Research
The Yildirim lab investigates brain complexity through the lenses of engineering, optics, photonics, behavioral and computational neuroscience to solve basic and translational neuroscience problems. We are creating cutting-edge, custom-made microscopes and computational techniques to perform large-scale fluorescent and label-free imaging, as well as perturbations of neural circuits that result in complex behavior in vivo and in vitro. We are developing diagnostic and therapeutic platforms for a variety of neurological disorders by developing these novel custom-made technologies.
Biography
Dr. Murat Yildirim holds a PhD degree from UT Austin and he received his B.Sc. and M.Sc. degrees from Middle East Technical University in Turkey, both in Mechanical Engineering. He performed his postdoctoral training at Picower Institute for Learning and Memory at the Massachusetts Institute of Technology where he developed custom-made optical systems to perform large scale optical recordings and perturbations in behaving mouse brain and cerebral organoids. His current research interests are developing next generation multiphoton systems to study the structure, function, and biomechanics of the brain with various biological systems and models spanning from cerebral organoids, to small animals, to humans through their interaction with femtosecond laser pulses for the diagnosis, characterization, and treatment of specific brain disorders to positively impact human mental health. He is the recipient of several prestigious awards such as a Pathway to Independence Award (K99/R00) from NIBIB. He is a member of SPIE, OSA, and SfN.
Education & Professional Highlights
Education & Fellowships
Ph.D. Mechanical Engineering – University of Texas at Austin
Austin, TX
2015
Postdoctoral Fellow – Picower Institute for Learning and Memory, Massachusetts Institute of Technology
Cambridge, MA
2022
Awards & Honors
National Institute of Biomedical Imaging and Bioengineering
Grant number: R00EB027706
National Institute of Biomedical Imaging and Bioengineering
Grant number: K99EB027706
Research
Neuroscience meets with Engineering, Optics, and Photonics
Our highly motivated multidisciplinary research team is dedicated to solving basic and translational neuroscience problems by examining brain complexity via the lenses of engineering, optics, photonics, behavioral, and computational neuroscience.
News
09/14/23
Our collaborative work on behavioral neuroscience is just publised in PlosOne Computational Biology
09/13/23
Our collaborative work with Harvard and MIT is just published in Light: Science and Applications Journal
08/15/23
Our postdoctoral scholar, Emre is accepted to participate Allen Brain Institute Workshop
We congratulate Emre for his acceptance to Allen Brain Institute Dynamic Brain Workshop which will be held in Friday Harbor, WA between August 20-September 3, 2023.
04/06/23
We are looking for a Research Technician
The successful applicant will be responsible for rodent handling and behavioral training, stereotaxic surgeries, brain slicing and histology, and carrying out experiments using systemic injections, microinjections, and/or electrophysiological recording. Some lab manager duties, such as rat colony maintenance and keeping track of lab supplies, will also be required, along with training and assisting undergraduate students in the lab. Experience in rodent handling or another of the above skills is desirable but not required.
04/04/23
Dr. Yildirim attended SUNPOSIUM and COSYNE conferences
Dr. Yildirim attended back-to-back two conferences. First one is the Sunposium Conference held in West Palm Beach and the second one is COSYNE Conference held in Montreal.
03/28/23
First social outing
We had our first social outing and our choice was bowling. We enjoyed celebrating our accomplishments in this event. Let's see what the next celebration is.
02/01/23
We have our first photo shoot with our lab members
We are so excited to welcome our new lab members. We had so much fun with our first photo shoot.
01/27/23
Our abstract is accepted at Optical Biophotonics Conference
We are so excited to present our work as an oral presentation at Optica Biophotonics Conference: Optics in the Life Sciences which will be held in Vancouver.
01/22/23
Our abstract is accepted at COSYNE 23
Our abstract is accepted as a poster presentation at COSYNE 23. We are delighted to join this prestigious conference.
10/30/22
Nicole presented our first research poster
Nicole did an outstanding job presenting her (and our lab's) first poster presentation at Kent State University's 10th Annual Neuroscience Symposium. Nicole, congratulations!
10/30/22
Dr. Yildirim gave an invited talk at the Mayo Clinic Science of Medicine Ground Rounds
Dr. Yildirim gave an invited talk entitled as " Of mice and men: Spatiotemporally-targeted high resolution brain imaging and optogenetics in health and disease" in the Mayo Clinic Science of Medicine Ground Rounds.
