Deconwolf enables high-performance deconvolution of widefield fluorescence microscopy images.

Microscopy-based spatially resolved omic methods are transforming the life sciences. However, these methods rely on high numerical aperture objectives and cannot resolve crowded molecular targets, limiting the amount of extractable biological information. To overcome these limitations, here we develop Deconwolf, an open-source, user-friendly software for high-performance deconvolution of widefield fluorescence microscopy images, which efficiently runs on laptop computers. Deconwolf enables accurate quantification of crowded diffraction limited fluorescence dots in DNA and RNA fluorescence in situ hybridization images and allows robust detection of individual transcripts in tissue sections imaged with ×20 air objectives. Deconvolution of in situ spatial transcriptomics images with Deconwolf increased the number of transcripts identified more than threefold, while the application of Deconwolf to images obtained by fluorescence in situ sequencing of barcoded Oligopaint probes drastically improved chromosome tracing. Deconwolf greatly facilitates the use of deconvolution in many bioimaging applications.

3D mapping and accelerated super-resolution imaging of the human genome using in situ sequencing.

There is a need for methods that can image chromosomes with genome-wide coverage, as well as greater genomic and optical resolution. We introduce OligoFISSEQ, a suite of three methods that leverage fluorescence in situ sequencing (FISSEQ) of barcoded Oligopaint probes to enable the rapid visualization of many targeted genomic regions. Applying OligoFISSEQ to human diploid fibroblast cells, we show how four rounds of sequencing are sufficient to produce 3D maps of 36 genomic targets across six chromosomes in hundreds to thousands of cells, implying a potential to image thousands of targets in only five to eight rounds of sequencing. We also use OligoFISSEQ to trace chromosomes at finer resolution, following the path of the X chromosome through 46 regions, with separate studies showing compatibility of OligoFISSEQ with immunocytochemistry. Finally, we combined OligoFISSEQ with OligoSTORM, laying the foundation for accelerated single-molecule super-resolution imaging of large swaths of, if not entire, human genomes.

Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling.

Chromosome organization is crucial for genome function. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. Using the super-resolution microscopy methods of OligoSTORM and OligoDNA-PAINT, we trace 8 megabases of human chromosome 19, visualizing structures ranging in size from a few kilobases to over a megabase. Focusing on chromosomal regions that contribute to compartments, we discover distinct structures that, in spite of considerable variability, can predict whether such regions correspond to active (A-type) or inactive (B-type) compartments. Imaging through the depths of entire nuclei, we capture pairs of homologous regions in diploid cells, obtaining evidence that maternal and paternal homologous regions can be differentially organized. Finally, using restraint-based modeling to integrate imaging and Hi-C data, we implement a method-integrative modeling of genomic regions (IMGR)-to increase the genomic resolution of our traces to 10 kb.

Probing the conformational dynamics of photosystem I in unconfined and confined spaces.

The fluorescence dynamics of Photosystem I (PSI) in bulk water and inside a confined environment like a liposome have been investigated using time resolved confocal microscopy. In bulk water, PSI exhibits a major emission peak at ∼680 nm, while in the liposome it exhibits a markedly blue shifted emission maximum at ∼485 nm. This is indicative of conformational changes due to entrapment and emergence of a stressed conformation of PSI inside the liposome. The observed time constants for the fluorescence lifetime of PSI inside the liposome are significantly high as opposed to PSI in bulk water. More interestingly, the fluorescence intensity of PSI in bulk water exhibits strong fluctuations with many high intensity jumps and these are anti-correlated with the fluorescence lifetime of PSI. In contrast, inside the liposome, no such anti-correlated behaviour is observed. We further demonstrated that PSI exhibits at least two conformational states in bulk water, whereas a single conformation is observed inside the liposome, indicating the conformational rigidity and locking of the PSI complex inside a liposome.

Cytochrome†c-Capped Fluorescent Gold Nanoclusters: Imaging of Live Cells and Delivery of Cytochrome†c.

