After studying Mechanical Engineering as an METU undergraduate, I made a turn toward the optical sciences as I sought out interesting optical measurement techniques to address different engineering questions. As I was performing my MSc degree at METU Mechanical Engineering, I was determined to develop fiber optic interferometry to determine micron level displacements for revealing the lubricant thickness in hermetically-sealed small compressor cylinder systems. This experience led me to learn more about lasers and their applications. After finishing my MSc degree, I found out biological applications of femtosecond lasers which can provide unprecedented precision in imaging and surgery applications while I was pursuing my PhD at UT Austin. Thus, I learned to develop bench-top and endoscopic systems to perform nonlinear imaging assisted microsurgery to understand the vocal fold scarring. As a post-doctoral associate at MIT, I am currently developing optical microscopy and stimulation tools to perform in vivo imaging and optogenetics with awake animal models. Therefore, I am having the ability of acquiring simultaneous image activity from multiple brain regions and activating/deactivating targeted specific brain regions to enhance my understanding of how task-relevant information is distributed and coded across the cortex. Supervisors: Adela Ben Yakar, Peter So, and Mriganka Sur Address: Cambridge, Massachusetts, United States
Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventric... more Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventricular zones from which neuronal progenitors differentiate and migrate radially. Analyses of cerebral organoids thus far have been performed in sectioned tissue or in superficial layers due to their high scattering properties. Here, we demonstrate label-free three-photon imaging of whole, uncleared intact organoids (~2 mm depth) to assess early events of early human brain development. Optimizing a custom-made three-photon microscope to image intact cerebral organoids generated from Rett Syndrome patients, we show defects in the ventricular zone volumetric structure of mutant organoids compared to isogenic control organoids. Longterm imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our labelfree imaging system constitutes a particularly useful platform for tracking normal and abnormal development in individual organoids, as well as for screening therapeutic molecules via intact organoid imaging. Editor's evaluation This manuscript will be of interest to stem cell and developmental biologists who aim to use newly emerging brain organoid models to understand the structure and function of the developing human brain. It presents a technological advance in imaging and describes an innovative method for labeling and tracking cells within organoids to enable the assessment of dynamic processes within the intact organoid. The method is validated in a disease model and addresses a challenge in the field of human stem cell modeling of assessing cells within the 3D structure.
Intrinsic neuronal variability significantly limits information encoding in the primary visual co... more Intrinsic neuronal variability significantly limits information encoding in the primary visual cortex (V1). However, under certain conditions, neurons can respond reliably with highly precise responses to the same visual stimuli from trial to trial. This suggests that there exist intrinsic neural circuit mechanisms that dynamically modulate the inter-trial variability of visual cortical neurons. Here, we sought to elucidate the role of different inhibitory interneurons in reliable coding in mouse V1. To study the interactions between somatostatin-expressing (SST) and parvalbumin-expressing (PV) interneurons, we used a dual-color calcium imaging technique that allowed us to simultaneously monitor these two neural ensembles while awake mice, of both sexes, passively viewed natural movies. SST neurons were more active during epochs of reliable pyramidal neuron firing whereas PV neurons were more active during epochs of unreliable firing. SST neuron activity lagged that of PV neurons, consistent with a feedback inhibitory SST→PV circuit. To dissect the role of this circuit in pyramidal neuron activity, we used temporally limited optogenetic activation and inactivation of SST and PV interneurons during periods of reliable and unreliable pyramidal cell firing. Transient firing of SST neurons increased pyramidal neuron reliability by actively suppressing PV neurons, a proposal that was supported by a rate-based model of V1 neurons. These results identify a cooperative functional role for the SST→PV circuit in modulating the reliability of pyramidal neuron activity.