10/22/22
New paper is posted on arxiv. org
We posted a new paper focusing on combining deep learning and compressive sensing in multiphoton microscopy. Limited throughput is a key challenge in in-vivo deep-tissue imaging using nonlinear optical microscopy. Point scanning multiphoton microscopy, the current gold standard, is slow especially compared to the wide-field imaging modalities used for optically cleared or thin specimens. We recently introduced 'De-scattering with Excitation Patterning or DEEP', as a widefield alternative to point-scanning geometries. Using patterned multiphoton excitation, DEEP encodes spatial information inside tissue before scattering. However, to de-scatter at typical depths, hundreds of such patterned excitations are needed. In this work, we present DEEP2, a deep learning based model, that can de-scatter images from just tens of patterned excitations instead of hundreds. Consequently, we improve DEEP's throughput by almost an order of magnitude. We demonstrate our method in multiple numerical and physical experiments including in-vivo cortical vasculature imaging up to four scattering lengths deep, in preclinical models.
10/21/22
Welcome Nicole
We are delighted to welcome Nicole, our lab technician. She'll be presenting a poster at the Kent State University Brain Symposium this week. What a fantastic start.
09/23/22
Dr. Yildirim was invited to talk at "Becoming a Faculty 101" Panel Discussion
Dr. Yildirim was invited to share his experience on moving from his dependent to independent career at "Becoming a Faculty 101" Panel Discussion organized by Inet NYC. Inet NYC is a international scholar community providing support and professional guidance for all international graduate students and research fellows in STEM fields, that are or will be affiliated to any institution in the NYC area, to expand their skill set and career development opportunities.
9/12/22
Our lab opens its doors today
We are so excited to open our doors officially today. We are so excited to welcome our new lab members and grow together while solving basic and translational neuroscience problems by examining brain complexity via the lenses of engineering, optics, photonics, behavioral, and computational neuroscience. Watch our growth all together.
09/02/22
New paper is published in Journal of Biomedical Optics
We are so glad to publish this paper in collaboration with researchers at UT Austin. In this paper, we showed proof-of-concept application of creating subepithelial voids, injecting and localizing biomaterial for the treatment of vocal fold scarring.
08/17/22
R00 Career Transition grant is approved by NIH
We are so happy to announce that Dr. Yildirim's R00 Career Transition grant is approved by NIH. Congratulations to Dr. Yildirim and the whole team.
08/10/22
New paper is highlighted in several outlets
Our seminal work on label-free three-photon imaging of intact cerebral organoids is highlighted at MIT, Brain Initiative, Medxpress and many other outlets.
MIT Picower Institute Highlight
07/29/22
New paper is published online at Elife journal.
Our seminal paper demonstrating label-free three-photon imaging of intact cerebral organoids derived from Rett syndrome patients for the first time is just published online at Elife.
Here is the paper link: Label-free three-photon imaging of intact human cerebral organoids for tracking early events in brain development and deficits in Rett syndrome | eLife (elifesciences.org)
Here is the video prepared by Picower Institute for Learning and Memory:
07/16/22
New paper is accepted in Elife
The first paper demonstrates the use of label-free three-photon imaging of intact cerebral organoids is accepted in Elife. In this paper, 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. Long-term imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our label-free 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.
06/23/22
Dr. Yildirim gave an invited talk at Brain Research Society of Turkey
On June 21, Dr. Yildirim gave an invited talk entitled "Of mice and men: Spatiotemporally targeted high-resolution imaging and optogenetics in health and disease" in the Young Investigator Seminar Series of Brain Research Society of Turkey.
04/27/22
Check out our open positions
We are looking for motivated technicians, graduate students, and postdoctoral scholars for joining our multidisciplinary lab. Please check our open positions, lab policy and diversity pages.
04/26/22
Our lab will be open on 09/01/22
Our lab will open its doors at the Neuroscience Department at Lerner Research Institute (LRI) in the Cleveland Clinic. We are looking for motivated technicians, graduate students, and postdocs to join our lab. We will be a multi-disciplinary group composed of neuroscientists, engineers, and medical doctors. If you are interested in making new discoveries in the brain through developing world-class technologies and working with world-class colleagues and collaborators, please check our open positions and lab policy/diversity pages.
