Peter TC So, Ph.D. - US grants

Development of fluorescence lifetime microscope. Two new time-resolved fluorescence microscopy techniques are being developed in the LFD. The first technique uses a frequency-modulated CCD camera to simultaneous determine the lifetime of chromophores over the whole image. The second technique uses two-photon scanning technology coupled with frequency-domain instrument to determine the fluorescence intensity and lifetime distribute in a three dimensional sample volume. These new techniques has been applied in collaborative studies such as DNA-probe interaction, antibody-cell receptor binding, drosophila, embryo development, and oxygen distribute in cells.

technology /technique development; lasers; biomedical resource; biomedical equipment development; bioengineering /biomedical engineering;

technology /technique development; biomedical resource; biomedical equipment development;

An understanding of the kinetics of transport processes is crucial in biology and medicine. On the cellular level, examples of important transport processes include endocytosis of extracellular protein, organelle transport during mitosis, phagocytosis of antigen material, mRNA nucleo-cytoplasmic transport, virus docking and infection. Trafficking inside a complex three-dimension environment is a shared common theme. These trafficking processes are rarely passive or diffusion-controlled in cellular system but are guided through active mechanisms including molecular motors and ion pumps. These complex sequences of events are difficult to resolve when the action of many cells are asynchronously averages. Singe particle tracking (SPT) was developed in the early 1980's to address this problem and has proved to be a powerful technique to further our understanding of membrane protein diffusion, membrane compartmentalization and protein-cytoskeleton interaction. However, the traditional SPT method which uses wide field to the study of two-dimensional systems. In this proposal, the applicant plans to develop a 3-D particle tracking technique utilizing the inherent sub-femtoliter localization of two photon excitation. Using a real-time feedback system, he can dynamically position this excitation volume to follow the position of a fluorescent particle under transport by maximizing detected fluorescent intensity. It has been well established that the sequence of cellular transport are often controlled by biochemical signals. Fluorescent spectroscopic method applied in microscopy setting can provide sensitive and specific sampling of the local cellular biochemical states. Dr. So proposes to integrate wavelength resolved spectroscopy with 3D-SPT to continuously monitor the particle micro- environment along its transpiration path. The first application of this instrument will be focused on the study of receptor-mediate protein endocytosis.

EIA-9871329 Lauffenburger, Douglas A. MIT MRI: Development of the MIT QMIP Network This request pertains to an innovative initiative that brings together investigators from HST, CBE and the School of Engineering to further support an MIT wide microscopy network. Three phases are involved: Phase I - The Light, fluorescent, confocal, atomic-force, and transmission electron microscopes will be connected to a central server/router. Acquired images will be transported via high-speed ATM connections and downloaded to large capacity storage systems so that more complex, computer-intensive processing methods can be employed. Phase II - An advanced object-oriented programming environment and associated database will be established. Phase III -The network will be extended to an even wider group of investigators both on and off campus. Accessibility of the network through the Internet will provide a powerful teaching tool that can be tapped by other less-advantaged institutions with a relatively small investment.

Fluorescence correlation spectroscopy studies with biologic materials.

This award will fund the development of a Standing Wave Total Internal Reflection Microscopy (SW-TIRM). This microscope has the potential to provide lateral resolution better than one twelfth of the excitation wavelength. This technique is based on the formation of high frequency standing wave excitation in a total internal reflection geometry. Using a simple algorithm, high-resolution images can be re-constructed from a series of images at different phases of the standing wave. Of particular promise is the ability to perform non-contact imaging of biological specimens at a resolution that is comparable to or better than that of scanning probe microscopy. A preliminary SW-TIRM has been constructed to demonstrate the principle of this technique in 1D. With funding from this award, the capabilities of this current instrument will be extended from 1D to 2D and 3D, use scattered light instead of fluorescence, and incorporate digital deconvolution and two photon methods to image analysis. Two graduate students will be directly involved in the development of this instrument.