Nonlinear optical microscopy has enabled in vivo deep tissue imaging on the millimeter scale. A k... more Nonlinear optical microscopy has enabled in vivo deep tissue imaging on the millimeter scale. A key unmet challenge is its limited throughput especially compared to rapid wide-field modalities that are used ubiquitously in thin specimens. Wide-field imaging methods in tissue specimens have found successes in optically cleared tissues and at shallower depths, but the scattering of emission photons in thick turbid samples severely degrades image quality at the camera. To address this challenge, we introduce a novel technique called De-scattering with Excitation Patterning or "DEEP," which uses patterned nonlinear excitation followed by computational imagingassisted wide-field detection. Multiphoton temporal focusing allows high-resolution excitation patterns to be projected deep inside specimen at multiple scattering lengths due to the use of long wavelength light. Computational reconstruction allows high-resolution structural features to be reconstructed from tens to hundreds of DEEP images instead of millions of point-scanning measurements.
The structure of brain regions is assumed to correlate with their function, but there are very fe... more The structure of brain regions is assumed to correlate with their function, but there are very few instances in which the relationship has been demonstrated in the live brain. This is due to the difficulty of simultaneously measuring functional and structural properties of brain areas, particularly at cellular resolution. Here, we performed label-free, third-harmonic generation (THG) microscopy to obtain a key structural signature of cortical areas, their effective attenuation lengths (EAL), in the vertical columns of functionally defined primary visual cortex and five adjacent visual areas in awake mice. EALs measured by THG microscopy in the cortex and white matter showed remarkable correspondence with the functional retinotopic sign map of each area. Structural features such as cytoarchitecture, myeloarchitecture and blood vessel architecture were correlated with areal EAL values, suggesting that EAL is a function of these structural features as an optical property of these areas. These results demonstrate for the first time a strong relationship between structural substrates of visual cortical areas and their functional representation maps in vivo. This study may also help in understanding the coupling between structure and function in other animal models as well as in humans.
Women harboring heterozygous germline mutations of BRCA2 have a 50-80% risk of developing
breast... more Women harboring heterozygous germline mutations of BRCA2 have a 50-80% risk of developing breast cancer, yet the early pathogenesis of these cancers is poorly understood. We sought to reveal early steps in BRCA2-associated carcinogenesis through analysis of sorted cell populations from freshly-isolated, non-cancerous breast tissues among a cohort of BRCA2 mutation carriers and matched controls. Single-cell whole-genome sequencing demonstrates that >25% of BRCA2 carrier (BRCA2mut/+) luminal progenitor (LP) cells exhibit sub-chromosomal copy number variations (CNVs), which are rarely observed in non-carriers. Correspondingly, primary BRCA2mut/+ breast epithelia exhibit spontaneous and replication stress-induced DNA damage together with attenuated replication checkpoint and apoptotic responses, associated with an age-associated expansion of the LP compartment in human carrier tissues. These phenotypes are not associated with loss of wild-type BRCA2. Collectively, these findings provide evidence for BRCA2 haploinsufficiency and associated DNA damage in vivo that precede histologic abnormalities. These results provide unanticipated opportunities for new cancer risk assessment and prevention strategies in high-risk patients.
Cortical neurons often respond to identical sensory stimuli with large variability. However, unde... more Cortical neurons often respond to identical sensory stimuli with large variability. However, under certain conditions, the same neurons can also respond highly reliably. The circuit mechanisms that contribute to this modulation, and their influence on behavior remains unknown. Here we used novel double transgenic mice, dual-wavelength calcium imaging and temporally selective optical perturbation to identify an inhibitory neural circuit in visual cortex that can modulate the reliability of pyramidal neurons to naturalistic visual stimuli. Our results, supported by computational models, suggest that somatostatin interneurons (SST-INs) increase pyramidal neuron reliability by suppressing parvalbumin interneurons (PV-INs) via the inhibitory SSTàPV circuit. Using a novel movie classification task, we further show that, by reducing variability, activating SST-INs can improve the ability of mice to discriminate between ambiguous stimuli.
Two-photon microscopy is used to image neuronal activity, but has severe limitations for studying... more Two-photon microscopy is used to image neuronal activity, but has severe limitations for studying deeper cortical layers. Here, we developed a custom three-photon microscope optimized to image a vertical column of the cerebral cortex>1mm in depth in awake mice with low (<20mW) average laser power. Our measurements of physiological responses and tissue-damage thresholds define pulse parameters and safety limits for damage-free threephoton imaging. We image functional visual responses of neurons expressing GCaMP6s across all layers of the primary visual cortex (V1) and in the subplate. These recordings reveal diverse visual selectivity in deep layers: layer 5 neurons are more broadly tuned to visual stimuli, whereas mean orientation selectivity of layer 6 neurons is slightly sharper, compared to neurons in other layers. Subplate neurons, located in the white matter below cortical layer 6 and characterized here for the first time, show low visual responsivity and broad orientation selectivity.