Cytochrome c-capped fluorescent gold nanoclusters (Au-NCs) are used for imaging of live lung and breast cells. Delivery of cytochrome c inside the cells is confirmed by covalently attaching a fluorophore (Alexa Fluor 594) to cytochrome c-capped Au-NCs and observing fluorescence from Alexa 594 inside the cell. Mass spectrometry studies suggest that in bulk water, addition of glutathione (GSH) to cytochrome c-capped Au-NCs results in the formation of glutathione-capped Au-NCs and free apo-cytochrome c. Thus glutathione displaces cytochrome c as a capping agent. Using confocal microscopy, the emission spectra and decay of Au-NCs are measured in live cells. From the position of the emission maximum it is shown that the Au-NCs exist as Au8 in bulk water and as Au13 inside the cells. Fluorescence resonance energy transfer from cytochrome c-Au-NC (donor) to Mitotracker Orange (acceptor) indicates that the Au-NCs localise in the mitochondria of live cells.

Cancer Cell Imaging Using in Situ Generated Gold Nanoclusters.

In situ generated fluorescent gold nanoclusters (Au-NCs) are used for bio-imaging of three human cancer cells, namely, lung (A549), breast (MCF7), and colon (HCT116), by confocal microscopy. The amount of Au-NCs in non-cancer cells (WI38 and MCF10A) is 20-40 times less than those in the corresponding cancer cells. The presence of a larger amount of glutathione (GSH) capped Au-NCs in the cancer cell is ascribed to a higher glutathione level in cancer cells. The Au-NCs exhibit fluorescence maxima at 490-530 nm inside the cancer cells. The fluorescence maxima and matrix-assisted laser desorption ionization (MALDI) mass spectrometry suggest that the fluorescent Au-NCs consist of GSH capped clusters with a core structure (Au8-13). Time-resolved confocal microscopy indicates a nanosecond (1-3 ns) lifetime of the Au-NCs inside the cells. This rules out the formation of aggregated Au-thiolate complexes, which typically exhibit microsecond (≈1000 ns) lifetimes. Fluorescence correlation spectroscopy (FCS) in live cells indicates that the size of the Au-NCs is ≈1-2 nm. For in situ generation, we used a conjugate consisting of a room-temperature ionic liquid (RTIL, [pmim][Br]) and HAuCl4. Cytotoxicity studies indicate that the conjugate, [pmim][AuCl4], is non-toxic for both cancer and non-cancer cells.

Selective Killing of Breast Cancer Cells by Doxorubicin-Loaded Fluorescent Gold Nanoclusters: Confocal Microscopy and FRET.

Fluorescent gold nanoclusters (AuNCs) capped with lysozymes are used to deliver the anticancer drug doxorubicin to cancer and noncancer cells. Doxorubicin-loaded AuNCs cause the highly selective and efficient killing (90 %) of breast cancer cells (MCF7) (IC50 =155 nm). In contrast, the killing of the noncancer breast cells (MCF10A) by doxorubicin-loaded AuNCs is only 40 % (IC50 =4500 nm). By using a confocal microscope, the fluorescence spectrum and decay of the AuNCs were recorded inside the cell. The fluorescence maxima (at ≈490-515 nm) and lifetime (≈2 ns), of the AuNCs inside the cells correspond to Au10-13 . The intracellular release of doxorubicin from AuNCs is monitored by Förster resonance energy transfer (FRET) imaging.

Direct observation of the growth and shrinkage of microtubules by single molecule Förster resonance energy transfer.

Single molecule Förster resonance energy transfer (FRET) has been applied, for the first time, to monitor the growth (polymerization) and the shrinkage (depolymerization) of the dynamic microtubules, employing EGFP (attached to Mal3) as a donor and alexa-568 bound to tubulin as an acceptor.

Interaction of ß-cyclodextrin with nile red in a single live CHO cell: an initiative towards developing a prospective strategy for the excretion of adsorbed drugs from the cell membrane.

