation (2PE) wavelengths are less concerned as 2PE uses wavelengths doubles the absorption wavelength of the probe, which means 2PE wavelengths for probes even with absorption at visible wavelength will fall into NIR region. Therefore, probes that fluoresce at near IR region with high quantum yields are needed. A series of dyes based on 5-thienyl-2, 1, 3-benzothiadiazole and 5-thienyl-2, 1, 3-benzoselenadiazole core were synthesized as near infrared two-photon fluorophores. Fluorescence maxima wavelengths as long as 714 nm and fluorescence quantum yields as high as 0.67 were achieved. The fluorescence quantum yields of the dyes were nearly constant, regardless of solvents polarity. These diazoles exhibited large Stokes shift (<114nm), high two-photon absorption cross sections (up to 2,800 GM), and high two-photon fluorescence figure of merit (FM , 1.04×10-2 GM). Cells incubated on a 3D scaffold with one of the new probes (encapsulated in Pluronic micelles) exhibited bright fluorescence, enabling 3D two-photon fluorescence imaging to a depth of 100 µm.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"8 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"121875172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
In this presentation, a review and quasioptical imaging system and design considerations for an off-axis parabolic mirror based THz imaging systems are presented. It is shown that off-axis parabolic mirrors introduce off-axis intensity and polarization distortion. When a train of OAPs are used to relay THz beam, each distortions rapidly stacks to produce quite ugly beam and polarization profile. We show that the distortion of field distribution and polarization as a function of mirror curvature and focusing parameters. A brief review of design rules are shown to eliminate these distortions by a symmetric configurations of off-axis parabolic mirror train. The detrimental distortion effects were cancelled out by orienting the final two mirrors in a way to that recovers the original source profile. Comparisons of field profiles between compensated and uncompensated design are shown and imaging performance on characterization targets presented. In addition to benefits in field and polarization distribution the improved design facilitates 1D scanning with minimal change to overall optical path length.
{"title":"Quasioptical imaging system design for THz medical imaging application (Conference Presentation)","authors":"S. Sung, Z. Taylor","doi":"10.1117/12.2218578","DOIUrl":"https://doi.org/10.1117/12.2218578","url":null,"abstract":"In this presentation, a review and quasioptical imaging system and design considerations for an off-axis parabolic mirror based THz imaging systems are presented. It is shown that off-axis parabolic mirrors introduce off-axis intensity and polarization distortion. When a train of OAPs are used to relay THz beam, each distortions rapidly stacks to produce quite ugly beam and polarization profile. We show that the distortion of field distribution and polarization as a function of mirror curvature and focusing parameters. A brief review of design rules are shown to eliminate these distortions by a symmetric configurations of off-axis parabolic mirror train. The detrimental distortion effects were cancelled out by orienting the final two mirrors in a way to that recovers the original source profile. Comparisons of field profiles between compensated and uncompensated design are shown and imaging performance on characterization targets presented. In addition to benefits in field and polarization distribution the improved design facilitates 1D scanning with minimal change to overall optical path length.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"109 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"127665292","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Dukho Do, S. Alali, Dongkyun Kang, N. Tabatabaie, Weina Lu, Catriona N. Grant, Amna R. Soomro, N. Nishioka, M. Rosenberg, P. Hesterberg, Qian Yuan, J. Garber, A. Katz, W. Shreffler, G. Tearney
Eosinophilic Esophagitis (EoE) is caused by food allergies, and defined by histological presence of eosinophil cells in the esophagus. The current gold standard for EoE diagnosis is endoscopy with pinch biopsy to detect more than 15 eosinophils/ High power field (HPF). Biopsy examinations are expensive, time consuming and are difficult to tolerate for patients. Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology capable of imaging individual eosinophils as highly scattering cells (diameter between 8 µm to 15 µm) in the epithelium. Our lab has developed a tethered SECM capsule that can be swallowed by unsedated patients. The capsule acquires large area confocal images, equivalent to more than 30,000 HPFs, as it traverses through the esophagus. In this paper, we present the outcome of a clinical study using the tethered SECM capsule for diagnosing EoE. To date, 32 subjects have been enrolled in this study. 88% of the subjects swallowed the capsules without difficulty and of those who swallowed the capsule, 95% preferred the tethered capsule imaging procedure to sedated endoscopic biopsy. Each imaging session took about 12 ± 2.4 minutes during which 8 images each spanning of 24 ± 5 cm2 of the esophagus were acquired. SECM images acquired from EoE patients showed abundant eosinophils as highly scattering cells in squamous epithelium. Results from this study suggest that the SECM capsule has the potential to become a less-invasive, cost-effective tool for diagnosing EoE and monitoring the response of this disease to therapy.
