R-3 Repository :: Browsing by Author "Tkaczyk, Tomasz"

Browsing by Author "Tkaczyk, Tomasz"

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Assessment of In Vivo Microscopy for Malaria Detection
(2014-10-23) Burnett, Jennifer; Richards-Kortum, Rebecca Rae; Drezek, Rebekah; Tkaczyk, Tomasz
Half of the world’s population is at risk for malaria, yet 90% of deaths due to malaria occur in sub-Saharan Africa, with the highest mortality rates occurring in children under 5. Due to this high mortality rate, children with common flu-like symptoms are often treated for malaria presumptively. This overtreatment with anti-malarial drugs can contribute to the emergence of drug resistant species. Commercially available diagnostic tools, such as blood smear microscopy and rapid diagnostic tests, offer greater specificity than presumptive diagnosis, but require the collection of a finger prick blood sample for diagnosis, generating biohazards and requiring consumables. This thesis introduces a new diagnostic concept to detect parasitized blood cells as they circulate in vivo, avoiding the requirement for blood collection. The work presented here investigates two major components of this concept: 1) develop and characterize a needle-free malaria diagnostic system, and 2) evaluate the performance of the system in biological environments of progressively increasing complexity. Results show promising optical signatures from two biomarkers for malaria detection. This work demonstrates the feasibility for imaging circulating blood cells in vivo through a non-invasive technique, warranting future investigations in a small malaria-infected sample population.
Confocal foveated endomicroscope for the detection of esophageal carcinoma
(The Optical Society, 2015) Shadfan, Adam; Hellebust, Anne; Richards-Kortum, Rebecca; Tkaczyk, Tomasz; Bioengineering
By mimicking the variable resolution of the human eye, a newly designed foveated endomicroscopic objective shows the potential to improve current endoscopic based techniques of identifying abnormal tissue in the esophagus and colon. The prototype miniature foveated objective is imaged with a confocal microscope to provide large field of view images combined with a high resolution central region to rapidly observe morphological structures associated with cancer development in a mouse model.
Design and Fabrication of Snapshot Imaging Spectrometers for Biomedical and Remote Sensing Applications
(2023-08-28) Lu, Jiawei; Tkaczyk, Tomasz
In this work, two advanced snapshot field-integral hyperspectral imaging spectrometers are presented. The first system is based on image mapping mirrors and achieves significantly increased datacube size and dynamic range compared to previous generations. This benefits from a novel design and fabrication method for making the large-format multifaceted mapping mirrors based on the two-photon grayscale lithography (2GL) mode from a commercial two-photon polymerization(2PP) printer. The new fabrication technique can accelerate the fabrication process, increase facet density, eliminate edge eating, and reduce shadowing effects. Additionally, images combining the IMS and dual inverted selective plane illumination microscope (diSPIM) are demonstrated for high spatial and spectral resolution 5D imaging (x,y,z,λ,t). The second system is a fiber-based snapshot hyperspectral imaging spectrometer which can work in both visible (490 nm-732 nm) and short-wave infrared (1090 nm - 1310 nm) wave ranges. A customized relay system with a high numerical aperture (NA) and large field of view (FOV) is designed, fabricated, and then implemented in the spectrometer to overcome the light collection eﬀiciency problems in previous systems. The new relay system enables imaging with a fast frame rate and/or under low illumination conditions. The aims of this work are to advance the two hyperspectral imaging techniques to broaden their applications in biomedical imaging and remote sensing.
Development of cancer screening technologies for imaging nuclear morphology across a large field-of-view
(2020-04-27) Gawedzinski, John Patrick; Tkaczyk, Tomasz
Identifying precancerous lesions in epithelial tissue requires identification of both the location of neoplastic tissue and microscopic examination of subcellular features for confirmation. The location of potential lesions is traditionally identified by performing a wide-field colposcopy, while the diagnosis is provided after histopathological examination of excised tissue. This process can be both time-consuming and invasive, requiring multiple follow up visits and time taken to perform these ex vivo examinations. Endomicroscopy is a technique in which an “optical biopsy” is obtained by imaging the tissue-of-interest in vivo with the ability to visualize subcellular features to achieve a diagnosis. While endomicroscopes are both less invasive and able to function as real-time point-of-care devices, they are inherently limited in field-of-view (FOV) by the size of the distal end of probe. This proposal describes the development of a cervical CAncer Screening Patch (CASP) that can image across the entire epithelium of cervical tissue (~25 mm diameter FOV) without sacrificing the ability to resolve subcellular features. The system utilizes an epifluorescence microscope coupled to a custom-machined, high-resolution polymer image guide in contact with the tissue-of-interest. The image guide will be illuminated by an excitation wavelength at the proximal end and relay the fluorescence signal to also be scanned an imaged at the proximal end, outside of the patient body. Here, the image is scanned and algorithmically stitched together to produce a full field-of-view of the tissue in contact with the image guide. Due to the inflexibility, low contrast, and requirement for custom machining of these image guides, we also explore the potential for manufacturing custom high-resolution waveguides using 3D printing technologies.
