Single cell analysis is critical for understanding cellular activities, diagnosing clinicaldiseases, and designing personalized treatments. However, the detection of single cells with high sensitivity has been challenging, especially for clinical samples, as targets of detection are often immersed in extremely complex background. Due to the lack of single cell sensitivity, current mainstream approaches isolate the cells and increase cell numbers by culturing, which is time consuming and often leads to the change of cellular population composition and the loss of native characteristics. In addition, the ensembled detection approaches provide only averaged information of the cell population, thereby missing vital cellular heterogeneity information. The applied probes during detection can also alter the native structures and influence the reliability of the results. In this dissertation, novel label- free optical imaging methods for single cell analysis of raw clinical samples are developed and described to address these challenges. First, a large volume imaging platform is developed for rapid diagnostics of clinical samples of critically low bacterial concentrations without enrichment. Both dual channel and multiplexed versions of the platform are introduced for continuous, detailed monitoring and high throughput minimum inhibitory concentration determination, respectively. With these platforms, the susceptibility of the pathogenic microorganisms in raw urine and blood samples are rapidly quantified within 90 minutes and 240 minutes, respectively, significantly improving the diagnostic time. Second, the large volume imaging platform is adapted for rapid drug susceptibility testing of multidrug-resistant mycobacterial species. Using this method, the susceptibility profile of Mycobacterium tuberculosis and nontuberculous mycobacteria are ascertained within a short timeframe - less than two proliferation cycles. By coupling with single particle tracking, the presence of resistant subpopulations at the therapeutic failure limit of 1% can be detected within one day. Last, by sensitively tracking the emergence of precipitation in various polymer solutions with an upper critical solution temperature upon heating using plasmonic scattering microscopy, precise temperature control over the highly localized plasmonic field is achieved. TRPV1 channel is accurately activated without the aid of an external temperature controlling platform, highlighting the capability of the method for single cell manipulation and in-depth analysis.

Chimeric antigen receptor (CAR) T-cell therapies present transformative potentials for progressive and refractory cancer treatment. However, therapy-associated neuronal toxicities, cytokine release syndromes, relapse rates, and the complex responses of patients and medical management have increased the cost of patient care. Prompt point-of-care (POC) quantification of circulating CAR T-cells and associated cytokines could enhance safety, simplify patients' management, and decrease patient care costs. While effective, existing standard detection methods, such as Enzyme-Linked Immunosorbent Assay (ELISA), quantitative Polymerase Chain Reaction(qPCR), and Flow cytometry, are not conducive to quick POC testing due to their complexity and expense. This research introduces a centrifuge-free Rapid Optical Imaging (ROI)-based platform to quantify CAR T-cells and therapy-related cytokine (Interleukin-6) from a single drop of whole blood. Through machine learning, label-free ROI-based CAR T-cell detection has been improved for accuracy compared with fluorescent staining results, and the morphological characteristics of CAR-T cells have been applied to attribute for differentiation and reduce false positives. This multi-layered microfluidic chip integrates cell and cytokines separation, collection, and detection steps, reducing the need for centrifugation or staining procedures. The microfluidic channel system separates white blood cells from whole blood after red blood cell agglutination and membrane filtration. The non-agglutinated samples are then extracted into a subchannel with a functionalized sensor surface for CAR-T-specific detection. Calibration curves were established using blood samples spiked with varying CAR-T cell concentrations. Another subchannel, featuring dual-layer membrane filtration, has been designed for cytokine detection using gold nanoparticle-labeled detection antibodies. Cytokine concentrations are digitally measured by tracking the number of gold nanoparticles in designated zones. This platform aims to offer a rapid and cost-efficient prognostic tool for timely assessment of key molecular and cellular biomarkers of CAR-T therapy patients, facilitating timely and evidence-based treatment adjustments.

Analysis of the characteristics of biomolecules, including size, charge and binding kinetics, is essential for biomedical and life science research and applications. State-of-the-art protein analysis methods rely on separate technologies to quantify these characteristics, and considerable time, cost and analytes are required. Lack of single molecule analysis capability in above methods also making them difficult to study heterogeneous processes and achieving precision diagnosis.To address these issues, several techniques based on surface sensitive optical imaging principles were developed. The first technique is evanescent scattering microscopy (ESM) with single molecule resolution, which is capable of imaging single immunoglobulin G with high signal-to-noise ratio. In addition, nano-oscillator was combined with the ESM to achieve the simultaneous size and charge detection of single proteins. Based on the unique high axial sensitivity of the surface plasmon resonance (SPR), a 3D tracking technique to study the motion and interaction of biomolecules was introduced. With the additional dimension, more information in particle motions can be revealed compared to conventional 2D bright field tracking. By tracking the motion of nanoparticles, motion pattern of tethered nanoparticles and interaction between double-stranded DNA and an enzyme can be visualized. The G protein-coupled receptors (GPCRs) expressed virion oscillator array for quantification of the binding kinetics of small molecule drugs and different GPCRs was attempted. Cross-talking signals between the array spots were discovered, and several control experiments were performed to explore the possible reason. As an alternative solution for multiplexing, DNA barcode technique was implemented with the GPCR virions and achieved with the ESM, which paved a way for multiplexed single molecule binding kinetics studies. Circular RNAs has been found as an important class of regulators at the transcriptional and posttranscriptional level and could be potential biomarkers for many diseases. However, determination of its existence from the linear RNAs is challenging for the tradition molecular detection methods. Due to the no ending feature, by designing a unique complementary probe sequence, hybridization affinity difference between circular and linear RNA can be distinguished. Affinities with different hybridization nucleotides number were measured and verified.