A central theme of this group of proposals is the use of quantifiable mechanical perturbation at the microscopic scale to elicit and study cellular mechano-responses. This requirement necessitates the development of quantitative microscopic force transduction methods to apply external forces at selected cell locations. Further, the magnitude and frequency of the applied force have to precisely controlled. The resultant cellular responses to be monitored non-invasively using high sensitivity fluorescence microscopy with 3-D resolution. We propose to establish a core fluorescence microscope facility housing a two-photon confocal microscope with quantitative micro-manipulation capability to serve this common need. The traditional approach to study mechanotransduction on the microscopic level is the use of gradient optical trap (optical tweezers). Optical tweezers have been used in studies ranging from monitoring the deformability of cellular cytoskeleton to the measurement of force generated by a single molecular motor. Optical force on the order of pico- newtons is generated on micro size particles held at the local point of light. After coupling these particles to the cell membrane surface receptors, mechanical perturbation can be applied at specific locations. Our projects further require generating different models of mechanical deformations of the cytoskeleton. A number of these deformation modes of mechanical deformations of the cytoskeleton. A number of these deformation modes are achieved by applying stress at multiple cellular locations and will require the simultaneous manipulation of multiple particles. We will develop a novel multiple trap optical tweezer system where up to four individual particles can be simultaneously manipulated.

Mechanical stimuli regulate many cellular responses, particularly within the cardiovascular system. Understanding the process of mechanotransduction has been challenging, in part because techniques for precisely controlled mechanical stimulation have not been widely used. We proposed that rigorous, highly specific and controllable methods of studying mechanotransduction are needed. By combining the sensitive reporting, we have developed a powerful system for studying mechanotransduction. We have identified several novel genes that are highly mechanoresponsive in human aortic smooth muscle cells. By cloning functional promoters for these genes 5' to the beta-lactamase reporter gene, we have created a powerful specific molecular assay for gene induction in living cells. We will perform mechanical stimulation of cells using magnetic force and follow specific promoter responses using two-photon 3D microscopy. Thus, this experimental system will permit the study of specific molecular responses by precisely controlled mechanostimulation. The specific hypotheses to be tested are highly relevant to a number of cardiovascular diseases, including hypertensive vasculopathy. However, we believe that the lessons from these studies will extend beyond the vascular smooth muscle cell and provide insight into tissue engineering principles. Aim 1) We will explore characteristics of the mechanical stimulus necessary to induce a molecular response. Aim 2) We will explore the role of mechanotransduction through specific integrin subunits in the molecular mechanoresponses of vascular smooth muscle cells. Aim 3) We will explore the relationship between the nature of the mechanical stimulus required to induce one gene may be greater than the stimulus required to induce another gene.

DESCRIPTION (provided by applicant): Two-photon microscope is a powerful tool for in vivo imaging of cellular and extracellular matrix structures with sub-micron resolution. Two-photon based non-invasive optical biopsy methods have the potential of being used as an adjunct to excisional biopsy and histopathology. While the utility of two-photon microscopy for biological studies has been clearly demonstrated in areas such as neurobiology and embryology, its clinical potential remains unrealized. The operational complexity, size, imaging speed and cost stand in the way of testing this new technology in a clinical setting. This proposal addresses these difficulties by engineering a compact two-photon optical biopsy probe. A hand-held device will be built to image tissue cellular structures at video rate (> 10 frames/sec) with sub-cellular resolution down to a depth of 150-200 -1. A number of preliminary studies have been completed to demonstrate the feasibility of this project. (1) The use of two- photon excitation to image tissue cellular structures and metabolism based on its autofluorescence was demonstrated, Preliminary data indicating this technique's potential to distinguish normal and malignant tissues are included. (2) A prototype video-rate two-photon microscope was constructed. (3) A confocal reflected-light imaging sub-system was incorporated into a two-photon microscope. (4) The distribution of tissue biochemical constituents has been resolved based on their two-photon spectra. (5) The key two-photon photodamage mechanisms were identified, The aim of this proposal is to develop a hand-held two-photon biopsy probe suitable for clinical research. We will study the engineering challenges associated with miniaturizing two-photon technology such as the delivep ultra-short light pulses. It is also critical to avoid tissue photodamage; the maximum perr&sibl.e laser power and dosage levels will be established. We will characterize the performance this device in tissue phantoms, animal models and excised human skin biopsy specimens. We hope that the successful completion of this project will result in a first generation device that will allow the evaluation of two-photon optical biopsy techniques in the clinics.