STLE/ASME 2010 International Joint Tribology Conference, 2010
ABSTRACT The aim of this study is to develop an interferometric technique to measure micron level... more ABSTRACT The aim of this study is to develop an interferometric technique to measure micron level clearances occurring in small piston-cylinder arrangements; such as hermetically sealed compressors. A Cartesian model of the piston cylinder arrangement is manufactured and lateral and vertical motions are generated via a step motor, and micrometers, respectively, to simulate the piston motion and the change of clearance during the motion. Experiments are conducted in air and a lubricant. Measurements show the important effect of surface waviness of the mirror and the effect of the motion of the carriage at micron level measurements.
Fiber optic interferometry has been used to detect small displacements in diverse applications. C... more Fiber optic interferometry has been used to detect small displacements in diverse applications. Counting the number of fringes in fiber-optic interferometry is challenging due to the external effects induced in dynamic systems. In this paper, a novel interference fringe counting technique is developed to convert the intensity of interference data into displacements in the range of micrometers to millimeters while simultaneously resolving external dynamic effects. This technique consists of filtering the rough experimental data, converting filtered optical interference data into displacements, and resolving dynamic effects of the experimental system. Filtering the rough data is performed in time by using the moving average method with a window size of 400 data points. Filtered optical data is further converted into displacement by calculating relative phase differences of each data point compared to local maximum and local minimum points. Next, a linear curve-fit is subtracted from the calculated displacement curve to reveal dynamic effects. Straightness error of the lead screw driven stage, dynamics of the stepper motor, and profile of the reflective surfaces are investigated as the external dynamic effects. Straightness error is characterized by a 9th order polynomial function, and the effect of the dynamics of the stepper motor is fitted using a sinusoidal function. The remaining part of the measurement is the effect of roughness and waviness of the reflective surfaces. As explained in the experimental setup part, two fiber-optic probes detect the vertical relative displacements in the range of 1-50 mm, and the encoder probe detects 13.5 mm horizontal displacement. Thus, this technique can detect three order of magnitude different dynamic displacements with sub-micrometer resolution. The current methodology can be utilized in different applications which require measuring straightness error of lead-screw driven stages, large area surface profile of specimens, and vibration of actuators such as stepper motors.
The aim of this study was to differentiate normal and scarred hamster cheek pouch samples by appl... more The aim of this study was to differentiate normal and scarred hamster cheek pouch samples by applying a quantitative image analysis technique for determining collagen fiber direction and density in second-harmonic generation microscopy images. This paper presents a collagen tissue analysis of scarred cheek pouches of four adult male Golden Syrian hamsters as an animal model for vocal fold scarring. One cheek pouch was scarred using an electrocautery unit and the other cheek was used as a control for each hamster. A home-built upright microscope and a compact ultrafast fiber laser were used to acquire depth resolved epi-collected second-harmonic generation images of collagen fibers. To quantify the average fiber direction and fiber density in each image, we applied two-dimensional Fourier analysis and intensity thresholding at five different locations for each control and scarred tissue sample, respectively. The resultant depth-resolved average fiber direction variance for scarred hamster cheek pouches (0.61 AE 0.03) was significantly lower (p < 0.05) than control tissue (0.73 AE 0.04), indicating increased fiber alignment within the scar. Depth-resolved average voxel density measurements indicated scarred tissues contained greater (p < 0.005) fiber density (0.72 AE 0.09) compared to controls (0.18 AE 0.03). In the present study, image analysis of both fiber alignment and density from depth-resolved second-harmonic generation images in epi-detection mode enabled the quantification of the increased collagen fiber deposition and alignment typically observed in fibrosis. The epi-detection geometry is the only viable method for in vivo imaging as well as imaging thick turbid tissues. These quantitative endpoints, clearly differentiating between control and scarred hamster cheek pouches, provide an objective means to characterize the extent of vocal fold scarring in vivo in preclinical and clinical research. In particular, this non-invasive method offers advantages for monitoring scar treatments in live animals and following the effects of scarring-related treatments such as application of steroids or drugs targeting pathways involved in fibrosis. SCANNING 9999:1–10, 2016.