{"title":"Clinical experience using the tethered capsule-based spectrally encoded confocal microendoscopy for diagnosis of eosinophilic esophagitis (Conference Presentation)","authors":"Dukho Do, S. Alali, Dongkyun Kang, N. Tabatabaie, Weina Lu, Catriona N. Grant, Amna R. Soomro, N. Nishioka, M. Rosenberg, P. Hesterberg, Qian Yuan, J. Garber, A. Katz, W. Shreffler, G. Tearney","doi":"10.1117/12.2218559","DOIUrl":"https://doi.org/10.1117/12.2218559","url":null,"abstract":"Eosinophilic Esophagitis (EoE) is caused by food allergies, and defined by histological presence of eosinophil cells in the esophagus. The current gold standard for EoE diagnosis is endoscopy with pinch biopsy to detect more than 15 eosinophils/ High power field (HPF). Biopsy examinations are expensive, time consuming and are difficult to tolerate for patients. Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology capable of imaging individual eosinophils as highly scattering cells (diameter between 8 µm to 15 µm) in the epithelium. Our lab has developed a tethered SECM capsule that can be swallowed by unsedated patients. The capsule acquires large area confocal images, equivalent to more than 30,000 HPFs, as it traverses through the esophagus. In this paper, we present the outcome of a clinical study using the tethered SECM capsule for diagnosing EoE. To date, 32 subjects have been enrolled in this study. 88% of the subjects swallowed the capsules without difficulty and of those who swallowed the capsule, 95% preferred the tethered capsule imaging procedure to sedated endoscopic biopsy. Each imaging session took about 12 ± 2.4 minutes during which 8 images each spanning of 24 ± 5 cm2 of the esophagus were acquired. SECM images acquired from EoE patients showed abundant eosinophils as highly scattering cells in squamous epithelium. Results from this study suggest that the SECM capsule has the potential to become a less-invasive, cost-effective tool for diagnosing EoE and monitoring the response of this disease to therapy.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"14 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"132770460","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Adam Eshein, The-Quyen Nguyen, A. Radosevich, Bradley Gould, Wenli Wu, V. Konda, Leslie W. Yang, A. Koons, Seth Feder, Vesta Valuckaite, H. Roy, V. Backman
While there are a plethora of in-vivo spectroscopic techniques that have demonstrated the ability to detect a number of diseases in research trials, very few techniques have successfully become a fully realized clinical technology. This is primarily due to the stringent demands on a clinical device for widespread implementation. Some of these demands include: simple operation requiring minimal or no training, safe for in-vivo patient use, no disruption to normal clinic workflow, tracking of system performance, warning for measurement abnormality, and meeting all FDA guidelines for medical use. Previously, our group developed a fiber optic probe-based optical sensing technique known as low-coherence enhanced backscattering spectroscopy (LEBS) to quantify tissue ultrastructure in-vivo. Now we have developed this technique for the application of prescreening patients for colonoscopy in a primary care (PC) clinical setting. To meet the stringent requirements for a viable medical device used in a PC clinical setting, we developed several novel components including an automated calibration tool, optical contact sensor for signal acquisition, and a contamination sensor to identify measurements which have been affected by debris. The end result is a state-of-the-art medical device that can be realistically used by a PC physician to assess a person’s risk for harboring colorectal precancerous lesions. The pilot study of this system shows great promise with excellent stability and accuracy in identifying high-risk patients. While this system has been designed and optimized for our specific application, the system and design concepts are universal to most in-vivo fiber optic based spectroscopic techniques.