[unreadable] DESCRIPTION (provided by applicant): The 2008 Gordon Research Conference on Lasers in Medicine and Biology will be held from July 20-27, 2008 at the Holderness School in Plymouth, NH. The intent of the conference is to assemble lead investigators from academia, clinics/hospitals, national laboratories, and industry to examine the future directions of biophotonics research with the goal of improving medical practices and biological understanding. A second goal of this conference is to provide an intellectually challenging, open, and convivial environment to assimilate young researchers into this exciting field. Nine technical sessions are scheduled to provide a broad and comprehensive coverage of the biophotonics field while giving in depth examination of several rapidly developing subfields. Four sessions provide general coverage: Novel biophotonic technologies in the macroscopic area, Novel biophotonic technologies in the microscopic/nanoscopic area, Clinical diagnosis based on endogenous signals, and Clinical diagnosis based on exogenous probes. Four sessions are more focused: Photodynamic therapy, Minimal invasive techniques, Intravital tumor biology investigations in animal models, and Image based bioinformatics. We further scheduled a technical session featuring interactive scientific discussions on two topical issues in the biophotics field; the participants of these debates are limited to graduates students and postdoctoral fellows who will be coached by dedicated faculty mentors. The main goal of these interactive discussion sessions is to further involve junior researchers in active scientific dialogs. We believe that the biophotoics community is an important growing component in our national's biomedical research portfolio and this Gordon Research Conference is vital to the health of our community. We seek funds from the National Institute of Health to support this important meeting. [unreadable] [unreadable] [unreadable]

DESCRIPTION: (provided by applicant): The Massachusetts Institute of Technology (MIT) Laser Biomedical Research Center (LBRC) requests NIH funding support for 26-30 years of operation. Under the leadership of its new Director, Dr. Peter So, LBRC will provide integrative photonic solutions to complex problems in biological research, pharmaceutical development, and medical diagnosis. Dr. So will be assisted by three Associated Directors: Drs. Ramachandra Dasari, Moungi Bawendi, and Andrie Tokmakoff. These senior investigators have a broad range of biophotonics expertise allowing LBRC to put forth three Technology Research and Development Projects proposing new biophotonic methods in the areas of (1) flurosecence instrumentation and probe, (2) phase-resolved spectroscopy and imaging, and (3) Raman and two-dimensional infrared spectroscopies. These TR&D projects are design to create new biophotonic tools for our collaborators to tackle important biomedical research problems in their respective fields. LBRC feature eight of these Driving Biomedical Projects covering the study of molecular and cell biology, neurobiology, cancer biology, diabetes diagnosis, and infectious disease treatment. In addition, LBRC also serve the biomedical community by disseminate matured technologies developed in the center. LBRC feature seven Collaborative Projects covering areas ranging from cancer, immunology, to tissue regeneration. Among these fifteen projects, half of these projects are led by investigators who have collaborated with LBRC in the past. The other half of these projects involves new researchers and new scientists. About 30 percent of these projects are conducted by teams led by clinicians. The rest of the projects are led by biologists or bioengineers. In additoin to research, collaboration and service, LBRC will also pursue a broad spectrum of activies in the dissemination of biophotonics technologies in the center and to train biomedical researcher in their uses. For the past 25 years, the success of LBRC has been built on research excellence and our ability to train stellar classes of biophotonics researchers. We look forward to carry on our mission and serve the biomedical community for the next five years.

? DESCRIPTION (provided by applicant): The goal of this proposal is to develop new methods for high speed monitoring of sensory-driven synaptic activity across all inputs to single living neurons in the context of the intact cerebral cortex. Although our focus is on understanding how synaptic inputs are integrated across a single neuron embedded in an intact circuit, the next generation random access imaging technology we propose is more broadly applicable for monitoring multi-cellular activity representing large intra-and inter areal neuronal networks. The approach improves on the speed and sensitivity of current random-access technology by nearly 2 orders of magnitude, enabling high- throughput interrogation of up to 104 independent locations within a fraction of a millisecond and compatible with imaging using next generation voltage sensitive indicators. In Aim 1 we propose to generate a comprehensive structural map that will allow random access scanning of all excitatory and inhibitory synapses on functionally defined pyramidal cell types expressing a genetically encoded Ca+2 indicator. The data generated in this Aim will be used to develop image segmentation algorithms to quickly convert structural images of the dendritic tree and the associated synapses into a 3D location map with grid coordinates for sparse sampling of activity patterns at known locations using a fast random access imaging approach described in Aim 2. In Aim 2 we will construct and develop an imaging system allowing high throughput, random addressing within 10-100 ms of approximately 10,000 locations corresponding to all excitatory synapses and other functionally relevant dendritic and somal sites on a single neuron. In Aim 3 we will test and validate the utility of our approach.

