Fluorescent Gold Nanocluster Inside a Live Breast Cell: Etching and Higher Uptake in Cancer Cell

Time-resolved confocal microscopy is applied to compare fluorescence properties of gold nanocluster (Au-NC) inside human breast cells with those in bulk water. In bulk water, Au-NC, coated with bovine serum albumin (BSA), displays a major emission peak at ∼640 nm, a minor peak at 460 nm and a very weak peak at 500 nm. The major peak is ascribed to an Au₂₅ cluster with an icosahedral Au₁₃ core, surrounded by six thiol (from BSA) mediated Au₂ staples. Inside the live cells, emission maximum of Au-NC exhibits a dramatic blue shift to 530 nm in normal breast cell (MCF10A) and 510 nm in breast cancer cell (MCF7). The 510–530 nm emission peak corresponds to an icosahedral Au₁₃ cluster. It appears that inside the cell, glutathione competes with and replaces BSA as a ligand of the Au-NC. This leads to etching of the Au-NC to Au₁₃. Confocal images indicate that the Au-NCs localize in the membrane of the normal breast cell, MCF10A. In the case of breast cancer cell MCF7, the Au-NCs localize in a much larger volume encompassing the cell membrane and the cytoplasm. This demonstrates higher uptake of Au-NCs by the cancer cell. Fluorescence correlation spectroscopy (FCS) is applied to measure viscosity inside the live cells, using Au-NC as a probe. For the cancer cell, the cytoplasmic viscosity is found to be 7 cP. The FCS data for the membrane is fitted to two-dimensional (2D) diffusion. From this the surface viscosity is obtained using Saffman–Stokes–Einstein theory. The surface viscosity in the cancer cell is ∼9-times higher than that in the normal cell

Dynamics in Cytoplasm, Nucleus, and Lipid Droplet of a Live CHO Cell: Time-Resolved Confocal Microscopy

Different regions of a single live Chinese hamster ovary (CHO) cell are probed by time-resolved confocal microscopy. We used coumarin 153 (C153) as a probe. The dye localizes in the cytoplasm, nucleus, and lipid droplets, as is clearly revealed by the image. The fluorescence correlation spectroscopy (FCS) data shows that the microviscosity of lipid droplets is ∼34 ± 3 cP. The microviscosities of nucleus and cytoplasm are found to be 13 ± 1 and 14.5 ± 1 cP, respectively. The average solvation time (⟨τ_s⟩) in the lipid droplets (3600 ± 50 ps) is slower than that in the nucleus (⟨τ_s⟩ = 750 ± 50 ps) and cytoplasm (⟨τ_s⟩ = 1100 ± 50 ps). From the position of emission maxima of C153, the polarity of the nucleus is estimated to be similar to that of a mixture containing 26% DMSO in triacetin (η ∼ 11.2 cP, ε ∼ 26.2). The cytoplasm resembles a mixture of 18% DMSO in triacetin (η ∼ 12.6 cP, ε ∼ 21.9). The polarity of lipid droplets is less than that of pure triacetin (η ∼ 21.7 cP, ε ∼ 7.11)

Dynamics of Gene Silencing in a Live Cell: Stochastic Resonance

Binding of a specific siRNA to the target mRNA in a live cell (human breast cancer cell, MCF-7) is studied by confocal microscopy. The specific siRNA (labeled with a fluorophore, alexa 488) exhibits much higher intensity of fluorescence in the bound state than in the free (unbound) state. It is observed that repeated unbinding and rebinding of siRNA (to target mRNA) occur before gene silencing. 16 273 on-time periods (residence or dwell time of siRNA in bound form) are detected. They follow a strikingly simple pattern. All of the on-time periods are odd-integral multiples of 5.5 ± 0.05 ms. This is ascribed to stochastic resonance

Dynamics of Gene Silencing in a Live Cell: Stochastic Resonance

Binding of a specific siRNA to the target mRNA in a live cell (human breast cancer cell, MCF-7) is studied by confocal microscopy. The specific siRNA (labeled with a fluorophore, alexa 488) exhibits much higher intensity of fluorescence in the bound state than in the free (unbound) state. It is observed that repeated unbinding and rebinding of siRNA (to target mRNA) occur before gene silencing. 16 273 on-time periods (residence or dwell time of siRNA in bound form) are detected. They follow a strikingly simple pattern. All of the on-time periods are odd-integral multiples of 5.5 ± 0.05 ms. This is ascribed to stochastic resonance