A successful endeavour has been made to develop a prospective strategy to oust the drug molecules adsorbed on the cell membranes by simply using the non-toxic ß-cyclodextrin (ß-CD). For this purpose, fluorescent probes of different geometries and charge characteristics, namely phenosafranin (PSF) and nile red (NR), were exploited for the in vitro studies using different lipids as model membranes. Considering the success of the in vitro study, the present work is extended to a live Chinese Hamster Ovary (CHO) cell using the dye NR acting as a model drug system. Steady state and time resolved confocal microscopic studies reveal that with the introduction of ß-CD, some of the adsorbed dye molecules are dislodged from the CHO cell membrane to the nanocavity of ß-CD resulting in the formation of dye-ß-CD inclusion complex. This study promises the development of a prospective strategy for the removal of the adsorbed drugs from the cell membranes.

Effect of ethanol-water mixture on the structure and dynamics of lysozyme: a fluorescence correlation spectroscopy study.

Effect of ethanol-water mixture on the hydrodynamic radius (r(H)) and conformational dynamics of lysozyme has been studied by circular dichroism, emission spectra, and fluorescence correlation spectroscopy. For this purpose, the protein lysozyme is covalently labeled near the active site with a fluorescent probe, alexa 488. The ethanol molecules are sequestered near the hydrophobic tryptophan residues as indicated by the blue shift of the emission maximum of tryptophan. It is observed that both size (r(H)) and time constant of conformational relaxation (τ(R)) of lysozyme oscillate with increase in ethanol concentration. The r(H) of the protein fluctuates from 19 Å in the native state, to a minimum of 13 Å, and a maximum of 29 Å. It is proposed that the oscillating behavior arises from competition between mutual interaction among protein, ethanol, and water. The fluorescence intensity fluctuates because of quenching of the fluorescence of the probe (alexa) by the free amino group of certain residues (e.g., tryptophan). Rate of inter-conversion (folding dynamics) between the open (fluorescent) and closed (non-fluorescent) form has been determined and is found to exhibit similar oscillation with variation in ethanol content.

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 (<τs>) in the lipid droplets (3600 ± 50 ps) is slower than that in the nucleus (<τs> = 750 ± 50 ps) and cytoplasm (<τs> = 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).

FRET between a donor and an acceptor covalently bound to human serum albumin in native and non-native states.

Fluctuation in the inter-domain distance of a protein, human serum albumin (HSA), in the native, molten globule and denatured states is studied by Förster resonance energy transfer (FRET). For this purpose, a donor (CPM) and an acceptor (Alexa Fluor 488) are covalently attached to HSA. Unfolding of the protein is induced by pH changes as well as by the addition of 6 M GdnHCl and addition of 1.5 M of a room temperature ionic liquid (RTIL, [pmim][Br]). The efficiency of FRET (εFRET) and hence donor (D) - acceptor (A) distances of protein molecules in the native and non-native states are determined using FRET. In the native state (N), there is only one value of εFRET and D-A distance. In the non-native states (molten globule and unfolded) there are multiple values of εFRET and D-A distances. This suggests the presence of multiple conformers in equilibrium in the non-native states. When the protein is unfolded (on addition of GdnHCl or RTIL), separation between the two domains (I and II) increases and as a result εFRET decreases. In the presence of both GdnHCl and RTIL, the protein undergoes compaction (to form N'). However, in spite of the decrease in the overall radius, the D-A distance in the compact state (N') is found to be larger than that in the native state (N) of the protein. In contrast, two acid induced molten globule states of HSA (formed at pH 2 and 4) exhibit high εFRET indicating short D-A distances. In summary, we show that under chemical denaturation HSA undergoes stepwise unfolding and different domains unfold independently.

Heterogeneity in binary mixtures of dimethyl sulfoxide and glycerol: fluorescence correlation spectroscopy.