The goal of this proposal is to elucidate the mechanisms of cortical structural plasticity by combining innovative in vivo imaging technology with classical visual manipulations. This integrative approach holds the potential to revolutionize our understanding of adaptive circuit modification, a fundamental aspect of brain function. Our previous findings show that while pyramidal neurons in layer 2/3 of adult visual cortex show little, if any, change in branch tip length over time, GABAergic non-pyramidal interneurons display significant dendritic branch tip remodelling driven by visual experience in an input and circuit-specific manner. The fact that structural plasticity of interneurons is continuous through adulthood raises the intriguing possibility that local remodelling of inhibitory connections may underlie adult cortical plasticity. Yet, how experience alters inhibitory circuitry is unclear, and how modifications to inhibitory and excitatory circuits are locally coordinated remains unaddressed. In the previous funding period we developed a method for labeling inhibitory synapses in vivo and simultaneously monitored inhibitory synapse and dendritic spine remodeling across the entire dendritic arbor of cortical layer 2/3 pyramidal neurons in vivo during normal and altered visual experience. We found that the rearrangements of inhibitory synapses and dendritic spines are locally clustered, mainly within 10 ¿m of each other, and that this clustering is influenced by experience. In this proposal we seek to characterize with high temporal resolution the nature of the coordinated insertion and removal of excitatory synapses and neighboring inhibitory synapses in the neocortical circuit. To this purpose we will implement a newly developed three-color labeling system to independently and simultaneously monitor the formation and disappearance of dendritic spines along with appearance or removal of the post-synaptic density in these spines, and the appearance and removal of inhibitory synapses along the same dendrites. Using spectrally resolved two-photon microscopy we will 1) monitor the temporal sequence of inhibitory and excitatory synapse remodeling in vivo across the full dendritic arbor of L2/3 pyramidal neurons at short time intervals; 2) monitor the effects of experience- dependent plasticity on coordination of inhibitory and excitatory synapse remodeling; 3) examine the specificity of afferent inputs to coordinated excitatory/inhibitory synaptic pairs. Further, 4) we will develop and implement spectrally resolved multifocal multiphoton microscopy to further enhance imaging speed and allow interrogation of synaptic dynamics at even shorter time intervals.

DESCRIPTION (provided by applicant): Sickle cell disease (SCD), known in the homozygous form as sickle cell anemia, affects 1 in 50 African Americans with debilitating, chronic, crisis episodes and reduced life expectancy. SCD is an inherited blood disorder caused by a single point mutation in the beta-globin gene. Sickle hemoglobin (HbS) has the unique property of polymerizing when deoxygenated, triggering red blood cell (RBC) sickling and dehydration, leading to vaso-occlusion and impaired blood flow in capillaries and small vessels. The biochemistry of HbS polymerization in vitro is well understood. However, inside RBC, the mechanism of underlying changes in cell mechanics and adhesion properties resulting from HbS polymerization is poorly understood due to a lack of appropriate measurement methods and realistic models. Three research teams with complementary expertise in bio-photonics (lead by Peter So, MIT), in biomechanics and microfluidics (lead by Ming Dao, MIT), and in SCD treatment (lead by Gregory Kato, UPMC) will join force to develop technologies that can quantify RBC biomechanics during RBC sickling. While there are many factors contributing to vaso-occlusion, RBC biomechanics is known to play a key role. The development of a predictive vaso-occlusion model will deepen our understanding of SCD etiology on a system level allowing the development of more effective drugs and treatments. Toward these goals, our team will develop reflection mode quantitative phase microscopy and a 3-D dissipative particle dynamics (DPD) multi-scale model. These technologies together will allow us to quantify RBC rheological properties with unprecedented accuracy during sickling transition inside microfluidic devices with precisely controlled oxygenation level. We will further develop complementary phase microscopy based spectroscopic methods to quantify HbS oxygenation and polymerization states. Simultaneous measurement of changes in RBC shape and rheology with changes in HbS biochemical states should allow us to better understand how intracellular molecular level variations drive RBC biomechanics, a key factor in vaso-occlusion and SCD crisis. The power of this approach will be evaluated in pilot studies to elucidate the therapeutic mechanisms of hydroxyurea, the only FDA approved drug specifically for SCD, and Aes -103, a new drug under development. These studies will develop proof of principle that this platform could be utilized in screening new anti-sickling drugs. The UPMC sickle cell disease registry will provide a rich clinical database to annotate the patient specimens that will be analyzed by advanced RBC biomechanics assays. This will allow preliminary exploratory statistical correlation of clinical characteristics to the potential biomarkers derived from the biomechanics assays.