Pseudohalide (SCN^–)‑Doped MAPbI₃ Perovskites: A Few Surprises

Pseudohalide thiocyanate anion (SCN^–) has been used as a dopant in a methylammonium lead tri-iodide (MAPbI₃) framework, aiming for its use as an absorber layer for photovoltaic applications. The substitution of SCN^– pseudohalide anion, as verified using Fourier transform infrared (FT-IR) spectroscopy, results in a comprehensive effect on the optical properties of the original material. Photoluminescence measurements at room temperature reveal a significant enhancement in the emission quantum yield of MAPbI_3–<i>x</i>(SCN)_<i>x</i> as compared to MAPbI₃, suggestive of suppression of nonradiative channels. This increased intensity is attributed to a highly edge specific emission from MAPbI_3–<i>x</i>(SCN)_<i>x</i> microcrystals as revealed by photoluminescence microscopy. Fluoresence lifetime imaging measurements further established contrasting carrier recombination dynamics for grain boundaries and the bulk of the doped material. Spatially resolved emission spectroscopy on individual microcrystals of MAPbI_3–<i>x</i>(SCN)_<i>x</i> reveals that the optical bandgap and density of states at various (local) nanodomains are also nonuniform. Surprisingly, several (local) emissive regions within MAPbI_3–<i>x</i>(SCN)_<i>x</i> microcrystals are found to be optically unstable under photoirradiation, and display unambiguous temporal intermittency in emission (blinking), which is extremely unusual and intriguing. We find diverse blinking behaviors for the undoped MAPbI₃ crystals as well, which leads us to speculate that blinking may be a common phenomenon for most hybrid perovskite materials

Pseudohalide (SCN^–)‑Doped MAPbI₃ Perovskites: A Few Surprises

Pseudohalide thiocyanate anion (SCN^–) has been used as a dopant in a methylammonium lead tri-iodide (MAPbI₃) framework, aiming for its use as an absorber layer for photovoltaic applications. The substitution of SCN^– pseudohalide anion, as verified using Fourier transform infrared (FT-IR) spectroscopy, results in a comprehensive effect on the optical properties of the original material. Photoluminescence measurements at room temperature reveal a significant enhancement in the emission quantum yield of MAPbI_3–<i>x</i>(SCN)_<i>x</i> as compared to MAPbI₃, suggestive of suppression of nonradiative channels. This increased intensity is attributed to a highly edge specific emission from MAPbI_3–<i>x</i>(SCN)_<i>x</i> microcrystals as revealed by photoluminescence microscopy. Fluoresence lifetime imaging measurements further established contrasting carrier recombination dynamics for grain boundaries and the bulk of the doped material. Spatially resolved emission spectroscopy on individual microcrystals of MAPbI_3–<i>x</i>(SCN)_<i>x</i> reveals that the optical bandgap and density of states at various (local) nanodomains are also nonuniform. Surprisingly, several (local) emissive regions within MAPbI_3–<i>x</i>(SCN)_<i>x</i> microcrystals are found to be optically unstable under photoirradiation, and display unambiguous temporal intermittency in emission (blinking), which is extremely unusual and intriguing. We find diverse blinking behaviors for the undoped MAPbI₃ crystals as well, which leads us to speculate that blinking may be a common phenomenon for most hybrid perovskite materials

Pseudohalide (SCN^–)‑Doped MAPbI₃ Perovskites: A Few Surprises

Pseudohalide thiocyanate anion (SCN^–) has been used as a dopant in a methylammonium lead tri-iodide (MAPbI₃) framework, aiming for its use as an absorber layer for photovoltaic applications. The substitution of SCN^– pseudohalide anion, as verified using Fourier transform infrared (FT-IR) spectroscopy, results in a comprehensive effect on the optical properties of the original material. Photoluminescence measurements at room temperature reveal a significant enhancement in the emission quantum yield of MAPbI_3–<i>x</i>(SCN)_<i>x</i> as compared to MAPbI₃, suggestive of suppression of nonradiative channels. This increased intensity is attributed to a highly edge specific emission from MAPbI_3–<i>x</i>(SCN)_<i>x</i> microcrystals as revealed by photoluminescence microscopy. Fluoresence lifetime imaging measurements further established contrasting carrier recombination dynamics for grain boundaries and the bulk of the doped material. Spatially resolved emission spectroscopy on individual microcrystals of MAPbI_3–<i>x</i>(SCN)_<i>x</i> reveals that the optical bandgap and density of states at various (local) nanodomains are also nonuniform. Surprisingly, several (local) emissive regions within MAPbI_3–<i>x</i>(SCN)_<i>x</i> microcrystals are found to be optically unstable under photoirradiation, and display unambiguous temporal intermittency in emission (blinking), which is extremely unusual and intriguing. We find diverse blinking behaviors for the undoped MAPbI₃ crystals as well, which leads us to speculate that blinking may be a common phenomenon for most hybrid perovskite materials

