Single cell analysis 2019

Why? Limited, hard to get cell samples Heterogeneity with in sample population: think biopsy, small organisms etc. Cell to cell variability due to genetic differences leads to differential activities, responsiveness, effect on environment Developmental biology Localization of cellular activities while they occur We already know about the heterogeneity of cancer cell population Identify clonal expansion Tumor development history

Semin Cell Dev Biol (2016),

Challenges Isolating a single cell w/o changing its characteristics Signal enhancement (fluorescent, molecular structure= DNA, quantifying+ RNA/ protein) Screening noise (technical, leakiness) Unbiased detection Localization per cell / time computation

Isolating a single cell(s) Reduce the sampling- few cells/ volume. Can be a “must” or a choice Disperse cells (disconnect, re-plate..) Immunoafinity on unique markers (FACS) Magnetic trapping Allow cells to “move” through separating matrix like soundwaves, ultrasound, traping into nano-wells

Lab on a chip- microfluidic separation https://www.youtube.com/watch?v=TF4NJRE4Xg4 https://www.nature.com/articles/srep46507#s2 http://news.mit.edu/2014/sorting-cancer-cells-with-sound-0825 Proc Natl Acad Sci U S A. 2015 Apr 21;112(16):4970-5. doi: 10.1073/pnas.1504484112

Technology Micro-electronics Especially designed platforms Micro-fluidics parameters Where to put the cellular assay? Application: CTC, Inflammation, Field diagnosis, Research

Molecular analysis Common approach= Sequencing DNA, RNA and protein detection Problems: identification on single molecules in a complex background, Signal, quantification Linking expression profile to genomics, Epigenomics etc. Transcriptome +/- direct genomics Barcoding the specific amplification/cell (see below) and article Linking Existing proteins to transcriptome/ Genome Use very specific Ab linked to strong “trapping” Split cells Cool trick: Ab connected to primers/ each protein- get qPCR from proximity In-House Validation, Repeating by others, Cost

Liquid Biopsy, CTC ECC Stem cells Micro- niche

Inserting constructs or engineering cells/ animals with appropriate sequences Level of endogenous expression Budget/ equipment / specificity

The Rac-FRET Mouse Reveals Tight Spatiotemporal Control of Rac Activity in Primary Cells and Tissues (Cell reports 2014)

Back to research How much data can we achieve/cell? Is one cell enough? How reliable is SCA, could SCA be carried out on tissue level? What is the relevance of strength in number? Microdots as a fluorescent vehicle.

QD (especially with cadmium selenide (CdSe) core and a zinc sulfide (ZnS) shell ) Bright fluorescence (x10) 10-40 nm Colour spectrum Non-bleaching (yet blinking) Surface functionalization/ non reactiveness FRET friendly/ single molecule tracking Size and chemistry are usually obstacles for crowded membrane pass cellular environments Non-reversal binding

QD-labeled Myosin V molecules in the cytoplasm of a HeLa cell observed by fluorescence microscopy A persistence filter was applied to highlight the trajectories of Myosin V driven quantum dots moving along the actin filaments (white arrows) Trajectory of a Myosin V-driven quantum dot moving along an actin filament (scale bar 1μm). And Quantum dot randomly diffusing through the cytoplasm