ABSTRACT We represent an ex-vivo study to investigate maximum imaging depth improvement of third-... more ABSTRACT We represent an ex-vivo study to investigate maximum imaging depth improvement of third-harmonic generation (THG) microscopy. We observed 2.5 times improvement with THG microscopy at 1552 nm wavelength compared to two-photon microscopy at 776 nm.
The growing interest in performing high-resolution, deep-tissue imaging has galvanized the use o... more The growing interest in performing high-resolution, deep-tissue imaging has galvanized the use of longer excitation wavelengths and three-photon-based techniques in nonlinear imaging modalities. This study presents a threefold improvement in maximum imaging depth of ex vivo porcine vocal folds using third-harmonic generation (THG) microscopy at 1552-nm excitation wavelength compared to two-photon microscopy (TPM) at 776-nm excitation wavelength. The experimental, analytical, and Monte Carlo simulation results reveal that THG improves the maximum imaging depth observed in TPM significantly from 140 to 420μm in a highly scattered medium, reaching the expected theoretical imaging depth of seven extinction lengths. This value almost doubles the previously reported normalized imaging depths of 3.5 to 4.5 extinction lengths using three-photon-based imaging modalities. Since tissue absorption is substantial at the excitation wavelength of 1552 nm, this study assesses the tissue thermal damage during imaging by obtaining the depth-resolved temperature distribution through a numerical simulation incorporating an experimentally obtained thermal relaxation time (τ). By shuttering the laser for a period of 2τ, the numerical algorithm estimates a maximum temperature increase of ∼2°C at the maximum imaging depth of 420μm. The paper demonstrates that THG imaging using 1552 nm as an illumination wavelength with effective thermal management proves to be a powerful deep imaging modality for highly scattering and absorbing tissues, such as scarred vocal folds.
OSA, along with co-sponsors SPIE and Material Research Society (MRS) hosts two Congressional fell... more OSA, along with co-sponsors SPIE and Material Research Society (MRS) hosts two Congressional fellows each year. Congressional Fellowships are designed to provide a unique public policy learning experience, demonstrate the value of science-government interaction and bring technical backgrounds and external perspectives to the decision making process in Congress. This program, as well as many other fellowship programs, is facilitated by the American Association for the Advancement of Science (AAAS) and includes an orientation on congressional and executive branch operations and a year long seminar program on issues involving science, technology, and public policy.
Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventric... more Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventricular zones from which neuronal progenitors differentiate and migrate radially. Analyses of cerebral organoids thus far have been performed in sectioned tissue or in superficial layers due to their high scattering properties. Here, we demonstrate label-free three-photon imaging of whole, uncleared intact organoids (~2 mm depth) to assess early events of early human brain development. Optimizing a custom-made three-photon microscope to image intact cerebral organoids generated from Rett Syndrome patients, we show defects in the ventricular zone volumetric structure of mutant organoids compared to isogenic control organoids. Longterm imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our labelfree imaging system constitutes a particularly useful platform for tracking normal and abnormal development in individual organoids, as well as for screening therapeutic molecules via intact organoid imaging. Editor's evaluation This manuscript will be of interest to stem cell and developmental biologists who aim to use newly emerging brain organoid models to understand the structure and function of the developing human brain. It presents a technological advance in imaging and describes an innovative method for labeling and tracking cells within organoids to enable the assessment of dynamic processes within the intact organoid. The method is validated in a disease model and addresses a challenge in the field of human stem cell modeling of assessing cells within the 3D structure.