{"title":"Creating an optical spectroscopy system for use in a primary care clinical setting (Conference Presentation)","authors":"Adam Eshein, The-Quyen Nguyen, A. Radosevich, Bradley Gould, Wenli Wu, V. Konda, Leslie W. Yang, A. Koons, Seth Feder, Vesta Valuckaite, H. Roy, V. Backman","doi":"10.1117/12.2211421","DOIUrl":"https://doi.org/10.1117/12.2211421","url":null,"abstract":"While there are a plethora of in-vivo spectroscopic techniques that have demonstrated the ability to detect a number of diseases in research trials, very few techniques have successfully become a fully realized clinical technology. This is primarily due to the stringent demands on a clinical device for widespread implementation. Some of these demands include: simple operation requiring minimal or no training, safe for in-vivo patient use, no disruption to normal clinic workflow, tracking of system performance, warning for measurement abnormality, and meeting all FDA guidelines for medical use. Previously, our group developed a fiber optic probe-based optical sensing technique known as low-coherence enhanced backscattering spectroscopy (LEBS) to quantify tissue ultrastructure in-vivo. Now we have developed this technique for the application of prescreening patients for colonoscopy in a primary care (PC) clinical setting. To meet the stringent requirements for a viable medical device used in a PC clinical setting, we developed several novel components including an automated calibration tool, optical contact sensor for signal acquisition, and a contamination sensor to identify measurements which have been affected by debris. The end result is a state-of-the-art medical device that can be realistically used by a PC physician to assess a person’s risk for harboring colorectal precancerous lesions. The pilot study of this system shows great promise with excellent stability and accuracy in identifying high-risk patients. While this system has been designed and optimized for our specific application, the system and design concepts are universal to most in-vivo fiber optic based spectroscopic techniques.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"30 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116349061","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The THz electromagnetic properties of rough surface are explored and their effect on the observed contrast in THz images is quantified. Rough surface scatter is a major source of clutter in THz imaging as the rough features of skin and other tissues result in non-trivial reflection signal modulation. Traditional approaches to data collection utilize dielectric windows to flatten surfaces for THz imaging. However, there is substantial interest surrounding window free imaging as contact measurements are not ideal for a range of candidate diseases and injuries. In this work we investigate the variation in reflected signal in the specular direction from rough surfaces targets with known roughness parameters. Signal to clutter ratios are computed and compared with that predicted by Rayleigh Rough surface scattering theory. It is shown that Rayleigh rough surface scattering theory, developed for rough features larger than the interacting wavelength, holds acceptable at THz frequencies with rough features much smaller than the wavelength. Additionally, we present some biological tissue imaging examples to illustrate the impact of rough surface scattering in image quality.
{"title":"Surface roughness limited contrast to clutter ratios THz medical imaging (Conference Presentation)","authors":"S. Sung, N. Bajwa, Jacob Goell, Z. Taylor","doi":"10.1117/12.2218571","DOIUrl":"https://doi.org/10.1117/12.2218571","url":null,"abstract":"The THz electromagnetic properties of rough surface are explored and their effect on the observed contrast in THz images is quantified. Rough surface scatter is a major source of clutter in THz imaging as the rough features of skin and other tissues result in non-trivial reflection signal modulation. Traditional approaches to data collection utilize dielectric windows to flatten surfaces for THz imaging. However, there is substantial interest surrounding window free imaging as contact measurements are not ideal for a range of candidate diseases and injuries. In this work we investigate the variation in reflected signal in the specular direction from rough surfaces targets with known roughness parameters. Signal to clutter ratios are computed and compared with that predicted by Rayleigh Rough surface scattering theory. It is shown that Rayleigh rough surface scattering theory, developed for rough features larger than the interacting wavelength, holds acceptable at THz frequencies with rough features much smaller than the wavelength. Additionally, we present some biological tissue imaging examples to illustrate the impact of rough surface scattering in image quality.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"47 7","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"114040327","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Aadhar Jain, Elizabeth G Rey, Seoho Lee, Dakota O'Dell, D. Erickson
Anxiety disorders are estimated to be the most common mental illness in US affecting around 40 million people and related job stress is estimated to cost US industry up to $300 billion due to lower productivity and absenteeism. A personal diagnostic device which could help identify stressed individuals would therefore be a huge boost for workforce productivity. We are therefore developing a point of care diagnostic device that can be integrated with smartphones or tablets for the measurement of cortisol - a stress related salivary biomarker, which is known to be strongly involved in body's fight-or-flight response to a stressor (physical or mental). The device is based around a competitive lateral flow assay whose results can then be read and quantified through an accessory compatible with the smartphone. In this presentation, we report the development and results of such an assay and the integrated device. We then present the results of a study relating the diurnal patterns of cortisol levels and the alertness of an individual based on the circadian rhythm and sleep patterns of the individual. We hope to use the insight provided by combining the information provided by levels of stress related to chemical biomarkers of the individual with the physical biomarkers to lead to a better informed and optimized activity schedule for maximized work output.