? DESCRIPTION (provided by applicant): Spectrally-resolved imaging is ubiquitous in numerous biological studies ranging from mapping synapse dynamics, to monitoring of intracellular signaling, and studying protein-protein interactions. The ability to independently monitor the lifetime and dynamics of cellular structures, such as the synapse, the nucleus, protein trafficking vesicles, and various other multicomponent complexes is critical to revealing their cellular function as well as their assembly and disassembly. While spectrally resolved visualization of 3-4 different proteins in the same cell is quite routine using confocal microscopy in fixed brain sections or in cell culture, dynamic multi-protein imaging in vivo remains a challenge, yet many intra- and inter- cellular interactions are dependent on the context of an intact tissue. Our goal is to develop and implement spectrally resolved technologies that are compatible with high throughput multiphoton microscopy to allow large volume, in vivo imaging of multicomponent subcellular structures. In the first two aims we propose testing two novel spectrometric approaches for large volume, high-speed imaging, with respective strengths and weaknesses, that can be tailored to tackle different imaging needs. In the third aim, we will develop a highly efficient wavelet based Poisson denoised spectral un-mixing algorithm that can potentially enhance both approaches by allowing accurate analysis of images with far lower SNR. Aim 1: Design a dispersive spectrometer (DS) to enable hyperspectral imaging in a multifocal multiphoton microscope (MMM) system utilizing multianode PMTs (MAPMT). Aim 2: Design a Fourier transform spectrometer (FTS) to enable hyperspectral imaging in MMM and wide-field multiphoton microscopy (WFMM) systems. Aim 3: Develop a morphology-guided, Poisson-denoised maximum likelihood (MLE) spectral decomposition algorithm to reduce SNR requirement of raw images.

TRD 1: FLUORESCENCE SPECTROSCOPY AND MICROSCOPY TECHNIQUES Investigators: P. So (1.1, 1.2) [lead]; M. Bawendi (1.2); G. Schlau-cohen (1.3) Collaborative Projects: Jain (CP1), Boyden (CP4), Campagnola (CP8), Coleman (CP9) Project Summary: Fluorescence spectroscopy and imaging are key techniques in the repertoire of the biomedical research community. In the LBRC, the investigators leverage their expertise in precision spectroscopy, contrast agent development, and coherent spatial and temporal control of ultrafast pulses to develop cutting-edge technologies for analyte-specific investigation of biological systems, from proteins to whole organisms. This fluorescence-based TRD builds upon 3D light sculpting techniques and short-wave infrared (SWIR) technologies developed in the current cycle with three exciting new directions: high-throughput deep SWIR imaging (TRD1.1), high-throughput, super-resolution 3D imaging (TRD1.2), and the nanometer- scale study of protein motions (TRD1.3). These directions are motivated by LBRC collaborations. Pushed by the study of cancer biology inside thick solid tumors in vivo, especially for monitoring dynamic events like blood flow and variations in oxygenation (CP1), TRD1.1 seeks to optimize both imaging speed and depth by combining patterned two-photon temporally focused wide-field excitation with compressive-sensing algorithms to image ultra-bright quantum dots (TRD4). Pushed also by Dr. Boyden's work to map the connection diagram of the brain (CP4), which in turn requires high-throughput identification of synaptic clefts at 50 nm resolution throughout a 0.5 cm3 volume. Based on our expertise in structured illumination (SI) and point spread function (PSF) engineering, TRD1.2 seeks to improve super-resolution imaging speed to approach 1G voxel/sec in order to map the whole brain within ~1 year. The same super-resolution approach is employed for high- throughput 3D microfabrication of an extracellular matrix to control cancer cell migration and tissue regeneration (CP8). Finally, pushed by the need for new insight into the signaling mechanisms of receptors, which are the targets of cancer therapeutics (CP9), TRD1.3 will develop fluorescence spectroscopy tools with nanometer spatial and sub-millisecond temporal resolution. In summary, this TRD further extends the core strength of the LBRC in fluorescence instrumentation by introducing these three new research directions.