Intrinsic neuronal variability significantly limits information encoding in the primary visual co... more Intrinsic neuronal variability significantly limits information encoding in the primary visual cortex (V1). However, under certain conditions, neurons can respond reliably with highly precise responses to the same visual stimuli from trial to trial. This suggests that there exist intrinsic neural circuit mechanisms that dynamically modulate the inter-trial variability of visual cortical neurons. Here, we sought to elucidate the role of different inhibitory interneurons in reliable coding in mouse V1. To study the interactions between somatostatin-expressing (SST) and parvalbumin-expressing (PV) interneurons, we used a dual-color calcium imaging technique that allowed us to simultaneously monitor these two neural ensembles while awake mice, of both sexes, passively viewed natural movies. SST neurons were more active during epochs of reliable pyramidal neuron firing whereas PV neurons were more active during epochs of unreliable firing. SST neuron activity lagged that of PV neurons, consistent with a feedback inhibitory SST→PV circuit. To dissect the role of this circuit in pyramidal neuron activity, we used temporally limited optogenetic activation and inactivation of SST and PV interneurons during periods of reliable and unreliable pyramidal cell firing. Transient firing of SST neurons increased pyramidal neuron reliability by actively suppressing PV neurons, a proposal that was supported by a rate-based model of V1 neurons. These results identify a cooperative functional role for the SST→PV circuit in modulating the reliability of pyramidal neuron activity.
Nonlinear optical microscopy has enabled in vivo deep tissue imaging on the millimeter scale. A k... more Nonlinear optical microscopy has enabled in vivo deep tissue imaging on the millimeter scale. A key unmet challenge is its limited throughput especially compared to rapid wide-field modalities that are used ubiquitously in thin specimens. Wide-field imaging methods in tissue specimens have found successes in optically cleared tissues and at shallower depths, but the scattering of emission photons in thick turbid samples severely degrades image quality at the camera. To address this challenge, we introduce a novel technique called De-scattering with Excitation Patterning or "DEEP," which uses patterned nonlinear excitation followed by computational imagingassisted wide-field detection. Multiphoton temporal focusing allows high-resolution excitation patterns to be projected deep inside specimen at multiple scattering lengths due to the use of long wavelength light. Computational reconstruction allows high-resolution structural features to be reconstructed from tens to hundreds of DEEP images instead of millions of point-scanning measurements.
The structure of brain regions is assumed to correlate with their function, but there are very fe... more The structure of brain regions is assumed to correlate with their function, but there are very few instances in which the relationship has been demonstrated in the live brain. This is due to the difficulty of simultaneously measuring functional and structural properties of brain areas, particularly at cellular resolution. Here, we performed label-free, third-harmonic generation (THG) microscopy to obtain a key structural signature of cortical areas, their effective attenuation lengths (EAL), in the vertical columns of functionally defined primary visual cortex and five adjacent visual areas in awake mice. EALs measured by THG microscopy in the cortex and white matter showed remarkable correspondence with the functional retinotopic sign map of each area. Structural features such as cytoarchitecture, myeloarchitecture and blood vessel architecture were correlated with areal EAL values, suggesting that EAL is a function of these structural features as an optical property of these areas. These results demonstrate for the first time a strong relationship between structural substrates of visual cortical areas and their functional representation maps in vivo. This study may also help in understanding the coupling between structure and function in other animal models as well as in humans.
Women harboring heterozygous germline mutations of BRCA2 have a 50-80% risk of developing
breast... more Women harboring heterozygous germline mutations of BRCA2 have a 50-80% risk of developing breast cancer, yet the early pathogenesis of these cancers is poorly understood. We sought to reveal early steps in BRCA2-associated carcinogenesis through analysis of sorted cell populations from freshly-isolated, non-cancerous breast tissues among a cohort of BRCA2 mutation carriers and matched controls. Single-cell whole-genome sequencing demonstrates that >25% of BRCA2 carrier (BRCA2mut/+) luminal progenitor (LP) cells exhibit sub-chromosomal copy number variations (CNVs), which are rarely observed in non-carriers. Correspondingly, primary BRCA2mut/+ breast epithelia exhibit spontaneous and replication stress-induced DNA damage together with attenuated replication checkpoint and apoptotic responses, associated with an age-associated expansion of the LP compartment in human carrier tissues. These phenotypes are not associated with loss of wild-type BRCA2. Collectively, these findings provide evidence for BRCA2 haploinsufficiency and associated DNA damage in vivo that precede histologic abnormalities. These results provide unanticipated opportunities for new cancer risk assessment and prevention strategies in high-risk patients.