Our Team
Selected Publications
View publications for Murat Yildirim, PhD
(Disclaimer: This search is powered by PubMed, a service of the U.S. National Library of Medicine. PubMed is a third-party website with no affiliation with Cleveland Clinic.)
2024
Cerebral organoids, self-organizing structures with increased cellular diversity and longevity, have addressed shortcomings in mimicking human brain complexity and architecture. However, imaging intact organoids poses challenges due to size, cellular density, and light-scattering properties. Traditional one-photon microscopy faces limitations in resolution and contrast, especially for deep regions. Here, we first discuss the fundamentals of multiphoton microscopy (MPM) as a promising alternative, leveraging non-linear fluorophore excitation and longer wavelengths for improved imaging of live cerebral organoids. Then, we review recent applications of MPM in studying morphogenesis and differentiation, emphasizing its potential for overcoming limitations associated with other imaging techniques. Furthermore, our paper underscores the crucial role of cerebral organoids in providing insights into human-specific neurodevelopmental processes and neurological disorders, addressing the scarcity of human brain tissue for translational neuroscience. Ultimately, we envision using multimodal multiphoton microscopy for longitudinal imaging of intact cerebral organoids, propelling advancements in our understanding of neurodevelopment and related disorders.
2023
Multiphoton microscopy (MPM) has emerged as a vital tool in neuroscience, enabling deeper imaging with a broader field of view, as well as faster and sub-cellular resolution. Recent innovations in ultrafast fiber laser technology have revolutionized MPM applications in living brains, offering advantages like cost-effectiveness and user-friendliness. In this review, we explore the progress in ultrafast fiber laser technology, focusing on its integration into MPM for neuroscience research. We also examine the utility of femtosecond fiber lasers in fluorescence and label-free two- and three-photon microscopy applications within the field. Furthermore, we delve into future possibilities, including next-generation fiber laser designs, novel laser characteristics, and their potential for achieving high spatial and temporal resolution imaging. We also discuss the integration of fiber lasers with implanted microscopes, opening doors for clinical and fundamental neuroscience investigations.
Limited throughput is a key challenge in in vivo deep tissue imaging using nonlinear optical microscopy. Point scanning multiphoton microscopy, the current gold standard, is slow especially compared to the widefield imaging modalities used for optically cleared or thin specimens. We recently introduced “De-scattering with Excitation Patterning” or “DEEP” as a widefield alternative to point-scanning geometries. Using patterned multiphoton excitation, DEEP encodes spatial information inside tissue before scattering. However, to de-scatter at typical depths, hundreds of such patterned excitations were needed. In this work, we present DEEP2, a deep learning-based model that can de-scatter images from just tens of patterned excitations instead of hundreds. Consequently, we improve DEEP’s throughput by almost an order of magnitude. We demonstrate our method in multiple numerical and experimental imaging studies, including in vivo cortical vasculature imaging up to 4 scattering lengths deep in live mice.
In reversal learning tasks, the behavior of humans and animals is often assumed to be uniform within single experimental sessions to facilitate data analysis and model fitting. However, behavior of agents can display substantial variability in single experimental sessions, as they execute different blocks of trials with different transition dynamics. Here, we observed that in a deterministic reversal learning task, mice display noisy and sub-optimal choice transitions even at the expert stages of learning. We investigated two sources of the sub-optimality in the behavior. First, we found that mice exhibit a high lapse rate during task execution, as they reverted to unrewarded directions after choice transitions. Second, we unexpectedly found that a majority of mice did not execute a uniform strategy, but rather mixed between several behavioral modes with different transition dynamics. We quantified the use of such mixtures with a state-space model, block Hidden Markov Model (block HMM), to dissociate the mixtures of dynamic choice transitions in individual blocks of trials. Additionally, we found that blockHMM transition modes in rodent behavior can be accounted for by two different types of behavioral algorithms, model-free or inference-based learning, that might be used to solve the task. Combining these approaches, we found that mice used a mixture of both exploratory, model-free strategies and deterministic, inference-based behavior in the task, explaining their overall noisy choice sequences. Together, our combined computational approach highlights intrinsic sources of noise in rodent reversal learning behavior and provides a richer description of behavior than conventional techniques, while uncovering the hidden states that underlie the block-by-block transitions.