Development of Fiber Arrays for Hyperspectral Imaging Spectroscopy Using 2-Photon Polymerization Technique
(2023-09-08) Cao, Haimu; Tkaczyk, Tomasz; Veeraraghavan, Ashok
Fiber-based snapshot imaging spectrometers have emerged as indispensable tools in hyperspectral imaging, finding widespread applications in diverse fields such as biomedical imaging, remote sensing, and astronomy. These spectrometers possess the unique ability to capture intricate spectral information from a scene, making them valuable assets in scientific research and industrial applications. However, their development and fabrication have traditionally been intricate, time-consuming, and costly endeavors, often involving manual assembly of commercial components, leading to large output areas in excess of 20 mm in diameter that only immense optics can accommodate. To address these limitations and push the boundaries of imaging spectrometer technology, this thesis aims to find a new approach that can simplify the fabrication process while further miniaturizing the fiber array. Therefore, I propose a design strategy and proof-of-concept snapshot imaging spectrometer using an array of optical fibers fabricated through the cutting-edge technique of Two-Photon Polymerization (2PP). Leveraging the capabilities of the Nanoscribe GmbH Quantum X system, which enables the precise 3D printing of optical quality structures with submicron resolution and exceptional smoothness (less than 5 nm roughness), this work aims to showcases the development of small core fibers and integrated arrays while demonstrating the functionality of the new snapshot imaging spectrometer. The prototype system features a 3D-printed fiber array with dimensions of 40x80, offering a pitch of 6 microns at the input and 80 microns at the output, with each core having a diameter of 5 µm. Incorporated into a prism-based imaging spectrometer, this fiber array facilitates the realization of 48 spectral channels, ranging from 465 nm to 700 nm, showcasing its ability for multi-spectral imaging. To validate the performance of the system, preliminary imaging results were obtained using a USAF target and a color printed letter C, alongside spectral comparisons to a commercial spectrometer. The obtained results unequivocally demonstrate the functionality and immense potential of the 3D-printed fiber-based snapshot imaging spectrometer. Further characterization tests, such as crosstalk and throughput, will be done in the future. We envision this imaging spectrometer to become a platform technology and encompass applications spanning from biomedical imaging, through environmental imaging and remote sensing, etc.
Development of Principles for the Design and Additive Manufacturing of Optical Systems for Biological and Biomedical Applications
(2022-04-12) Berglund, Gregory; Tkaczyk, Tomasz
While 3D printing has seen increased prevalence in a widespread array of applications, adaptation to optical fabrication has been limited due to strict requirements for surface roughness and shape. However, as in other applications, the ability to 3D print optics has numerous benefits; including the ability to quickly change designs, ability to design parts in freeform geometries, print completed systems with components already in alignment, and expand access to optical fabrication beyond specialized fabrication facilities. Presently, higher end printing options, such as two-photon printing, can create optical elements that can be used directly following the printing process. But these options are limited in terms of both printing sizes and times, as well as having costs that restrict access to fabrication. Consumer-grade options, such as fused deposition modeling or stereolithographic printing, are more affordable and allow for quicker printing times and larger printing volumes. However, the resolutions of these printers are insufficient to meet surface requirement needs for optical parts. This thesis focuses on developing methods to adapt consumer-grade stereolithographic printing to create optical elements and systems within the context of fabricating components for biomedical diagnostic systems for underserved medical populations. We also investigate the ability to fabricate full scale components and systems using two-photon printing, discussing ear imaging as a model application.