{"title":"StressPhone: smartphone based platform for measurement of cortisol for stress detection (Conference Presentation)","authors":"Aadhar Jain, Elizabeth G Rey, Seoho Lee, Dakota O'Dell, D. Erickson","doi":"10.1117/12.2212056","DOIUrl":"https://doi.org/10.1117/12.2212056","url":null,"abstract":"Anxiety disorders are estimated to be the most common mental illness in US affecting around 40 million people and related job stress is estimated to cost US industry up to $300 billion due to lower productivity and absenteeism. A personal diagnostic device which could help identify stressed individuals would therefore be a huge boost for workforce productivity. We are therefore developing a point of care diagnostic device that can be integrated with smartphones or tablets for the measurement of cortisol - a stress related salivary biomarker, which is known to be strongly involved in body's fight-or-flight response to a stressor (physical or mental). The device is based around a competitive lateral flow assay whose results can then be read and quantified through an accessory compatible with the smartphone. In this presentation, we report the development and results of such an assay and the integrated device. We then present the results of a study relating the diurnal patterns of cortisol levels and the alertness of an individual based on the circadian rhythm and sleep patterns of the individual. We hope to use the insight provided by combining the information provided by levels of stress related to chemical biomarkers of the individual with the physical biomarkers to lead to a better informed and optimized activity schedule for maximized work output.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"65 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"126610447","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
J. Jo, Dae Yon Hwang, J. Palma, Shuna Cheng, Rodrigo Cuenca, Bilal H. Malik, Joey M. Jabbour, Lisa Cheng, John M. Wright, Kristen C. Maitland
Endogenous fluorescence lifetime imaging (FLIM) provides direct access to the concomitant functional and biochemical changes accompanying tissue transition from benign to precancerous and cancerous. Since FLIM can noninvasively measure different and complementary biomarkers of precancer and cancer, we hypothesize that it will aid in clinically detecting early oral epithelial cancer. Our group has recently demonstrated the detection of benign from premalignant and malignant lesions based on endogenous multispectral FLIM in the hamster cheek-pouch model. Encouraged by these positive preliminary results, we have developed a handheld endoscope capable of acquiring multispectral FLIM images in real time from the oral mucosa. This novel FLIM endoscope is being used for imaging clinically suspicious pre-malignant and malignant lesions from patients before undergoing tissue biopsy for histopathological diagnosis of oral epithelial cancer. Our preliminary results thus far are already suggesting the potential of endogenous FLIM for distinguishing a variety of benign lesions from advanced dysplasia and squamous cell carcinoma (SCC). To the best of out knowledge, this is the first in vivo human study aiming to demonstrate the ability to predict the true malignancy of clinically suspicious lesions using endogenous FLIM. If successful, the resulting clinical tool will allow noninvasive real-time detection of epithelial precancerous and cancerous lesions in the oral mucosa and could potentially be used to assist at every step involved on the clinical management of oral cancer patients, from early screening and diagnosis, to treatment and monitoring of recurrence.