Diffusion of four coumarin dyes in a binary mixture of dimethyl sulfoxide (DMSO) and glycerol is studied using fluorescence correlation spectroscopy (FCS). The coumarin dyes are C151, C152, C480, and C481. In pure DMSO, all the four dyes exhibit a very narrow (almost uni-modal) distribution of diffusion coefficient (Dt). In contrast, in the binary mixtures all of them display a bimodal distribution of Dt with broadly two components. One of the components of D(t) corresponds to the bulk viscosity. The other one is similar to that in pure DMSO. This clearly indicates the presence of two distinctly different nano-domains inside the binary mixture. In the first, the micro-environment of the solute consists of both DMSO and glycerol approximately at the bulk composition. The other corresponds to a situation where the first layer of the solute consists of DMSO only. The burst integrated fluorescence lifetime (BIFL) analysis also indicates presence of two micro-environments one of which resembles DMSO. The relative contribution of the DMSO-like environment obtained from the BIFL analysis is much larger than that obtained from FCS measurements. It is proposed that BIFL corresponds to an instantaneous environment in a small region (a few nm) around the probe. FCS, on the contrary, describes the long time trajectory of the probes in a region of dimension ~200 nm. The results are explained in terms of the theory of binary mixtures and recent simulations of binary mixtures containing DMSO.

Effect of ionic liquid on the native and denatured state of a protein covalently attached to a probe: solvation dynamics study.

Effect of a room temperature ionic liquid (RTIL, [pmim][Br]) on the solvation dynamics of a probe covalently attached to a protein (human serum albumin (HSA)) has been studied using femtosecond up-conversion. For this study, a solvation probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) has been covalently attached to the lone cysteine group (cys-34) of the protein HSA. Addition of 1.5 M RTIL or 6 M GdnHCl causes a red shift of the emission maxima of CPM bound to HSA by 3 nm and 12 nm, respectively. The average solvation time 〈τ(s)〉 decreases from 650 ps (in native HSA) to 260 ps (~2.5 times) in the presence of 1.5 M RTIL and to 60 ps (~11 times) in the presence of 6 M GdnHCl. This is ascribed to unfolding of the protein by RTIL or GdnHCl and therefore making the probe CPM more exposed. When 1.5 M RTIL is added to the protein denatured by 6 M GdnHCl in advance, a further ~5 nm red shift along with further ~2 fold faster solvent relaxation (<τ> ~30 ps) is observed. Our previous fluorescence correlation spectroscopy study [D. K. Sasmal, T. Mondal, S. Sen Mojumdar, A. Choudhury, R. Banerjee, and K. Bhattacharyya, J. Phys. Chem. B 115, 13075 (2011)] suggests that addition of RTIL to the protein denatured by 6 M GdnHCl causes a reduction in hydrodynamic radius (r(h)). It is demonstrated that in the presence of RTIL and GdnHCl, though the protein is structurally more compact, the local environment of CPM is very different from that in the native state.

Differential role of nonmuscle myosin II isoforms during blebbing of MCF-7 cells.

Bleb formation has been correlated with nonmuscle myosin II (NM-II) activity. Whether three isoforms of NM-II (NM-IIA, -IIB and -IIC) have the same or differential roles in bleb formation is not well understood. Here we report that ectopically expressed, GFP-tagged NM-II isoforms exhibit different types of membrane protrusions, such as multiple blebs, lamellipodia, combinations of both, or absence of any such protrusions in MCF-7 cells. Quantification suggests that 50% of NM-IIA-GFP-, 29% of NM-IIB-GFP-, and 19% of NM-IIC1-GFP-expressing MCF-7 cells show multiple bleb formation, compared with 36% of cells expressing GFP alone. Of interest, NM-IIB has an almost 50% lower rate of dissociation from actin filament than NM-IIA and -IIC1 as determined by FRET analysis both at cell and bleb cortices. We induced bleb formation by disruption of the cortex and found that all three NM-II-GFP isoforms can reappear and form filaments but to different degrees in the growing bleb. NM-IIB-GFP can form filaments in blebs in 41% of NM-IIB-GFP-expressing cells, whereas filaments form in only 12 and 3% of cells expressing NM-IIA-GFP and NM-IIC1-GFP, respectively. These studies suggest that NM-II isoforms have differential roles in the bleb life cycle.