Hyperspectral imaging for simultaneous measurements of two FRET biosensors in pancreatic β-cells
(Public Library of Science, 2017) Elliott, Amicia D.; Bedard, Noah; Ustione, Alessandro; Baird, Michelle A.; Davidson, Michael W.; Tkaczyk, Tomasz; Piston, David W.; Bioengineering
Fluorescent protein (FP) biosensors based on Förster resonance energy transfer (FRET) are commonly used to study molecular processes in living cells. There are FP-FRET biosensors for many cellular molecules, but it remains difficult to perform simultaneous measurements of multiple biosensors. The overlapping emission spectra of the commonly used FPs, including CFP/YFP and GFP/RFP make dual FRET measurements challenging. In addition, a snapshot imaging modality is required for simultaneous imaging. The Image Mapping Spectrometer (IMS) is a snapshot hyperspectral imaging system that collects high resolution spectral data and can be used to overcome these challenges. We have previously demonstrated the IMS’s capabilities for simultaneously imaging GFP and CFP/YFP-based biosensors in pancreatic β-cells. Here, we demonstrate a further capability of the IMS to image simultaneously two FRET biosensors with a single excitation band, one for cAMP and the other for Caspase-3. We use these measurements to measure simultaneously cAMP signaling and Caspase-3 activation in pancreatic β-cells during oxidative stress and hyperglycemia, which are essential components in the pathology of diabetes.
Kagome fiber based ultrafast laser microsurgery probe delivering micro-Joule pulse energies
(The Optical Society, 2016) Subramanian, Kaushik; Gabay, Ilan; Ferhanoğlu, Onur; Shadfan, Adam; Pawlowski, Michal; Wang, Ye; Tkaczyk, Tomasz; Ben-Yakar, Adela; Bioengineering
We present the development of a 5 mm, piezo-actuated, ultrafast laser scalpel for fast tissue microsurgery. Delivery of micro-Joules level energies to the tissue was made possible by a large, 31 μm, air-cored inhibited-coupling Kagome fiber. We overcome the fiber’s low NA by using lenses made of high refractive index ZnS, which produced an optimal focusing condition with 0.23 NA objective. The optical design achieved a focused laser spot size of 4.5 μm diameter covering a 75 × 75 μm2 scan area in a miniaturized setting. The probe could deliver the maximum available laser power, achieving an average fluence of 7.8 J/cm2 on the tissue surface at 62% transmission efficiency. Such fluences could produce uninterrupted, 40 μm deep cuts at translational speeds of up to 5 mm/s along the tissue. We predicted that the best combination of speed and coverage exists at 8 mm/s for our conditions. The onset of nonlinear absorption in ZnS, however, limited the probe’s energy delivery capabilities to 1.4 μJ for linear operation at 1.5 picosecond pulse-widths of our fiber laser. Alternatives like broadband CaF2 crystals should mitigate such nonlinear limiting behavior. Improved opto-mechanical design and appropriate material selection should allow substantially higher fluence delivery and propel such Kagome fiber-based scalpels towards clinical translation.
Optical Contrast Agents to Distinguish Benign Inflammation from Neoplasia in Epithelial Tissues
(2016-01-29) Hellebust, Anne E; Richards-Kortum, Rebecca Rae; Farach-Carson, Mary; Gillenwater, Ann; Sikora, Andrew; Tkaczyk, Tomasz
A minimally-invasive, optical strategy to detect and discriminate between inflammation and neoplasia could improve early cancer detection by reducing the number of false positive exams due to benign inflammation. This thesis describes research to optimize optical molecular contrast agents to observe architectural, metabolic, and biomolecular changes from inflammation and cancer in the gastrointestinal tract. My goal was to: 1) understand the limitations of autofluorescence imaging for cancer detection, 2) image exogenous fluorescent contrast agents specific to inflammation and neoplasia in rodent models, and 3) topically deliver a contrast agent cocktail in vivo in a mouse model. Wide field autofluorescence imaging of oral tissue utilizes endogenous tissue contrast to discriminate neoplastic from normal tissue; clinical studies of this technique show good sensitivity but poor specificity. I conducted a confocal microscopy study of 47 biopsies from 20 patients; results showed a similar decrease in autofluorescence in the stroma of inflamed and neoplastic tissue. This finding helps explain the low specificity of wide field autofluorescence imaging. Topically applied exogenous contrast agents could be used to improve discrimination between neoplasia and inflammation. I tested individual fluorescent contrast agents and contrast agent cocktails in chemically induced rodent models of inflammation and neoplasia. The first model used autofluorescence imaging with fluorescence imaging of proflavine to highlight cell nuclei and 2-NBDG to assess metabolic activity for oral cancer detection. A classification algorithm based on proflavine and 2-NBDG staining separated neoplastic from non-neoplastic areas on the tongue with 91% sensitivity and specificity. In the second model, a contrast agent cocktail composed of proflavine, a fluorescently labelled CD45-targeted antibody to identify inflammatory cells, and permeation enhancers was evaluated for topical in vivo delivery to image ulcerative colitis. The antibody identified the presence of inflammation and established topical delivery of antibody sized agents in vivo. These results provide evidence that topically applied contrast agent cocktails could improve discrimination between inflammation and neoplasia when endogenous contrast is insufficient. An optical-based strategy utilizing contrast agent cocktails to observe architectural, metabolic, and biomolecular changes associated with inflammation and cancer could improve early cancer detection by reducing the number of false positives from inflammation.
