Label-free, image-based cellular mechanophenotyping in microfluidic devices provides a high-throughput method for single-cell profiling. However, while complex microchannels (e.g., hyperbolic geometries) reveal transient deformation dynamics under continuous extensional stress, the resulting high-dimensional feature spaces are highly susceptible to hydrodynamic artifacts. Flow rate variations often distort discriminative boundaries, linking feature distributions to fluid conditions rather than intrinsic biology. To overcome this, we introduce a stability-guided analytical framework that decouples flow-induced noise from authentic mechanobiological signatures. We tracked the morphodynamic, kinematic, and intracellular optical-density trajectories of healthy and malignant ovarian cells to build a 93-dimensional feature space. Using a cross-flow screening strategy based on structural consistency and statistical persistence, we isolated robust descriptors, creating task-adapted subsets (20 features for binary classification; 25 for cancer subtyping). Variance-attribution analysis confirmed the neutralization of flow-conditioned artifacts; notably, flow-associated variance in the primary principal component fell from 69.9% to 9.3% in the subtyping task. We also found that macroscopic binary discrimination depends on bulk kinematic transitions, while clonal subtyping requires localized intracellular optical heterogeneity. These optimized subsets maintained diagnostic fidelity across multiple machine learning architectures and restricted sampling conditions. This framework establishes a robust, flow-independent foundation for continuous dynamic phenotyping.
Label-free single-cell imaging offers a scalable, non-invasive alternative to fluorescence-based cytometry, yet inferring molecular phenotypes directly from bright-field morphology remains challenging. We present a unified Deep Learning (DL) framework that jointly performs White Blood Cell (WBC) classification and continuous protein-expression regression from label-free Differential Phase Contrast (DPC) images. Our model employs a Hybrid architecture that fuses convolutional fine-grained texture features with transformer-based global representations through a learnable cross-branch gating module, enabling robust morpho-molecular inference from DPC images. To support downstream interpretability, we further incorporate a Large Language Model (LLM) that generates concise, biologically grounded summaries of the predicted cell states. Experiments on the Berkeley Single Cell Computational Microscopy (BSCCM) and Blood Cells Image benchmarks demonstrate strong performance, achieving a 91.3% WBC classification accuracy and a 0.72 Pearson correlation for CD16 expression regression on BSCCM. These results underscore the promise of label-free single-cell imaging for cost-effective hematological profiling, enabling simultaneous phenotype identification and quantitative biomarker estimation without fluorescent staining. The source code is available at https://github.com/saqibnaziir/Single-Cell-Phenotyping.
Deformability cytometry (DC) is a type of imaging flow cytometry, which uses a camera-equipped device to measure cellular stiffness in addition to other cellular properties at high throughput. Cellular properties such as area and elongation can identify cell types, but this requires prior knowledge of distinguishing properties and cannot be applied to clinically important cell aggregates. Using DC data, we evaluated conventional multi-class (MC) classification and introduced a multi-label (ML) approach for identifying blood cells and their aggregates. In particular, an ML classifier can simultaneously assign multiple cell-type labels to a single imaged event. We show that, unlike MC classification, ML classification can identify cell aggregates not represented in the training data. It also avoids the need for exhaustive, strictly defined aggregate labels, thereby simplifying and speeding up annotation. Since automated blood analyzers do not reliably analyze cell aggregates, our approach may help address this clinical gap.
In histopathology, human experts primarily rely on color as a means of enhancing contrast to interpret tissue morphology, whereas machine vision models process color as raw statistical information. This distinction raises a fundamental question: to what extent can pixel intensity alone, independent of structural and morphological cues, support cancer classification? To address this question, we systematically evaluated the standalone discriminative power of global color features while deliberately excluding all morphological information. Specifically, we extracted statistical color moments and discretized RGB and HSV color histograms, and assessed their performance across ten diverse experimental settings using classical machine learning classifiers. Our results demonstrate that color features alone can achieve strong performance in binary diagnostic tasks (e.g., benign versus malignant), with classification accuracies reaching up to 89%. This performance is likely attributable to global chromatic shifts associated with malignancy. Importantly, these simple color-based representations consistently outperformed random baselines by a substantial margin, indicating that raw color distributions encode a non-random and diagnostically relevant signal for cancer detection. Consequently, this study suggests that simple, computationally efficient color features can serve as an effective pre-screening tool. By identifying samples with strong chromatic indicators of malignancy, these lightweight models could function as a first-pass triage system, reducing the computational burden on complex deep learning architectures.