2022
Limited throughput is a key challenge in in-vivo deep-tissue imaging using nonlinear optical microscopy. Point scanning multiphoton microscopy, the current gold standard, is slow especially compared to the wide-field imaging modalities used for optically cleared or thin specimens. We recently introduced 'De-scattering with Excitation Patterning or DEEP', as a widefield alternative to point-scanning geometries. Using patterned multiphoton excitation, DEEP encodes spatial information inside tissue before scattering. However, to de-scatter at typical depths, hundreds of such patterned excitations are needed. In this work, we present DEEP2, a deep learning based model, that can de-scatter images from just tens of patterned excitations instead of hundreds. Consequently, we improve DEEP's throughput by almost an order of magnitude. We demonstrate our method in multiple numerical and physical experiments including in-vivo cortical vasculature imaging up to four scattering lengths deep, in alive mice.
Significance: The creation of subepithelial voids within scarred vocal folds via ultrafast laser ablation may help in localization of injectable biomaterials toward a clinically viable therapy for vocal fold scarring.
Aim: We aim to prove that subepithelial voids can be created in a live preclinical model and that the ablation process does not engender additional scar formation. We demonstrate localization and long-term retention of an injectable biomaterial within subepithelial voids.
Approach: A benchtop nonlinear microscope was used to create subepithelial voids within healthy and scarred cheek pouches of four Syrian hamsters. A model biomaterial, polyethylene glycol tagged with rhodamine dye, was then injected into these voids using a custom injection setup. Follow-up imaging studies at 1- and 2-week time points were performed using the same benchtop nonlinear microscope. Subsequent histology assessed void morphology and biomaterial retention.
Results: Focused ultrashort pulses can be used to create large subepithelial voids in vivo. Our analysis suggests that the ablation process does not introduce any scar formation. Moreover, these studies indicate localization, and, more importantly, long-term retention of the model biomaterial injected into these voids. Both nonlinear microscopy and histological examination indicate the presence of biomaterial-filled voids in healthy and scarred cheek pouches 2 weeks postoperation.
Conclusions: We successfully demonstrated subepithelial void formation, biomaterial injection, and biomaterial retention in a live preclinical model. This pilot study is an important step toward clinical acceptance of a new type of therapy for vocal fold scarring. Future long-term studies on large preclinical models will utilize a miniaturized surgical probe to further assess the clinical viability of such a therapy.
Multiphoton microscopy is the gold standard for deep tissue fluorescence imaging. Long wavelengths enable hundreds of microns deep penetration of excitation light, but the emission fluorescence at shorter wavelengths encounters scattering before detection. While not being an issue for point scanning geometries, for wide-field geometries emission light scattering degrades the image quality. In this work, we use temporally focused pattered excitations to spatially encode image information before emission light scattering. Upon detection, images are reconstructed computationally by solving a linear inverse problem. We further improve our results by learning inverse solvers and optimal patterns through physics-based deep learning.
In volatile foraging environments, animals need to adapt their learning in accordance with the uncertainty of the environment and knowledge of the hidden structure of the world. In these contexts, previous studies have distinguished between two types of strategies, model-free learning, where reward values are updated locally based on external feedback signals, and inference-based learning, where an internal model of the world is used to make optimal inferences about the current state of the environment. Distinguishing between these strategies during the dynamic foraging behavioral paradigm has been a challenging problem for studies of reward-guided decisions, due to the diversity in behavior of model-free and inference-based agents, as well as the complexities that arise when animals mix between these types of strategies. Here, we developed two solutions that jointly tackle these problems. First, we identified four key behavioral features that together benchmark the switching dynamics of agents in response to a change in reward contingency. We performed computational simulations to systematically measure these features for a large ensemble of model-free and inference-based agents, uncovering an organized structure of behavioral choices where observed behavior can be reliably classified into one of six distinct regimes in the two respective parameter spaces. Second, to address the challenge that arises when animals use multiple strategies within single sessions, we developed a novel state-space method, block Hidden Markov Model (blockHMM), to infer switches in discrete latent states that govern the choice sequences across blocks of trials. Our results revealed a remarkable degree of mixing between different strategies even in expert animals, such that model-free and inference-based learning modes often co-existed within single sessions. Together, these results invite a re-evaluation of the stationarity of behavior during dynamic foraging, provide a comprehensive set of tools to characterize the evolution of learning strategies, and form the basis of understanding neural circuits involved in different modes of behavior within this domain.
