3D Amplified Single-Cell RNA and Protein Imaging Identifies Oncogenic Transcript Subtypes in B-Cell Acute Lymphoblastic Leukemia.

Simultaneous in situ detection of transcript and protein markers at the single-cell level is essential for gaining a better understanding of tumor heterogeneity and for predicting and monitoring treatment responses. However, the limited accessibility to advanced 3D imaging techniques has hindered their rapid implementation. Here, we present a 3D single-cell imaging technique, termed 3D digital rolling circle amplification (4DRCA), capable of the multiplexed and amplified simultaneous digital quantification of single-cell RNAs and proteins using standard fluorescence microscopy and off-the-shelf reagents. We generated spectrally distinguishable DNA amplicons from molecular markers through an integrative protocol combining single-cell RNA and protein assays and directly enumerated the amplicons by leveraging an open-source algorithm for 3D deconvolution with a custom-built automatic gating algorithm. With 4DRCA, we were able to simultaneously quantify surface protein markers and cytokine transcripts in T-lymphocytes. We also show that 4DRCA can distinguish BCR-ABL1 fusion transcript positive B-cell acute lymphoblastic leukemia cells with or without CD19 protein expression. The accessibility and extensibility of 4DRCA render it broadly applicable to other cell-based diagnostic workflows, enabling sensitive and accurate single-cell RNA and protein profiling.

Integrative Magneto-Microfluidic Separation of Immune Cells Facilitates Clinical Functional Assays.

Accurately analyzing the functional activities of natural killer (NK) cells in clinical diagnosis remains challenging due to their coupling with other immune effectors. To address this, an integrated immune cell separator is required, which necessitates a streamlined sample preparation workflow including immunological cell isolation, removal of excess red blood cells (RBCs), and buffer exchange for downstream analysis. Here, a self-powered integrated magneto-microfluidic cell separation (SMS) chip is presented, which outputs high-purity target immune cells by simply inputting whole blood. The SMS chip intensifies the magnetic field gradient using an iron sphere-filled inlet reservoir for high-performance immuno-magnetic cell selection and separates target cells size-selectively using a microfluidic lattice for RBC removal and buffer exchange. In addition, the chip incorporates self-powered microfluidic pumping through a degassed polydimethylsiloxane chip, enabling the rapid isolation of NK cells at the place of blood collection within 40 min. This chip is used to isolate NK cells from whole blood samples of hepatocellular cancer patients and healthy volunteers and examined their functional activities to identify potential abnormalities in NK cell function. The SMS chip is simple to use, rapid to sort, and requires small blood volumes, thus facilitating the use of immune cell subtypes for cell-based diagnosis.

Real-time monitoring of mono- and dual-species biofilm formation and eradication using microfluidic platform.

In a human host, bacterial Staphylococcus aureus and fungal Candida albicans pathogens form a mixed biofilm that causes severe mortality and morbidity. However, research on the formation and eradication of mixed biofilms under dynamic conditions is lacking. Thus, this study employed a microfluidic technique to analyze the real-time formation of mono- and dual-species (S. aureus and C. albicans) biofilms and noninvasive optical treatment of the established mature biofilm using 405-nm laser light. A herringbone mixer thoroughly mixed both bacterial and fungal cells in the growth media before being injected into the observation channels on the microfluidic chip. At a flow rate of 1.0 µL/min of growth media for 24 h, the bacterial biofilm coverage was up to 15% higher than that of the fungal biofilm (50% for bacteria vs. 35% for fungus). On the other hand, the dual-species biofilm yielded the highest coverage of ~ 96.5% because of the collective interaction between S. aureus and C. albicans. The number of cell proliferation events in S. aureus was higher than that of C. albicans for 12 h, which indicates that the S. aureus biofilm was developed faster than C. albicans. The novel in situ test platform showed a significant bactericidal effect (80%) of the 405-nm laser light at 1080 J/cm2 towards the established S. aureus biofilm, whereas the same treatment removed approximately 69% of the mixed cells in the dual-species biofilm. This study revealed that the developed microfluidic platform could be utilized to monitor the formation of dual-species biofilms in real-time and laser-induced antimicrobial effects on dual-species biofilms.

Rotation angle measurement based on white-light interferometry with a standard optical flat.

We propose a simple white-light interferometric method of measuring a one-dimensional rotation angle with use of an optical plane parallel plate of standard refractive index. The phase change of the interference pattern of the interferometer during the rotation of the flat plate of known refractive index and thickness placed in one of the interferometer's arms is used for determination of the rotation angle. This method has been demonstrated for an accurate angle measurement over the angle range from 0° to 40° within a maximum uncertainty of 0.057°.