Pseudohalide (SCN^–)‑Doped MAPbI₃ Perovskites: A Few Surprises

Pseudohalide thiocyanate anion (SCN^–) has been used as a dopant in a methylammonium lead tri-iodide (MAPbI₃) framework, aiming for its use as an absorber layer for photovoltaic applications. The substitution of SCN^– pseudohalide anion, as verified using Fourier transform infrared (FT-IR) spectroscopy, results in a comprehensive effect on the optical properties of the original material. Photoluminescence measurements at room temperature reveal a significant enhancement in the emission quantum yield of MAPbI_3–<i>x</i>(SCN)_<i>x</i> as compared to MAPbI₃, suggestive of suppression of nonradiative channels. This increased intensity is attributed to a highly edge specific emission from MAPbI_3–<i>x</i>(SCN)_<i>x</i> microcrystals as revealed by photoluminescence microscopy. Fluoresence lifetime imaging measurements further established contrasting carrier recombination dynamics for grain boundaries and the bulk of the doped material. Spatially resolved emission spectroscopy on individual microcrystals of MAPbI_3–<i>x</i>(SCN)_<i>x</i> reveals that the optical bandgap and density of states at various (local) nanodomains are also nonuniform. Surprisingly, several (local) emissive regions within MAPbI_3–<i>x</i>(SCN)_<i>x</i> microcrystals are found to be optically unstable under photoirradiation, and display unambiguous temporal intermittency in emission (blinking), which is extremely unusual and intriguing. We find diverse blinking behaviors for the undoped MAPbI₃ crystals as well, which leads us to speculate that blinking may be a common phenomenon for most hybrid perovskite materials

Homolog-specific Oligopaints for PGP1f.

(A) Violin plots of the ratios of 19 pairs of ellipticity scores, each pair representing the two homologs of one of the 19 imaged nuclei (Sample) or of 1,000 pairs of ellipticity scores, each pair representing two chromosomes chosen at random from the 38 representing the 19 imaged nuclei (Random). Boundaries of the black box-plot represent 1st and 3rd quartiles, white dot represents the median, and whiskers extend to 1.5 times the interquartile range (Mann-Whitney rank test, *: p −2). (B) The HOP-M and HOP-P probes for chromosome 19 each encompass the entire chromosome and contain 11,259 oligos that cover ≥ 1 SNV per oligo. Thus, they are in contrast to Oligopaint oligos used to image CS1-9, these latter probes being “interstitial” in nature, as they avoid SNVs. HOP-M and HOP-P probes are visualized with secondary oligos labeled with different dyes, such that the two probes can be distinguished. (C-G) Images of a nucleus visualized with DAPI (C, blue), an interstitial probe targeting just CS3 (D, grey), HOP-M (E, green, Atto488N), HOP-P (F, magenta, Atto565N), and all probes (G), the latter demonstrating co-localization of all signals (n = 128); percentages show efficiency of each probe configuration, with a combined efficiency of 96.5%. (H) Ellipticity for the maternal (green) and paternal (magenta) homologs of the 6 nuclei for which HOPs had been applied.</p

Tracing PGP1f chromosomes.

(A) OligoSTORM image of a diploid nucleus showing CS1-9 (Bottom, rounds 1 through 9; 1.28, 1.24, 1.80, 1.04, 0.56, 0.52, 0.84, 0.52, and 0.36 Mb, respectively) and four subregions of CS7 (top, rounds 10 through 13; 140, 260, 350, and 90 kb, respectively) of both homologs. In this and all panels excepting F, radius of spheres represents the localization precision in the axial dimension. Note, the coordinates of the four subregions, selected based on the IMR-90 Hi-C map, correspond well to contact domains. Sequencing depth of the PGP1f Hi-C map was not, however, able to confirm these as contact domains in PGP1f, and thus they are referred to simply as subregions. (B) Same as in A with respect to CS1-9, but a different nucleus. (C) Loop (290 kb), including loop anchors (rounds 14 and 16; pink and orange; 10 kb each) and loop body (round 15; blue; 270 kb); *, we also imaged the upstream and downstream regions flanking the loop (rounds 17 and 18; 20 and 80 kb, respectively), but these are not shown so that the structure of the loop can be more apparent. (D) DNMT1 gene (round 19; 59.5 kb) and its DMR (round 20; 2.9 kb). (E) Walking along chromosome 19 in variable step sizes (see A) while also walking along chromosomes 3 and 5 in uniform step sizes (500 and 250 kb, respectively). (F) OligoDNA-PAINT images of CS7-9 of one homolog showing each chromosomal segment by itself (top; color scale represents depth in z) or merged (bottom; color scale represents different chromosomal segments). In contrast to other panels, localizations are blurred according to precision. (G) Superimposition of two images, taken 90 hours apart, each of both homologs of CS1 in (red, first image; blue, later image). Average overlap is 80 ± 9% and 68 ± 12% (n = 20) when images are aligned based on their centers of mass (left) or not (right), respectively. (H) Superimposition of two images from one nucleus of both homologs of CS7, one image encompassing all of CS7 (blue) and the other being a composite of the four subregions (green; 140, 260, 350, and 90 kb) comprising CS7. With alignment based on centers of mass, 85% of the single image overlapped the composite image, and 76% of the latter overlapped the former (left). Without alignment, the analogous values were 84% and 75% (right).</p