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. Long-term imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our label-free 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.
- Article highlighted on MIT’s Picower Institute website
- Article highlighted on MIT's website
2021
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 exists intrinsic neural circuit mechanisms that dynamically modulate the intertrial variability of visual cortical neurons. Here, we sought to elucidate the role of different inhibitory interneurons (INs) in reliable coding in mouse V1. To study the interactions between somatostatin-expressing interneurons (SST-INs) and parvalbumin-expressing interneurons (PV-INs), 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.
- Our paper demonstrates a novel 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.
- Article highlighted in MIT’s Picower Institute website
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 imaging–assisted 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.
- Our paper for the first time demonstrates a novel technique called “DEEP’ which uses patterned nonlinear excitation followed by computational imaging-assisted widefield detection to image deeper and faster in the biological tissues such as blood vessels in the mouse brain.
- Article highlighted on MIT’s website
2020
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 preclinical models as well as in humans .
- Article highlighted on National Eye Institute (NEI)’s website
- Article highlighted on MIT’s website
- Article appeared on the cover of Biomedical Optics Express website
Women harboring heterozygous germline mutations of BRCA2 have a 50 to 80% risk of developing breast cancer, yet the pathogenesis of these cancers is poorly understood. To reveal early steps in BRCA2-associated carcinogenesis, we analyzed sorted cell populations from freshly-isolated, non-cancerous breast tissues of BRCA2 mutation carriers and matched controls. Single-cell whole-genome sequencing demonstrates that greater than 25% of BRCA2 carrier (BRCA2mut/+) luminal progenitor (LP) cells exhibit sub-chromosomal copy number variations, which are rarely observed in non-carriers. Correspondingly, primary BRCA2mut/+ breast epithelia exhibit DNA damage together with attenuated replication checkpoint and apoptotic responses, and an age-associated expansion of the LP compartment. We provide evidence that these phenotypes do not require loss of the wild-type BRCA2 allele. Collectively, our findings suggest that BRCA2 haploinsufficiency and associated DNA damage precede histologic abnormalities in vivo. Using these hallmarks of cancer predisposition will yield unanticipated opportunities for improved risk assessment and prevention strategies in high-risk patients.
2019
The first observation of liquid crystalline dispersions of liquid phase-exfoliated tungsten disulfide flakes is reported in a range of organic solvents. The liquid crystals demonstrate significant birefringence as observed in the linear and circular dichroism measurements respectively. In particular, linear dichroism is observed throughout the visible range while broad-band circular dichroism can be observed in the range from 500–800 nm. Under an applied magnetic field of ±1.5 T the circular dichroism can be switched ON/OFF, while the wavelength range for switching can be tuned from large to narrow range by the proper selection of the host solvent. In combination with photoluminescence capabilities of WS2, this opens a pathway to a wide variety of applications, such as deposition of highly uniform films over large areas for photovoltaic and terahertz devices.
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 greater than 1 mm in depth in awake mice with low (less than 20 mW) average laser power. Our measurements of physiological responses and tissue-damage thresholds define pulse parameters and safety limits for damage-free three-photon 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.
- Article highlighted in NIH Director Francis Collins’s blog
- Article highlighted on MIT’s website
- Article highlighted in many scientific magazines such as BioOpticsWorld
2016
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 preclinical 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 ± 0.03) was significantly lower (p less than 0.05) than control tissue (0.73 ± 0.04), indicating increased fiber alignment within the scar. Depth-resolved average voxel density measurements indicated scarred tissues contained greater (p less than 0.005) fiber density (0.72 ± 0.09) compared to controls (0.18 ± 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
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 µm, 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.
2015
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 420 μm 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 420 μm. 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.