Optically-sectioned two-shot structured illumination microscopy with Hilbert-Huang processing
(Optical Society of America, 2014) Patorski, Krzysztof; Trusiak, Maciej; Tkaczyk, Tomasz; Bioengineering
We introduce a fast, simple, adaptive and experimentally robust method for reconstructing background-rejected optically-sectioned images using two-shot structured illumination microscopy. Our innovative data demodulation method needs two grid-illumination images mutually phase shifted by π (half a grid period) but precise phase displacement between two frames is not required. Upon frames subtraction the input pattern with increased grid modulation is obtained. The first demodulation stage comprises two-dimensional data processing based on the empirical mode decomposition for the object spatial frequency selection (noise reduction and bias term removal). The second stage consists in calculating high contrast image using the two-dimensional spiral Hilbert transform. Our algorithm effectiveness is compared with the results calculated for the same input data using structured-illumination (SIM) and HiLo microscopy methods. The input data were collected for studying highly scattering tissue samples in reflectance mode. Results of our approach compare very favorably with SIM and HiLo techniques.
Two photon imaging probe with highly efficient autofluorescence collection at high scattering and deep imaging conditions
(Optica Publishing Group, 2024) Camli, Berk; Andrus, Liam; Roy, Aditya; Mishra, Biswajit; Xu, Chris; Georgakoudi, Irene; Tkaczyk, Tomasz; Ben-Yakar, Adela; Bioengineering
In this paper, we present a 2-photon imaging probe system featuring a novel fluorescence collection method with improved and reliable efficiency. The system aims to miniaturize the potential of 2-photon imaging in the metabolic and morphological characterization of cervical tissue at sub-micron resolution over large imaging depths into a flexible and clinically viable platform towards the early detection of cancers. Clinical implementation of such a probe system is challenging due to inherently low levels of autofluorescence, particularly when imaging deep in highly scattering tissues. For an efficient collection of fluorescence signals, our probe employs 12 0.5 NA collection fibers arranged around a miniaturized excitation objective. By bending and terminating a multitude of collection fibers at a specific angle, we increase collection area and directivity significantly. Positioning of these fibers allows the collection of fluorescence photons scattered away from their ballistic trajectory multiple times, which offers a system collection efficiency of 4%, which is 55% of what our bench-top microscope with 0.75 NA objective achieves. We demonstrate that the collection efficiency is largely maintained even at high scattering conditions and high imaging depths. Radial symmetry of arrangement maintains uniformity of collection efficiency across the whole FOV. Additionally, our probe can image at different tissue depths via axial actuation by a dc servo motor, allowing depth dependent tissue characterization. We designed our probe to perform imaging at 775 nm, targeting 2-photon autofluorescence from NAD(P)H and FAD molecules, which are often used in metabolic tissue characterization. An air core photonic bandgap fiber delivers laser pulses of 100 fs duration to the sample. A miniaturized objective designed with commercially available lenses of 3 mm diameter focuses the laser beam on tissue, attaining lateral and axial imaging resolutions of 0.66 µm and 4.65 µm, respectively. Characterization results verify that our probe achieves collection efficiency comparable to our optimized bench-top 2-photon imaging microscope, minimally affected by imaging depth and radial positioning. We validate autofluorescence imaging capability with excised porcine vocal fold tissue samples. Images with 120 µm FOV and 0.33 µm pixel sizes collected at 2 fps confirm that the 300 µm imaging depth was achieved.