{"title":"In vivo detection of oral epithelial cancer using endogenous fluorescence lifetime imaging: a pilot human study (Conference Presentation)","authors":"J. Jo, Dae Yon Hwang, J. Palma, Shuna Cheng, Rodrigo Cuenca, Bilal H. Malik, Joey M. Jabbour, Lisa Cheng, John M. Wright, Kristen C. Maitland","doi":"10.1117/12.2213846","DOIUrl":"https://doi.org/10.1117/12.2213846","url":null,"abstract":"Endogenous fluorescence lifetime imaging (FLIM) provides direct access to the concomitant functional and biochemical changes accompanying tissue transition from benign to precancerous and cancerous. Since FLIM can noninvasively measure different and complementary biomarkers of precancer and cancer, we hypothesize that it will aid in clinically detecting early oral epithelial cancer. Our group has recently demonstrated the detection of benign from premalignant and malignant lesions based on endogenous multispectral FLIM in the hamster cheek-pouch model. Encouraged by these positive preliminary results, we have developed a handheld endoscope capable of acquiring multispectral FLIM images in real time from the oral mucosa. This novel FLIM endoscope is being used for imaging clinically suspicious pre-malignant and malignant lesions from patients before undergoing tissue biopsy for histopathological diagnosis of oral epithelial cancer. Our preliminary results thus far are already suggesting the potential of endogenous FLIM for distinguishing a variety of benign lesions from advanced dysplasia and squamous cell carcinoma (SCC). To the best of out knowledge, this is the first in vivo human study aiming to demonstrate the ability to predict the true malignancy of clinically suspicious lesions using endogenous FLIM. If successful, the resulting clinical tool will allow noninvasive real-time detection of epithelial precancerous and cancerous lesions in the oral mucosa and could potentially be used to assist at every step involved on the clinical management of oral cancer patients, from early screening and diagnosis, to treatment and monitoring of recurrence.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"142 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123457153","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
R. Pal, Jinping Yang, S. Qiu, S. McCammon, V. Resto, G. Vargas
Volumetric Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) show promise for revealing indicators of neoplasia representing the complex microstructural organization of mucosa, potentially providing high specificity for detection of neoplasia, but is limited by small imaging area. Large area fluorescence methods on the other hand show high sensitivity appropriate for screening but are hampered by low specificity. In this study, we apply MPAM-SHGM following guidance from large area fluorescence, by either autofluorescence or a targeted metabolic fluorophore, as a potentially clinically viable approach for detection of oral neoplasia. Sites of high neoplastic potentially were identified by large area red/green autofluorescence or by a fluorescently labelled deoxy-glucose analog, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) to highlight areas of high glucose uptake across the buccal pouch of a hamster model for OSCC. Follow-up MPAM-SHGM was conducted on regions of interests (ROIs) to assess whether microscopy would reveal microscopic features associated with neoplasia to confirm or exclude large area fluorescence findings. Parameters for analysis included cytologic metrics, 3D epithelial connective tissue interface metrics (MPAM-SHGM) and intensity of fluorescence (widefield). Imaged sites were biopsied and processed for histology and graded by a pathologist. A small sample of human ex vivo tissues were also imaged. A generalized linear model combining image metrics from large area fluorescence and volumetric MPAM-SHGM indicated the ability to delineate normal and inflammation from neoplasia.
