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WIREs Nanomed Nanobiotechnol
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Inside single cells: quantitative analysis with advanced optics and nanomaterials

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Single‐cell explorations offer a unique window to inspect molecules and events relevant to mechanisms and heterogeneity constituting the central dogma of biology. A large number of nucleic acids, proteins, metabolites, and small molecules are involved in determining and fine‐tuning the state and function of a single cell at a given time point. Advanced optical platforms and nanotools provide tremendous opportunities to probe intracellular components with single‐molecule accuracy, as well as promising tools to adjust single‐cell activity. To obtain quantitative information (e.g., molecular quantity, kinetics, and stoichiometry) within an intact cell, achieving the observation with comparable spatiotemporal resolution is a challenge. For single‐cell studies, both the method of detection and the biocompatibility are critical factors as they determine the feasibility, especially when considering live‐cell analysis. Although a considerable proportion of single‐cell methodologies depend on specialized expertise and expensive instruments, it is our expectation that the information content and implication will outweigh the costs given the impact on life science enabled by single‐cell analysis. WIREs Nanomed Nanobiotechnol 2015, 7:387–407. doi: 10.1002/wnan.1321 This article is categorized under: Diagnostic Tools > Biosensing Nanotechnology Approaches to Biology > Cells at the Nanoscale Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
The spatiotemporal scales expected for single‐cell studies.
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Label‐free intracellular fingerprinting. (a) One‐dimensional nanoneedle sensors for single‐cell probing. (i, Reprinted with permission from Ref . Copyright 2000 Nature Publishing Group) Gold nanoparticle (GNP)‐coated nanoneedle was used for surface‐enhanced Raman spectroscopy (SERS). (ii, Reprinted with permission from Ref . Copyright 2010 John Wiley & Sons, Inc.) (b) SERS mapping of trivalent and hexavalent chromium was facilitated by intracellular growth of gold nanoislands. (Reprinted with permission from Ref . Copyright 2011 American Chemical Society) (c) 3,3′‐Dihexyloxacarbocyanine iodide [DIOC6(3)] was imaged by MALDI‐MSI at 7‐µm resolution. (Reprinted with permission from Ref . Copyright 2012 American Chemical Society) (d) Coherent anti‐stokes Raman spectroscopy (CARS) for probing intracellular lipid contents and poly(lactic‐co‐glycolic acid) (PLGA) polymers. (Reprinted with permission from Ref . Copyright 2009 American Chemical Society)
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Single‐molecule dynamics inside cells. (a) The rate of messenger RNA (mRNA) transcription was quantified by MS2 labeling and fluorescence recovery after photobleaching (FRAP). (Reprinted with permission from Ref . Copyright 2010 The Company of Biologists Ltd.) (b) The diffusion property of Oct4 variants in mouse embryos was profiled by fluorescence correlation spectroscopy (FCS). D, diffusion coefficient; α, degree of anomalous diffusion; % free, percentage of the free component. (Reprinted with permission from Ref . Copyright 2013 Nature Publishing Group) (c) Dark‐field illumination‐based scattering correlation spectroscopy (DFSCS) enables the monitoring of intracellular dynamics of gold nanoparticles (GNPs). (Reprinted with permission from Ref . Copyright 2014 American Chemical Society)
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Multimodal characterization of breast cancer surface markers. (a) HER2 clusters on SKBR3 cell membrane can be resolved by super‐resolution microscopy. (Reprinted with permission from Ref . Copyright 2010 John Wiley & Sons, Inc.) (b) The density of HER2 clusters can also be evaluated with gold nanoparticle (GNP) labels by scattering microscopy. The density positively correlates with the plasmonic peak (λmax). (Reprinted with permission from Ref . Copyright 2012 American Chemical Society) (c) HER2‐mediated cellular uptake was tracked by H‐GNRs and fluorescence correlation spectroscopy (FCS). (Reprinted with permission from Ref . Copyright 2009 American Chemical Society) (d) The ratio of CD44/CD24 was quantified by surface‐enhanced Raman spectroscopy (SERS) to identify breast cancer stem cells (CSCs). (Reprinted with permission from Ref . Copyright 2011 American Chemical Society)
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Single‐cell studies at the RNA level. (a) HT1080‐GFP‐96mer cells were incubated with ratiometric bimolecular beacons (RBMBs) targeting green fluorescent protein (GFP) messenger RNAs (mRNAs). Pink signal (i) is from the reference dye. Green fluorescence is from the reporter dye (ii). Single‐molecule fluorescence in situ hybridization (FISH) was performed (red in iii) to validate the targeting efficiency of RBMBs (overlay in iv). (Reprinted with permission from Ref . Copyright 2013 Oxford University Press) (b) Alternative splicing of BRCA1 was assessed by hyperspectral dark‐field microscopy. Gold nanoparticle (GNP) dimers exhibit a red‐shifted color compared with monomers (i). GNP‐based probes flanking distant mRNA regions enable the identification of variants with spliced‐out exons (ii). (Reprinted with permission from Ref . Copyright 2014 Nature Publishing Group)
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Single‐cell studies at the DNA and chromatin levels. (a) Label‐free stimulated Raman spectroscopy (SRS) imaging of nucleic acids in live cells. (Reprinted with permission from Ref . Copyright 2012 John Wiley & Sons, Inc.) (b) Spectral precision distance/position determination microscopy (SPDM) was applied to map the nanostructure of centromere 9 with an accuracy of 10–20 nm. (Reprinted with permission from Ref . Copyright 2010 MDPI AG, Basel, Switzerland) (c) Super‐resolution imaging of DNA replication factories based on proliferating cell nuclear antigen (PCNA) and replication protein A (RPA). HU, hydroxyurea treatment. (Reprinted with permission from Ref . Copyright 2009 BioMed Central Ltd)
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Fluorescence correlation spectroscopy (FCS) techniques for single‐cell analysis. (a) For fluorescent molecules without interaction, one‐component FCS can be used to determine molecular dynamics, number, size, and stoichiometry. (b) For molecules with higher degrees of interaction, two‐component fluorescence cross‐correlation spectroscopy (FCCS) or fluorescence lifetime cross‐correlation spectroscopy (FLCS) [in combination with Förster resonance energy transfer (FRET)] can be applied. In FCCS, the association (cross‐correlation) is determined when signals from different molecules are simultaneously detected within a diffraction limited spot. In FLCS, molecules with different fluorescence lifetimes can be separated, for example, distinguishing the FRET molecules from non‐FRET ones.
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Fluorescence recovery after photobleaching (FRAP) for measuring molecular dynamics inside live single cells. (a) Recombinant YFP‐GL‐GPI plasmid was transfected into COS‐7 cells. After photobleaching, the fluorescence recovery profile was recorded for 80 seconds. (b) Recovery curves of three regions are presented. A standard FRAP curve contains several critical points (right panel): initial intensity prebleach (Fi), starting point for postbleach (F0), half maximal fluorescence recovery (F1/2), and ultimate recovered fluorescence (F). (c) Effective diffusion coefficients of EGFP, LC3, tfLC3, and p53 in live COS‐7 cells were obtained. (Reprinted with permission from Ref . Copyright 2012 John Wiley & Sons, Inc.)
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Nanomaterials, ranging from 1 to approximately 100 nm, are broadly applied in single‐cell studies.
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Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
Diagnostic Tools > Biosensing
Nanotechnology Approaches to Biology > Cells at the Nanoscale

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