Cortical neurons often respond to identical sensory stimuli with large variability. However, unde... more Cortical neurons often respond to identical sensory stimuli with large variability. However, under certain conditions, the same neurons can also respond highly reliably. The circuit mechanisms that contribute to this modulation, and their influence on behavior remains unknown. Here we used novel double transgenic mice, dual-wavelength calcium imaging and temporally selective optical perturbation to identify an inhibitory neural circuit in visual cortex that can modulate the reliability of pyramidal neurons to naturalistic visual stimuli. Our results, supported by computational models, suggest that somatostatin interneurons (SST-INs) increase pyramidal neuron reliability by suppressing parvalbumin interneurons (PV-INs) via the inhibitory SSTàPV circuit. Using a novel movie classification task, we further show that, by reducing variability, activating SST-INs can improve the ability of mice to discriminate between ambiguous stimuli.
Two-photon microscopy is used to image neuronal activity, but has severe limitations for studying... more Two-photon microscopy is used to image neuronal activity, but has severe limitations for studying deeper cortical layers. Here, we developed a custom three-photon microscope optimized to image a vertical column of the cerebral cortex>1mm in depth in awake mice with low (<20mW) average laser power. Our measurements of physiological responses and tissue-damage thresholds define pulse parameters and safety limits for damage-free threephoton imaging. We image functional visual responses of neurons expressing GCaMP6s across all layers of the primary visual cortex (V1) and in the subplate. These recordings reveal diverse visual selectivity in deep layers: layer 5 neurons are more broadly tuned to visual stimuli, whereas mean orientation selectivity of layer 6 neurons is slightly sharper, compared to neurons in other layers. Subplate neurons, located in the white matter below cortical layer 6 and characterized here for the first time, show low visual responsivity and broad orientation selectivity.
STLE/ASME 2010 International Joint Tribology Conference, 2010
ABSTRACT The aim of this study is to develop an interferometric technique to measure micron level... more ABSTRACT The aim of this study is to develop an interferometric technique to measure micron level clearances occurring in small piston-cylinder arrangements; such as hermetically sealed compressors. A Cartesian model of the piston cylinder arrangement is manufactured and lateral and vertical motions are generated via a step motor, and micrometers, respectively, to simulate the piston motion and the change of clearance during the motion. Experiments are conducted in air and a lubricant. Measurements show the important effect of surface waviness of the mirror and the effect of the motion of the carriage at micron level measurements.
Fiber optic interferometry has been used to detect small displacements in diverse applications. C... more Fiber optic interferometry has been used to detect small displacements in diverse applications. Counting the number of fringes in fiber-optic interferometry is challenging due to the external effects induced in dynamic systems. In this paper, a novel interference fringe counting technique is developed to convert the intensity of interference data into displacements in the range of micrometers to millimeters while simultaneously resolving external dynamic effects. This technique consists of filtering the rough experimental data, converting filtered optical interference data into displacements, and resolving dynamic effects of the experimental system. Filtering the rough data is performed in time by using the moving average method with a window size of 400 data points. Filtered optical data is further converted into displacement by calculating relative phase differences of each data point compared to local maximum and local minimum points. Next, a linear curve-fit is subtracted from the calculated displacement curve to reveal dynamic effects. Straightness error of the lead screw driven stage, dynamics of the stepper motor, and profile of the reflective surfaces are investigated as the external dynamic effects. Straightness error is characterized by a 9th order polynomial function, and the effect of the dynamics of the stepper motor is fitted using a sinusoidal function. The remaining part of the measurement is the effect of roughness and waviness of the reflective surfaces. As explained in the experimental setup part, two fiber-optic probes detect the vertical relative displacements in the range of 1-50 mm, and the encoder probe detects 13.5 mm horizontal displacement. Thus, this technique can detect three order of magnitude different dynamic displacements with sub-micrometer resolution. The current methodology can be utilized in different applications which require measuring straightness error of lead-screw driven stages, large area surface profile of specimens, and vibration of actuators such as stepper motors.