TRD 2. QUANTITATIVE PHASE MICROSCOPY AND SPECTROSCOPY TECHNIQUES Investigators: Z. Yaqoob (2.1, 2.2, 2.3) [Lead], P. So (2.1, 2.2, 2.3); C.L. Evans (2.3) Collaborative Projects: G. Kato, U. Pittsburgh (CP2); J. Lammerding, Cornell (CP3); E. Boyden, MIT (CP4); Raman, John Hopkins (CP5); D. Fisher, MGH (CP6); P. Krauledat, PNP Research (CP7); P. Campagnola (CP8). PROJECT SUMMARY: The LBRC has been one of the leaders in interferometric imaging, including wide-field quantitative phase microscopy (QPM) and tomographic phase microscopy (TPM), with applications in label-free quantification of cellular morphology, biomechanics, and cell mass/cycle control. During the next cycle, the LBRC will push this technology forward in three fronts. First, the LBRC has successfully developed novel reflection mode QPMs based on temporal and spatial coherence. While these systems show promise to elucidate the biomechanical changes in red blood cells (RBCs), they do not have the necessary depth resolution and sensitivity to study eukaryotic cells. Given that nuclear rheology is important in diseases such as progeria and in cancer metastasis (CP3,5), we push to develop a next generation reflection-mode QPM to quantify biomechanical factors in diseases of nucleated cells (TRD2.1). Second, we have developed several generations of TPMs that provide exquisite 3D maps of the cellular refractive index (RI) distribution, but they offer low throughput and suffer from the ?missing cone? problem. Given the need to study shape variations during RBC sickling (CP2) and cancer cell migration (CP5,8), we push to explore novel tomographic reconstruction based on reflection mode QPM (TRD2.2). Third, while our interferometric imaging work has been mostly label-free, its usage is partly limited by its lack of molecular specificity. As a completely new direction, driven by the need to study (a) sickle cell disease requiring absorption contrast (CRP2), (b) melanomagenesis mechanisms requiring absorption and Raman contrast (CRP6), and (c) binding of cancer antigens to immune cells (CRP7), we push to explore the possibility of wide-field interferometric imaging with molecular specific contrast mechanisms (TRD2.3).

Many mutations resulting in neurodevelopmental and neuropsychiatric disorders target synaptic proteins. Synapse remodeling and loss precedes cell death in neurodegenerative diseases, such as Alzheimer's or Parkinson's, and addictive drugs are known to alter synaptic function. The convergence of many brain disorders at the synapse, indicate that its integrity is critical to normal brain function. Monitoring synapse lifetime and assembly/disassembly in vivo has been hampered by the difficulty of discretely labeling and simultaneously tracking the recruitment and assembly of its individual components. Technology for robust, real-time visualization of synapse formation and loss in vivo would enable the exploration of this fundamental feature of brain development and plasticity, and its dysfunction in brain disease. Our goal is to address this need by developing high-resolution, high throughput temporal focusing (TF) two-photon microscopy for large-volume imaging of synapse assembly-disassembly in the living mouse brain. We propose two aims: 1) Design and implement TF two-photon microscopy for imaging an entire neocortical neuron at synaptic resolution in vivo in less than one minute. Imaging small structures in vivo, especially within the context of the full dendritic arbor, imposes significant time demands due to the need for increased sampling and longer dwell times. Currently, imaging an entire neuron at synaptic resolution takes 60-90 minutes. It is impossible to track events on the order of hours or minutes with such long scan times, or to stably image in awake mice. We propose a novel parallelized approach, line scanning TF two-photon microscopy, to enable in vivo imaging with throughput at least two orders of magnitude higher than point scanning, but with comparable resolution and signal-to-noise ratio. We will test the feasibility of this approach for imaging synaptic structural dynamics in real time, in the awake mouse. 2) Incorporate multi-spectral capabilities into a TF imaging system to enable in vivo tracking of multiple synaptic labels across a single neuron. Visualizing multiple discrete subcellular structures in vivo requires methods for efficient spectrally resolved imaging in deep tissue. We have achieved simultaneous three-color imaging with a single focus scanning multiphoton microscope using Ti-Sapphire lasers and an optical parametric amplifier (OPA) as light sources. However, these devices do not provide light pulses with sufficient peak power for highly parallelized imaging. We will extend the capability of the high-throughput line-scan TF imaging system by implementing multi-color excitation using a regenerative amplifier delivering femtosecond pulses at 1040 nm combined with a tunable OPA providing additional pulses in the 650-1600 nm range. This will allow simultaneous excitation of a broad palette of fluorescent proteins, enabling the tracking of multiple synaptic components at once. The technology we propose will provide a new and powerful tool for dissecting the synaptic roots of many disorders that affect formation, stability, and plasticity of excitatory and inhibitory synapses.