Ultrafast laser surgery probe for sub-surface ablation to enable biomaterial injection in vocal folds
(Springer Nature, 2022) Andrus, Liam; Jeon, Hamin; Pawlowski, Michal; Debord, Benoit; Gerome, Frederic; Benabid, Fetah; Mau, Ted; Tkaczyk, Tomasz; Ben-Yakar, Adela; Bioengineering
Creation of sub-epithelial voids within scarred vocal folds via ultrafast laser ablation may help in localization of injectable therapeutic biomaterials towards an improved treatment for vocal fold scarring. Several ultrafast laser surgery probes have been developed for precise ablation of surface tissues; however, these probes lack the tight beam focusing required for sub-surface ablation in highly scattering tissues such as vocal folds. Here, we present a miniaturized ultrafast laser surgery probe designed to perform sub-epithelial ablation in vocal folds. The requirement of high numerical aperture for sub-surface ablation, in addition to the small form factor and side-firing architecture required for clinical use, made for a challenging optical design. An Inhibited Coupling guiding Kagome hollow core photonic crystal fiber delivered micro-Joule level ultrashort pulses from a high repetition rate fiber laser towards a custom-built miniaturized objective, producing a 1/e2 focal beam radius of 1.12 ± 0.10 μm and covering a 46 × 46 μm2 scan area. The probe could deliver up to 3.8 μJ pulses to the tissue surface at 40% transmission efficiency through the entire system, providing significantly higher fluences at the focal plane than were required for sub-epithelial ablation. To assess surgical performance, we performed ablation studies on freshly excised porcine hemi-larynges and found that large area sub-epithelial voids could be created within vocal folds by mechanically translating the probe tip across the tissue surface using external stages. Finally, injection of a model biomaterial into a 1 × 2 mm2 void created 114 ± 30 μm beneath the vocal fold epithelium surface indicated improved localization when compared to direct injection into the tissue without a void, suggesting that our probe may be useful for pre-clinical evaluation of injectable therapeutic biomaterials for vocal fold scarring therapy. With future developments, the surgical system presented here may enable treatment of vocal fold scarring in a clinical setting.
Ultrafast laser surgery probe with a calcium fluoride miniaturized objective for bone ablation
(Optical Society of Americ, 2021) Subramanian, Kaushik; Subramanian, Kaushik; Andrus, Liam; Andrus, Liam; Pawlowski, Michal; Wang, Ye; Tkaczyk, Tomasz; Ben-Yakar, Adela; Ben-Yakar, Adela; Ben-Yakar, Adela; Bioengineering
We present a miniaturized ultrafast laser surgery probe with improved miniaturized optics to deliver higher peak powers and enable higher surgical speeds than previously possible. A custom-built miniaturized CaF2 objective showed no evidence of the strong multiphoton absorption observed in our previous ZnS-based probe, enabling higher laser power delivery to the tissue surface for ablation. A Kagome fiber delivered ultrashort pulses from a high repetition rate fiber laser to the objective, producing a focal beam radius of 1.96 μm and covering a 90×90 μm2 scan area. The probe delivered the maximum available fiber laser power, providing fluences >6 J/cm2 at the tissue surface at 53% transmission efficiency. We characterized the probe’s performance through a parametric ablation study on bovine cortical bone and defined optimal operating parameters for surgery using an experimental- and simulation-based approach. The entire opto-mechanical system, enclosed within a 5-mm diameter housing with a 2.6-mm diameter probe tip, achieved material removal rates >0.1 mm3/min, however removal rates were ultimately limited by the available laser power. Towards a next generation surgery probe, we simulated maximum material removal rates when using a higher power fiber laser and found that removal rates >2 mm3/min could be attained through appropriate selection of laser surgery parameters. With future development, the device presented here can serve as a precise surgical tool with clinically viable speeds for delicate applications such as spinal decompression surgeries.

Browsing by Author "Tkaczyk, Tomasz"

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