体积多光子自体荧光显微镜(MPAM)和二次谐波生成显微镜(SHGM)有望揭示代表粘膜复杂微观结构组织的肿瘤指标，可能为肿瘤的检测提供高特异性，但受成像面积小的限制。另一方面，大面积荧光法具有适合筛选的高灵敏度，但特异性较低。在这项研究中，我们应用MPAM-SHGM在大面积荧光的指导下，通过自身荧光或靶向代谢荧光团，作为一种潜在的临床可行的口腔肿瘤检测方法。通过大面积的红/绿色自体荧光或荧光标记的脱氧葡萄糖类似物2-脱氧-2-[(7-硝基-2,1,3-苯并恶二唑-4-基)氨基]- d -葡萄糖(2-NBDG)来识别高肿瘤潜在部位，以突出OSCC仓鼠模型颊袋内的高葡萄糖摄取区域。对感兴趣区域(roi)进行随访MPAM-SHGM，以评估显微镜是否能显示与瘤变相关的显微特征，以确认或排除大面积荧光发现。分析参数包括细胞学指标、3D上皮结缔组织界面指标(MPAM-SHGM)和荧光强度(宽视场)。成像的部位被活检和处理的组织学和分级由病理学家。一小部分人体离体组织也进行了成像。结合大面积荧光和体积MPAM-SHGM图像指标的广义线性模型表明能够从肿瘤中描绘正常和炎症。
{"title":"Combining large area fluorescence with multiphoton microscopy for improved detection of oral epithelial neoplasia (Conference Presentation)","authors":"R. Pal, Jinping Yang, S. Qiu, S. McCammon, V. Resto, G. Vargas","doi":"10.1117/12.2213927","DOIUrl":"https://doi.org/10.1117/12.2213927","url":null,"abstract":"Volumetric Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) show promise for revealing indicators of neoplasia representing the complex microstructural organization of mucosa, potentially providing high specificity for detection of neoplasia, but is limited by small imaging area. Large area fluorescence methods on the other hand show high sensitivity appropriate for screening but are hampered by low specificity. In this study, we apply MPAM-SHGM following guidance from large area fluorescence, by either autofluorescence or a targeted metabolic fluorophore, as a potentially clinically viable approach for detection of oral neoplasia. Sites of high neoplastic potentially were identified by large area red/green autofluorescence or by a fluorescently labelled deoxy-glucose analog, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) to highlight areas of high glucose uptake across the buccal pouch of a hamster model for OSCC. Follow-up MPAM-SHGM was conducted on regions of interests (ROIs) to assess whether microscopy would reveal microscopic features associated with neoplasia to confirm or exclude large area fluorescence findings. Parameters for analysis included cytologic metrics, 3D epithelial connective tissue interface metrics (MPAM-SHGM) and intensity of fluorescence (widefield). Imaged sites were biopsied and processed for histology and graded by a pathologist. A small sample of human ex vivo tissues were also imaged. A generalized linear model combining image metrics from large area fluorescence and volumetric MPAM-SHGM indicated the ability to delineate normal and inflammation from neoplasia.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"19 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"126771641","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
E. van der Pol, Stefan Weidlich, Y. Lahini, F. Coumans, A. Sturk, R. Nieuwland, M. Schmidt, S. Faez, T. V. van Leeuwen
Background: Extracellular vesicles, such as exosomes, are abundantly present in human body fluids. Since the size, concentration and composition of these vesicles change during disease, vesicles have promising clinical applications, including cancer diagnosis. However, since ~70% of the vesicles have a diameter <70 nm, detection of single vesicles remains challenging. Thus far, vesicles <70 nm have only be studied by techniques that require the vesicles to be adhered to a surface. Consequently, the majority of vesicles have never been studied in their physiological environment. We present a novel label-free optical technique to track single vesicles <70 nm in suspension. Method: Urinary vesicles were contained within a single-mode light-guiding silica fiber containing a 600 nm nano-fluidic channel. Light from a diode laser (660 nm wavelength) was coupled to the fiber, resulting in a strongly confined optical mode in the nano-fluidic channel, which continuously illuminated the freely diffusing vesicles inside the channel. The elastic light scattering from the vesicles, in the direction orthogonal to the fiber axis, was collected using a microscope objective (NA=0.95) and imaged with a home-built microscope. Results: We have tracked single urinary vesicles as small as 35 nm by elastic light scattering. Please note that vesicles are low-refractive index (n<1.4) particles, which we confirmed by combining data on thermal diffusion and light scattering cross section. Conclusions: For the first time, we have studied vesicles <70 nm freely diffusing in suspension. The ease-of-use and performance of this technique support its potential for vesicle-based clinical applications.