PROJECT SUMMARY TRD 3: RAMAN SPECTROSCOPY AND IMAGING Investigators: R.R. Dasari (3.1) [co-lead]; I. Barman (3.2); C.L. Evans (3.3) [co-lead] Raman technologies offer the ability to detect, quantify, and visualize molecular species with high sensitivity and high resolution via their unique vibrational fingerprints, enabling a host of biomedical applications ranging from bench to the bedside. The LBRC has been a leader in Raman technology development with contributions such as: blood glucose sensing toolkits, chemometric algorithms, and clinical Raman spectroscopy systems. In this next cycle, the LBRC pushes technologies in three exciting areas: developing a high-speed multimodal confocal Raman and phase microscopy with enhanced resolution to enhance cellular mechanobiology studies (Aim 3.1), developing technologies for probing cellular nanomechanics and the accompanying biological responses (Aim 3.2), and developing a portable, robust clinical coherent Raman imaging system for the assessment of melanoma and other diseases of the skin in vivo (Aim 3.3). The collaboration with Dr. Kato investigates how sickle red cells aging affects their mechanical properties using Raman imaging of long term glycemic markers to quantify cell age and phase imaging of membrane fluctuations to quantify cell mechanics (CP2). The collaboration with Dr. Raman seeks to dissect organ-specific differences in metastatic breast cancer by determining microenvironment induced biophysical and molecular adaptations (CP5). This collaborative effort will offer mechanistic insights into metastasis organotropism, a critical step towards discovering optimal treatment strategies for specific metastatic lesions to improve overall survival. The ongoing collaborative project with Dr. Fisher will translate current findings on the oncogenic nature of the natural pigment pheomelanin in animal models to human studies focused on the identification and therapy of amelanotic melanomas (CP6).

The Massachusetts Institute of Technology (MIT) Laser Biomedical Research Center (LBRC) is a NIH NIBIB research resource. The LBRC is also a part of the G.R. Harrison Spectroscopy Laboratory (SpecLab) administrated by the Department of Chemistry. The LBRC will enter its next funding cycle with seven senior investigators with approximately 10-15 junior staff members managing approximately 40 collaborative and service projects. Comparatively speaking, the LBRC is not a large facility; however, given that the center lies at the intersection seven senior investigator laboratories and interactions with a web of national and international collaborations with over 100 associated personnel, the LBRC is a fairly complex enterprise that needs an efficient management plan with a clear chain of responsibilities to properly function. The leadership team of the LBRC further believes that the majority of the financial resource of the LBRC should be devoted to research, collaborations, and training/dissemination. Therefore, the center's guiding management philosophy is best encapsulated by Albert Einstein's saying: ?Make everything as simple as possible, but not simpler.? We have been and will continue to keep a very lean budget for administration. We are able to function well with minimal administrative expenditure for two reasons: First, as a part of MIT Department of Chemistry and the School of Science, we can leverage the very well-run, larger administrative infrastructure for many LBRC operations. Second, and probably most importantly, we have a highly energetic and collaborative group of senior investigators. While all of us have research and teaching roles beyond our involvement in the LBRC, having an enthusiastic group of senior investigators who are willing to take up administrative and management roles within their busy schedule is partly the reason for success of our center so far. Given our success in the last four years, we see no reason for radical changes. In summary, the LBRC administrative plan has five components: (1) LBRC organization: Describe relationship of LBRC within grantee institution and delineate administrative and operational responsibilities among senior investigators and key staff members. (2) LBRC External Advisory Committee (EAC): Review LBRC EAC structure and membership renewal plan. (3) LBRC operating procedures: Layout / review procedures for CP an SP management, equipment and facility usages, and facility user training. (4) Internal communication and networking plans: (a) Establish new TRD-wide and Center-wide regular meetings, (b) Establish a LBRC Retreat (5) Transition plan towards maintaining operations and service after ?sunset?.

TRD 4: Next-generation nanoprobe toolkit for biomedical applications Investigators: Moungi Bawendi (MIT) (4.1), Ishan Barman (JHU) (4.2), Conor Evans (MGH) (4.2,4.3), Gabriela Schlau-Cohen (MIT) (4.3) Collaborative Projects: Rakesh Jain (MGH) (CP1), Saraswati Sukumar, Johns Hopkins (CP10); PnP Research Corporation(CP7), Matthew Coleman, UC Davis (CP9). This TRD adds a synergistic focus on the development of novel molecular probes to complement and enhance the strong spectroscopy, imaging hardware and label-free assay developments of the LBRC. Molecular probes, tailored to our hardware development projects provide powerful toolkits that meet the demands of our collaborators for ultrasensitive, gene- and protein-specific analysis. We seek to develop nanostructured probes that enable new modes of visualization of biological function in real time. We are pulled by our collaborators to develop engineered nanoprobes to study metastasis and treatment response (Jain, CP1), to understand epigenetics factors (Sukumar, CP10), and to optimize cancer therapeutics (Coleman, CP9; PnP Research Corporation, CP7). We will leverage our bright nanoparticles (NPs) in combination with our advances in the other TRDs to improve our understanding, diagnosis, and monitoring of critical pathological conditions. Ultimately, conjugation of these nanoprobes to targeting entities will enable facile detection of numerous orthogonal biological processes.