2014
Towards developing precise microsurgery tools for the clinic, we previously developed image-guided miniaturized devices using low repetition rate amplified ultrafast lasers for surgery. To improve the speed of tissue removal while reducing device diameter, here we present a new 5-mm diameter device that delivers high-repetition rate laser pulses for high speed ultrafast laser microsurgery. The device consists of an air-core photonic bandgap fiber (PBF) for the delivery of high energy pulses, a piezoelectric tube actuator for fiber scanning, and two aspheric lenses for focusing the light. Its inline optical architecture provides easy alignment and substantial size reduction to 5 mm diameter as compared to our previous MEMS-scanning devices while realizing improved intensity squared (two-photon) lateral and axial resolutions of 1.16 μm and 11.46 μm, respectively. Our study also sheds light on the maximum pulse energies that can be delivered through the air-core PBF and identifies cladding damage at the input facet of the fiber as the limiting factor. We have achieved a maximum energy delivery larger than 700 nJ at 92% coupling efficiency. An in depth analysis reveals how this value is greatly affected by possible slight misalignments of the beam during coupling and the measured small beam pointing fluctuations. In the absence of these imperfections, self-phase modulation becomes the limiting factor for the maximum energy delivery, setting the theoretical upper bound to near 2 μJ for a 1-m long, 7-μm, air-core PBF. Finally, the use of a 300 kHz repetition rate fiber laser enabled rapid ablation of 150 µm x 150 µm area within only 50 ms. Such ablation speeds can now allow the surgeons to translate the surgery device as fast as ~4 mm/s to continuously remove a thin layer of a 150 µm wide tissue. Thanks to a high optical transmission efficiency of the in-line optical architecture of the device and improved resolution, we could successfully perform ablation of scarred cheek pouch tissue, drilling through a thin slice. With further development, this device can serve as a precise and high speed ultrafast laser scalpel in the clinic.
2013
Ultrafast pulsed lasers can be used to achieve remarkable precision during surgical ablation. Through nonlinear interactions with tissue, ultrafast lasers can provide a largely non-thermal mechanism of ablation and a unique ability to create targeted damage within bulk tissue. These advantages have made ultrafast lasers the ideal surgical tool for various novel applications in ophthalmology. Clinical adoption of ultrafast lasers in other surgical applications remains limited in part due to the lack of a means for fiber delivery of ultrafast laser pulses as a flexible, hand-held surgical endoscope. This review provides an overview of the recent advances in bringing this unique surgical tool into the clinic. We discuss fundamental mechanisms and limitations of ultrafast laser ablation, novel techniques for overcoming these limitations, the current state of clinical applications, and conclude with our recent efforts in developing fiber-coupled probes for flexible ultrafast laser surgery and imaging.
Toward developing a new method for restoring tissue viscoelasticity in scarred vocal folds, we previously proposed a method to localize biomaterials within subepithelial voids ablated using ultrafast laser pulses. The clinical implementation of this method necessitates the quantification of the laser parameters for ablating scarred tissue. Here, we present a comprehensive study of these parameters including ablation threshold and bubble lifetime in healthy and scarred tissues. We also present a new method for extracting tissue-specific ablation threshold and scattering lengths of different tissue layers. This method involves finding the ablation threshold at multiple depths and solving the equations based on Beer’s law of light attenuation for each depth to estimate the unknown parameters. Measured threshold fluences were 1.75 J/cm 2 for vocal folds and 0.5 J/cm 2 for cheek pouches for 3-ps, 776-nm laser pulses. Scarred pouches exhibited 30% lower threshold than healthy pouches, possibly due to the degraded mechanical properties of scarred collagen during wound healing. The analysis of tissue architecture indicated a direct correlation between the ablation threshold and tissue tensile strength and that the bubble lifetime is inversely related to tissue stiffness. Overall, this study sheds light on the required laser parameters for successful implementation of ultrafast laser ablation for phonosurgery.