The aim of this study was to differentiate normal and scarred hamster cheek pouch samples by appl... more The aim of this study was to differentiate normal and scarred hamster cheek pouch samples by applying a quantitative image analysis technique for determining collagen fiber direction and density in second-harmonic generation microscopy images. This paper presents a collagen tissue analysis of scarred cheek pouches of four adult male Golden Syrian hamsters as an animal model for vocal fold scarring. One cheek pouch was scarred using an electrocautery unit and the other cheek was used as a control for each hamster. A home-built upright microscope and a compact ultrafast fiber laser were used to acquire depth resolved epi-collected second-harmonic generation images of collagen fibers. To quantify the average fiber direction and fiber density in each image, we applied two-dimensional Fourier analysis and intensity thresholding at five different locations for each control and scarred tissue sample, respectively. The resultant depth-resolved average fiber direction variance for scarred hamster cheek pouches (0.61 AE 0.03) was significantly lower (p < 0.05) than control tissue (0.73 AE 0.04), indicating increased fiber alignment within the scar. Depth-resolved average voxel density measurements indicated scarred tissues contained greater (p < 0.005) fiber density (0.72 AE 0.09) compared to controls (0.18 AE 0.03). In the present study, image analysis of both fiber alignment and density from depth-resolved second-harmonic generation images in epi-detection mode enabled the quantification of the increased collagen fiber deposition and alignment typically observed in fibrosis. The epi-detection geometry is the only viable method for in vivo imaging as well as imaging thick turbid tissues. These quantitative endpoints, clearly differentiating between control and scarred hamster cheek pouches, provide an objective means to characterize the extent of vocal fold scarring in vivo in preclinical and clinical research. In particular, this non-invasive method offers advantages for monitoring scar treatments in live animals and following the effects of scarring-related treatments such as application of steroids or drugs targeting pathways involved in fibrosis. SCANNING 9999:1–10, 2016.
ABSTRACT We represent an ex-vivo study to investigate maximum imaging depth improvement of third-... more ABSTRACT We represent an ex-vivo study to investigate maximum imaging depth improvement of third-harmonic generation (THG) microscopy. We observed 2.5 times improvement with THG microscopy at 1552 nm wavelength compared to two-photon microscopy at 776 nm.
The growing interest in performing high-resolution, deep-tissue imaging has galvanized the use o... more The growing interest in performing high-resolution, deep-tissue imaging has galvanized the use of longer excitation wavelengths and three-photon-based techniques in nonlinear imaging modalities. This study presents a threefold improvement in maximum imaging depth of ex vivo porcine vocal folds using third-harmonic generation (THG) microscopy at 1552-nm excitation wavelength compared to two-photon microscopy (TPM) at 776-nm excitation wavelength. The experimental, analytical, and Monte Carlo simulation results reveal that THG improves the maximum imaging depth observed in TPM significantly from 140 to 420μm in a highly scattered medium, reaching the expected theoretical imaging depth of seven extinction lengths. This value almost doubles the previously reported normalized imaging depths of 3.5 to 4.5 extinction lengths using three-photon-based imaging modalities. Since tissue absorption is substantial at the excitation wavelength of 1552 nm, this study assesses the tissue thermal damage during imaging by obtaining the depth-resolved temperature distribution through a numerical simulation incorporating an experimentally obtained thermal relaxation time (τ). By shuttering the laser for a period of 2τ, the numerical algorithm estimates a maximum temperature increase of ∼2°C at the maximum imaging depth of 420μm. The paper demonstrates that THG imaging using 1552 nm as an illumination wavelength with effective thermal management proves to be a powerful deep imaging modality for highly scattering and absorbing tissues, such as scarred vocal folds.
OSA, along with co-sponsors SPIE and Material Research Society (MRS) hosts two Congressional fell... more OSA, along with co-sponsors SPIE and Material Research Society (MRS) hosts two Congressional fellows each year. Congressional Fellowships are designed to provide a unique public policy learning experience, demonstrate the value of science-government interaction and bring technical backgrounds and external perspectives to the decision making process in Congress. This program, as well as many other fellowship programs, is facilitated by the American Association for the Advancement of Science (AAAS) and includes an orientation on congressional and executive branch operations and a year long seminar program on issues involving science, technology, and public policy.