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 MAPbI3-x(SCN)x as compared to MAPbI3, suggestive of suppression of nonradiative channels. This increased intensity is attributed to a highly edge specific emission from MAPbI3-x(SCN)x 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 MAPbI3-x(SCN)x reveals that the optical bandgap and density of states at various (local) nanodomains are also nonuniform. Surprisingly, several (local) emissive regions within MAPbI3-x(SCN)x 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.

Role of Red-Ox Cycle in Structural Oscillations and Solvation Dynamics in the Mitochondria of a Live Cell.

Structural oscillations and solvation dynamics in the mitochondria of a live cell are studied by time-resolved microscopy using a covalent fluorescence probe. We compared the dynamics in a human breast cancer cell (MCF-7) with that in a normal breast cell MCF-10A. The probe, CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin), binds with the free thiol groups. In MCF-10A cell, CPM binds with the discrete mitochondria. In MCF-7, CPM labels the clustered mitochondria in the peri-nuclear region. Location of the CPM in the mitochondria is confirmed by colocalization with a mitochondria-tracker dye. The red-ox cycle in the mitochondria causes periodic fluctuation in the microenvironment in the discrete mitochondria. This is manifested in fluctuations in fluorescence intensity of CPM bound to mitochondria. The magnitude of oscillation is much less for CPM bound to the clustered mitochondria (in which the red-ox cycle is inefficient) in the cancer cell (MCF-7). In both of the cells (MCF-10A and MCF-7) CPM bound to thiol-containing proteins in mitochondria exhibits ultraslow response with average solvation time (⟨τs⟩) of 850 and 1400 ps in MCF-10A and MCF-7, respectively.

Solvation dynamics and intermittent oscillation of cell membrane: live Chinese hamster ovary cell.

Dynamics of the exofacial thiols (i.e., cell surface thiol containing membrane proteins) of a live Chinese hamster ovary (CHO) cell is probed by time-resolved confocal microscopy. For this purpose, a fluorescent probe, 7-(diethylamino)-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) is covalently attached to the exofacial thiols. The emission maximum of CPM bound exofacial thiols indicates a highly exposed and polar environment. Using CPM, we studied solvation dynamics, for the first time, at the membrane of a live cell. The thiol containing membrane proteins shows ultraslow response with average solvation time, ⟨τs⟩ = 475 ps. CPM labeled exofacial thiols also show spontaneous, intermittent oscillation in fluorescence intensity with a period of 0.5-1.0 s. This is ascribed to reversible, intermittent changes in the structure and conformation of the membrane proteins.

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.

Role of ionic liquid on the conformational dynamics in the native, molten globule, and unfolded states of cytochrome c: a fluorescence correlation spectroscopy study.

The role of a room temperature ionic liquid (RTIL, [pmim][Br]) on the size and conformational dynamics of a protein, horse heart cytochrome c (Cyt C) in its native, molten globule (MG-I and II), and unfolded states is studied using fluorescence correlation spectroscopy (FCS). For this purpose, the protein was covalently labeled by a fluorescent dye, Alexa Fluor 488. It is observed that the addition of the RTIL leads to an increase in the hydrodynamic radius (r(H)) of the protein, Cyt C in the native or MG-I state. In contrast, the addition of RTIL causes a decrease in the size (hydrodynamic radius, r(H)) of Cyt C unfolded by GdnHCl or MG-II state. The decrease in size indicates the formation of a relatively compact structure. We detected two types of conformational relaxation of the protein. The shorter relaxation time component (~3-5.5 µs) corresponds to the protein folding or intrachain contact formation, while the relatively longer time component (~63-122 µs) may be assigned to the motion of the protein side chains or concerted chain dynamics. The burst integrated fluorescence lifetime histograms indicate that the increase in size of the protein is accompanied by an increase in the contribution of the shorter component (~0.3-0.4 ns) with a concomitant decrease of the contribution of the longer component (~2.8-3.6 ns). An opposite trend is observed during the decrease in size of the protein.