{"title":"Label-free tracking of single extracellular vesicles in a nano-fluidic optical fiber (Conference Presentation)","authors":"E. van der Pol, Stefan Weidlich, Y. Lahini, F. Coumans, A. Sturk, R. Nieuwland, M. Schmidt, S. Faez, T. V. van Leeuwen","doi":"10.1117/12.2210876","DOIUrl":"https://doi.org/10.1117/12.2210876","url":null,"abstract":"Background: Extracellular vesicles, such as exosomes, are abundantly present in human body fluids. Since the size, concentration and composition of these vesicles change during disease, vesicles have promising clinical applications, including cancer diagnosis. However, since ~70% of the vesicles have a diameter <70 nm, detection of single vesicles remains challenging. Thus far, vesicles <70 nm have only be studied by techniques that require the vesicles to be adhered to a surface. Consequently, the majority of vesicles have never been studied in their physiological environment. We present a novel label-free optical technique to track single vesicles <70 nm in suspension. Method: Urinary vesicles were contained within a single-mode light-guiding silica fiber containing a 600 nm nano-fluidic channel. Light from a diode laser (660 nm wavelength) was coupled to the fiber, resulting in a strongly confined optical mode in the nano-fluidic channel, which continuously illuminated the freely diffusing vesicles inside the channel. The elastic light scattering from the vesicles, in the direction orthogonal to the fiber axis, was collected using a microscope objective (NA=0.95) and imaged with a home-built microscope. Results: We have tracked single urinary vesicles as small as 35 nm by elastic light scattering. Please note that vesicles are low-refractive index (n<1.4) particles, which we confirmed by combining data on thermal diffusion and light scattering cross section. Conclusions: For the first time, we have studied vesicles <70 nm freely diffusing in suspension. The ease-of-use and performance of this technique support its potential for vesicle-based clinical applications.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"15 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125953885","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Zachary J Smith, Changwon Lee, Tatu Rojalin, R. Carney, S. Hazari, A. Knudson, K. Lam, H. Saari, Elisa Lazaro Ibañez, T. Viitala, T. Laaksonen, M. Yliperttula, S. Wachsmann-Hogiu
Exosomes are small (~100nm) membrane bound vesicles excreted by cells as part of their normal biological processes. These extracellular vesicles are currently an area of intense research, since they were recently found to carry functional mRNA that allows transfer of proteins and other cellular instructions between cells. Exosomes have been implicated in a wide range of diseases, including cancer. Cancer cells are known to have increased exosome production, and may use those exosomes to prepare remote environments for metastasis. Therefore, there is a strong need to develop characterization methods to help understand the structure and function of these vesicles. However, current techniques, such as proteomics and genomics technologies, rely on aggregating a large amount of exosome material and reporting on chemical content that is averaged over many millions of exosomes. Here we report on the use of laser-tweezers Raman spectroscopy (LTRS) to probe individual vesicles, discovering distinct heterogeneity among exosomes both within a cell line, as well as between different cell lines. Through principal components analysis followed by hierarchical clustering, we have identified four “subpopulations” of exosomes shared across seven cell lines. The key chemical differences between these subpopulations, as determined by spectral analysis of the principal component loadings, are primarily related to membrane composition. Specifically, the differences can be ascribed to cholesterol content, cholesterol to phospholipid ratio, and surface protein expression. Thus, we have shown LTRS to be a powerful method to probe the chemical content of single extracellular vesicles.
{"title":"Raman spectroscopy of single extracellular vesicles reveals subpopulations with varying membrane content (Conference Presentation)","authors":"Zachary J Smith, Changwon Lee, Tatu Rojalin, R. Carney, S. Hazari, A. Knudson, K. Lam, H. Saari, Elisa Lazaro Ibañez, T. Viitala, T. Laaksonen, M. Yliperttula, S. Wachsmann-Hogiu","doi":"10.1117/12.2212914","DOIUrl":"https://doi.org/10.1117/12.2212914","url":null,"abstract":"Exosomes are small (~100nm) membrane bound vesicles excreted by cells as part of their normal biological processes. These extracellular vesicles are currently an area of intense research, since they were recently found to carry functional mRNA that allows transfer of proteins and other cellular instructions between cells. Exosomes have been implicated in a wide range of diseases, including cancer. Cancer cells are known to have increased exosome production, and may use those exosomes to prepare remote environments for metastasis. Therefore, there is a strong need to develop characterization methods to help understand the structure and function of these vesicles. However, current techniques, such as proteomics and genomics technologies, rely on aggregating a large amount of exosome material and reporting on chemical content that is averaged over many millions of exosomes. Here we report on the use of laser-tweezers Raman spectroscopy (LTRS) to probe individual vesicles, discovering distinct heterogeneity among exosomes both within a cell line, as well as between different cell lines. Through principal components analysis followed by hierarchical clustering, we have identified four “subpopulations” of exosomes shared across seven cell lines. The key chemical differences between these subpopulations, as determined by spectral analysis of the principal component loadings, are primarily related to membrane composition. Specifically, the differences can be ascribed to cholesterol content, cholesterol to phospholipid ratio, and surface protein expression. Thus, we have shown LTRS to be a powerful method to probe the chemical content of single extracellular vesicles.","PeriodicalId":227483,"journal":{"name":"SPIE BiOS","volume":"16 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2016-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"122212269","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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