I received the Best Student Poster Award from Dr. Mike Downer at the International Conference on ... more I received the Best Student Poster Award from Dr. Mike Downer at the International Conference on Optics of Surface and Interfaces (OSI-11) held in Austin, TX on July 28-July 3. OSI-11 highlights new developments of optical techniques for probing interfaces and nanostructures , and explore new advances in basic and applied science of surface optics.
I was the runner up for the Jenlab Young Investigator Award for Best Paper presented in the Multi... more I was the runner up for the Jenlab Young Investigator Award for Best Paper presented in the Multiphoton Microscopy in the Biomedical Sciences in SPIE Photonics West Conference. Conference chairs were Ammasi Periasamy (Univ. of Virginia), Karsten Konig (Jenlab GMbH), and Peter So (MIT). I was invited to give a talk about my research at the 8th Workshop on Advanced Multiphoton and Fluorescence Lifetime Imaging Techniques held in Saarland University, Saarbrucken, Germany, on July 1-2 2013.
Throughout the year, OSA offers its members the opportunity to communicate directly with US senat... more Throughout the year, OSA offers its members the opportunity to communicate directly with US senators and representatives. These meetings give the scientific community a voice in the halls of Congress-calling on elected officials to recognize the importance of science and technology, keep federal R&D funding levels consistent and sustainable, and support science, technology, engineering and math (STEM) education programs. OSA organizes all aspects of visits, from scheduling individual appointments to providing participants with detailed background information and talking points.
MIT has a long history, reputation, and strength in providing innovative engineering and technolo... more MIT has a long history, reputation, and strength in providing innovative engineering and technological solutions to the world's biggest problems. As part of this grant, we hope to expand our " out-of-the-box " neuroscience thinking to include opportunities to work closely with engineering experts to tackle the biggest issues in understanding the brain with clear, implementable collaborations.
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breast cancer, yet the early pathogenesis of these cancers is poorly understood. We sought to
reveal early steps in BRCA2-associated carcinogenesis through analysis of sorted cell
populations from freshly-isolated, non-cancerous breast tissues among a cohort of BRCA2
mutation carriers and matched controls. Single-cell whole-genome sequencing demonstrates that
>25% of BRCA2 carrier (BRCA2mut/+) luminal progenitor (LP) cells exhibit sub-chromosomal
copy number variations (CNVs), which are rarely observed in non-carriers. Correspondingly,
primary BRCA2mut/+ breast epithelia exhibit spontaneous and replication stress-induced DNA
damage together with attenuated replication checkpoint and apoptotic responses, associated with
an age-associated expansion of the LP compartment in human carrier tissues. These phenotypes
are not associated with loss of wild-type BRCA2. Collectively, these findings provide evidence
for BRCA2 haploinsufficiency and associated DNA damage in vivo that precede histologic
abnormalities. These results provide unanticipated opportunities for new cancer risk assessment
and prevention strategies in high-risk patients.
breast cancer, yet the early pathogenesis of these cancers is poorly understood. We sought to
reveal early steps in BRCA2-associated carcinogenesis through analysis of sorted cell
populations from freshly-isolated, non-cancerous breast tissues among a cohort of BRCA2
mutation carriers and matched controls. Single-cell whole-genome sequencing demonstrates that
>25% of BRCA2 carrier (BRCA2mut/+) luminal progenitor (LP) cells exhibit sub-chromosomal
copy number variations (CNVs), which are rarely observed in non-carriers. Correspondingly,
primary BRCA2mut/+ breast epithelia exhibit spontaneous and replication stress-induced DNA
damage together with attenuated replication checkpoint and apoptotic responses, associated with
an age-associated expansion of the LP compartment in human carrier tissues. These phenotypes
are not associated with loss of wild-type BRCA2. Collectively, these findings provide evidence
for BRCA2 haploinsufficiency and associated DNA damage in vivo that precede histologic
abnormalities. These results provide unanticipated opportunities for new cancer risk assessment
and prevention strategies in high-risk patients.