2012
Vocal fold scarring is a predominant cause of voice disorders yet lacks a reliable treatment method. The injection of soft biomaterials to improve mechanical compliance of the vocal folds has emerged as a promising treatment. Here, we study the use of precise femtosecond laser microsurgery to ablate subsurface voids, with a goal of eventually creating a plane in dense subepithelial scar tissue into which biomaterials can be injected for their improved localization. Specifically, we demonstrate the ablation of small subepithelial voids in porcine vocal fold tissue up to 120 µm below the surface such that larger voids in the active area of vocal fold mucosa ( ∼ 3×10 mm2) can eventually be ablated in about 3 min. We use sub-µJ, 776-nm pulses from a compact femtosecond fiber laser system operating at a 500-kHz repetition rate. The use of relatively high repetition rates, with a small number of overlapping pulses, is critical to achieving ablation in a very short time while still avoiding significant heat deposition. Additionally, we use the same laser for nonlinear optical imaging to provide visual feedback of tissue structure and to confirm successful ablation. The ablation parameters, including pulse duration, pulse energy, spot size, and scanning speed, are comparable to the specifications in our recently developed miniaturized femtosecond laser surgery probes, illustrating the feasibility of developing an ultrafast laser surgical instrument.
2011
We present the optical design of a 9.6-mm diameter fiber-coupled probe for combined femtosecond laser microsurgery and nonlinear optical imaging. Towards enabling clinical use, we successfully reduced the dimensions of our earlier 18-mm microsurgery probe by half, while improving optical performance. We use analytical and computational models to optimize the miniaturized lens system for off-axis scanning aberrations. The optimization reveals that the optical system can be aberration-corrected using simple aspheric relay lenses to achieve diffraction-limited imaging resolution over a large field of view. Before moving forward with custom lenses, we have constructed the 9.6-mm probe using off-the-shelf spherical relay lenses and a 0.55 NA aspheric objective lens. In addition to reducing the diameter by nearly 50% and the total volume by 5 times, we also demonstrate improved lateral and axial resolutions of 1.27 µm and 13.5 µm, respectively, compared to 1.64 µm and 16.4 µm in our previous work. Using this probe, we can successfully image various tissue samples, such as rat tail tendon that required 2-3 × lower laser power than the current state-of-the-art. With further development, image-guided, femtosecond laser microsurgical probes such as this one can enable physicians to achieve the highest level of surgical precision anywhere inside the body.
Careers
Postdoctoral Scholar
Computational Neuroscientist for Analyzing and Modeling Large Scale Recordings and Manipulations in Health and Disease
We are looking for a postdoctoral scholar who will analyze in vivo large-scale optogenetics and imaging dataset with behaving preclinical models in health and disease. With these analysis pipelines, we would like to understand how neural circuits generate patterned activity that gives rise to complex behaviors and phenotypes in health and disease states. It is a fully-funded and three-years long postdoctoral position. Its duration can be extended further.
Who we are: We are highly-motivated multi-disciplinary research group focuses on cracking brain complexity in the lens of engineering, optics, photonics, behavioral and computational neuroscience for basic and translational neuroscience questions. We are developing cutting edge microscopes which allows us to record and manipulate neuronal activity in various spatial and temporal scales in health and disease. Please visit our website, and check our lab policy and DEI (yildirimlab.org).
Who we are looking for: We are looking for a curious computational neuroscientist who is interested in working in a team-oriented environment and interested in making new discoveries in the basic and translational neuroscience. These positions are ideal for someone with a desire to develop skillsets with analyzing large scale recordings and manipulations and dissect neural circuits responsible for preclinical behavior in health and disease states.
Responsibilities: It is required to have a doctoral degree in Neuroscience, Engineering, or related disciplines, and to have expertise on one of these following topics: modeling preclinical behavior such as reinforcement learning and spatial learning, generalized linear model (GLM) for analyzing neural recordings, machine learning such as recurrent neural networks (RNN) for preclinical behavior and neural recordings, and Hidden Markov Modeling (HMM) for analyzing preclinical behavior. It is also required to have experience with coding (MATLAB, R, or Phyton), excellent organizational, analytical, and oral and written communication skills, ability to analyze data and present it in a format suitable for publication, self-motivation, and ability to function effectively in a teamoriented environment.
Preferences: It is preferred to have a doctoral degree in Neuroscience, and to have an expertise in GLM, HMM, and RNN.
Interested candidates should submit CV, cover letter, and contact information of two-three references to Dr. Murat Yildirim, [email protected].
Research News
Research aims to extend observations of reversal learning in preclinical models to human neurological disorders
An ongoing collaboration between Dr. Murat Yildirim and colleagues at Harvard and MIT aims to make it easier to track neurological activity deep within brain tissue.