Browsing by Author "Wong, Cynthia"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Development of a universal, tunable, miniature fluorescence microscope for use at the point of care
    (The Optical Society, 2018) Wong, Cynthia; Pawlowski, Michal E.; Forcucci, Alessandra; Majors, Catherine E.; Richards-Kortum, Rebecca; Tkaczyk, Tomasz S.
    Fluorescence microscopy can be a powerful tool for cell-based diagnostic assays; however, imaging can be time consuming and labor intensive to perform. Tunable systems give the ability to electronically focus at user selected depths inside an object volume and may simplify the opto-mechanical design of the imaging system. We present a prototype of a universal, tunable, miniature fluorescence microscope built from poly(methyl methacrylate) singlets that incorporates miniature, electrowetted lenses for electronic focusing. We demonstrate the ability of this system to perform clinically relevant differential white blood cell counts using single use custom cartridges pre-loaded with the fluorescent dye acridine orange.
  • Thumbnail Image
    Item
    Development of Fluorescence-based Optical Detection Techniques for Accessible and Efficient Point-of-Care Diagnostics
    (2019-04-12) Wong, Cynthia; Tkaczyk, Tomasz S
    Diagnostics performed at the point of care need to provide portable, rapid, inexpensive, and accurate results while overcoming challenges not typically seen in a central laboratory setting. Fluorescence microscopy gives a unique opportunity to address these needs. Since fluorescence looks for the presence of a target rather than morphological features in a sample, optical performance requirements may be reduced and medical device designs may be simplified. In this dissertation, the development of two classes of devices are presented. The first application targets white blood cell (WBC) differential counting, which can be used to determine bacterial or viral infections, evaluate allergic conditions, diagnose and monitor malignant diseases such as leukemia, and stage HIV infections. Two devices were developed to perform WBC differential counting: (1) a tunable fluorescence microscope using electrowetted lenses and (2) a fluorescence microscope using an ultraviolet (UV) LED as an excitation source. The electrowetted lenses incorporated in the tunable microscope were used to sharply focus on a specific wavelength at a time, simplifying the optical design as chromatic aberrations did not need to be corrected. The use of UV in the second system allowed for the removal of excitation, emission, and dichroic filters, as UV is absorbed by glass components, detectors are typically not sensitive to UV, and many fluorescent dyes are excitable by UV. Thus, this microscope could be fabricated out of commercial optics and use commercially available, low cost sample cartridges. The second application in the dissertation targets patient monitoring for tuberculosis (TB) treatment. Mycobacteria have an intrinsic molecule (thought to be used during metabolism) that is autofluorescent. This molecule had been previously shown in methanobacteria to have different photobleaching rates based on whether the molecule resided in live or dead organisms. Tracking of changes in autofluorescence decay may be used to augment current TB patient monitoring methods that cannot determine viability of organisms and may additionally reduce the time to determining viability from weeks to minutes.
  • Thumbnail Image
    Item
    Differentiating between live and deadﾠMycobacterium smegmatisﾠusing autofluorescence
    (Elsevier, 2016) Wong, Cynthia; Ha, Ngan P.; Pawlowski, Michal E.; Graviss, Edward A.; Tkaczyk, Tomasz S.
    While there have been research efforts to find faster and more efficient diagnostic techniques for tuberculosis (TB), it is equally important to monitor a patient's response to treatment over time, especially with the increasing prevalence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) TB. Between sputum smear microscopy, culture, and GeneXpert, only culture can verify viability of mycobacteria. However, it may take up to six weeks to grow Mycobacterium tuberculosis (Mtb), during which time the patient may have responded to treatment or the mycobacteria are still viable because the patient has MDR or XDR TB. In both situations, treatment incurs increased patient costs and makes them more susceptible to host-drug effects such as liver damage. Coenzyme Factor 420 (F420) is a fluorescent coenzyme found naturally in mycobacteria, with an excitation peak around 420 nm and an emission peak around 470 nm. Using Mycobacterium smegmatis, we show that live and dead mycobacteria undergo different rates of photobleaching over a period of 2 min. These preliminary experiments suggest that the different photobleaching rates could be used to help monitor a patient's response to TB treatment. In future studies, we propose to describe these experiments with Mtb as both M. smegmatis and Mtb use F420.
  • Thumbnail Image
    Item
    Simple ultraviolet microscope using off-the-shelf components for point-of-care diagnostics
    (Public Library of Science, 2019) Wong, Cynthia; Pawlowski, Michal E.; Tkaczyk, Tomasz S.
    At the primary care setting, where there are often no or minimal laboratories, examinations often consist of self-testing and rapid diagnostics. Because of this, medical devices must be simple, robust, and easy to operate. To address these concerns, an alternate fluorescence microscope design uses ultraviolet (UV) excitation, since fluorescent dyes that are excitable in the visible region are also excitable by UV. This may allow for the removal of typical excitation, emission, and dichroic filters as optical components absorb UV wavelengths and UV is not detected by silicon based detectors. Additionally, UV has a very low penetration into samples, which may allow for controlling the depth of excitation, and thus the imaging volume. Based on these ideas, we developed a simple fluorescence microscope built completely from off-the-shelf components that uses UV to image fluorescently stained samples. The simple opto-mechanical design of the system may allow it to be more compact and easy to use, as well as decrease the overall cost of the diagnostic device. For biological validation, we imaged whole blood stained with acridine orange and performed a two-part white blood cell differential count.