Vaso-occlusive crises (VOC) are ultimately responsible for the majority of morbidity and mortality in sickle cell disease (SCD). The initiation of VOC is not fully understood. For RBCs with sickle hemoglobin (HbS), deoxygenation induces polymerization, reducing cellular mechanical deformability, among other biophysical changes, and increasing VOC risk. By utilizing a recently developed interferometric phase and amplitude microscopy (iPAM) technique, we found a subpopulation of ?unfit? RBCs in the blood of SCD patients with altered material properties including shape and viscosity. In a parallel study using a novel microfluidic assay for sickling kinetics (MASK), we found that cellular defects appear to accumulate after either repeated sickling or mechanical stress cycles, resulting in faster sickling, reduced deformability, and significant shape changes in sickle cells. These observations suggest an overarching hypothesis that mechanical fatigue of sickle RBCs by repeated sickling or mechanical loading in circulation causes ?defects? to accumulate, producing an ?unfit? subpopulation of RBCs that is responsible for VOC initiation. This subpopulation of ?unfit? RBCs can be distinguished by iPAM. This proposal will examine this hypothesis by designing a next-generation iPAM platform integrated with MASK, elucidating how repeated mechanical stress affects sickle RBC properties and influences VOC propensity. We have assembled a team of investigators with relevant expertise to tackle this problem. These include Dr. So who is an expert in bioimaging, Dr. Dao who is an expert in microfluidics and biomechanics, and Dr. Higgins who is an expert in sickle cell disease pathophysiology. This team of investigators has worked together for over five years with several joint publications. The work in this proposal is divided into four aims. Aim 1 focuses on developing an extinction-based iPAM that will allow quantification of sickle RBC rheology in addition to fitness index. The RBCs from sickle patients will be studied in a novel microfluidic platform that will enable amplitude- modulated electrodeformation as well as repeated deoxygenation-oxygenation cycles for the cells under study. These technological innovations will allow us to evaluate whether unfit RBCs are mechanically compromised due to the accumulation of mechanical defects and whether these unfit cells sickle faster upon deoxygenation. In Aim 2, we will add the ability to measure both oxy- and deoxy-Hb concentration in iPAM, allowing us to explore whether mechanical cycling affects oxygen transport through the RBC membrane and its effect on HbS polymerization. In Aim 3, polarization-resolved capability will be added to iPAM enabling us to detect whether remnant polymerized HbS may persist inside unfit cells in the normoxic state acting as nuclei to promote polymerization. We will evaluate this possibility as a complementary mechanism beside accumulated membrane defects to explain why unfit cells may sickle faster. Finally, Aim 4 will correlate baseline patient clinical outcome with the level of unfit cells. In this aim, we will further evaluate the effect of hydroxyurea and voxelotor treatment on unfit cell fraction in SCD patients.

Abstract The goal of this proposal to develop a first of its kind acoustically driven quantitative phase microscopy (QPM) rheology system. Mechanical forces play an important role in physiology and pathology. Mechanical, electrical, and chemical phenomena govern all processes in cellular systems such as signaling, differentiation, migration, and apoptosis. Cellular mechanics play important roles in normal physiological processes but perhaps more importantly in numerous abnormal pathological processes. Mechanical environment have been demonstrated to regulate stem cell differentiation; embryo development is guided by many mechanical clues. In pathological processes, stiffening of red blood cell membrane is a factor in driving vaso-occlusive crisis in sickle cell disease and malaria patients. Mechanical forces dysregulation can lead to hypertrophy of cardiomyocytes that can cause sudden cardiac death, most commonly in young patients. Inflammation is known to be regulated by mechanical factors and is related to difficulties in treating chronic wounds. The stiffness of extracellular matrix environment is important in regulating cancer progression and the measurement of cell/tissue mechanical properties has been proposed as a way to identify resection margins during cancer surgery. Mechanopharmacology is also known to modulate cellular drug responses. The importance of cellular biomechanics is well recognized; however, the ability to investigate cellular scale mechanical factors in biology and medicine is limited by the available measurement tools.