Cell Imaging Research Articles

Exploring the Impact of Variability in Cell Segmentation and Tracking Approaches.

Segmentation and tracking are essential preliminary steps in the analysis of almost all live cell imaging applications. Although the number of open-source software systems that facilitate automated segmentation and tracking continue to evolve, many researchers continue to opt for manual alternatives for samples that are not easily auto-segmented, tracing cell boundaries by hand and reidentifying cells on consecutive frames by eye. Such methods are subject to inter-user variability, introducing idiosyncrasies into the results of downstream analysis that are a result of subjectivity and individual expertise. The methods are also susceptible to intra-user variability, meaning findings are challenging to reproduce. In this pilot study, we demonstrate and quantify the degree of intra- and inter-user variability in manual cell segmentation and tracking by comparing the phenotypic metrics extracted from cells segmented and tracked by different members of our research team. Furthermore, we compare the segmentation results for a ptychographic cell image obtained using different automated software and demonstrate the high dependence of performance on the imaging modality they were developed to handle. Our results show that choice of segmentation and tracking methods should be considered carefully in order to enhance the quality and reproducibility of results.

Impact of antiplatelets, anticoagulants and cyclic nucleotide stimulators on neutrophil extracellular traps (NETs) and inflammatory markers during COVID-19.

While the association between coronavirus disease-19 (COVID-19) and neutrophils extracellular traps (NETs) is recognized, uncertainties remain regarding its precise onset, timing of resolution and target therapy. To assess changes in inflammatory and NET markers during the first week of COVID-19 hospitalization, and the association with disease severity. "In vitro" experiments investigated the effect of antiplatelets, anticoagulants, and cyclic nucleotide stimulators on NETs release. Prospective cohort study, changes in interleukin (IL)-6, IL-8, IL-17, TNF-α, RANTES, PF4, and citrullinated-H3 (citH3) levels within each outcome group was evaluated using ANOVA. Differences between moderately ill, critically ill, and non-survivors were determined using Kruskal-Wallis and logistic regression. Healthy neutrophils were stimulated with phorbol-12-myristate-13-acetate (PMA) or COVID-19 sera and treated with unfractionated heparin (UFH), low molecular weight heparin (LMWH), aspirin (ASA), ticagrelor, cinaciguat, sildenafil, and milrinone. The proportion of NETosis was assessed using IncuCyte Cell Imager. Of the 125 patients, 40.8% had moderate COVID-19, 40.8% had critical COVID-19 but recovered, and 18.4% died. From admission to hospitalization day 8, IL-6 levels decreased in moderately and critically ill, but not in non-survivors, while citH3 levels increased in critically ill and non-survivors. IL-6, IL-8, and TNF-α levels were associated with critical and fatal COVID-19. The release of NETs by neutrophils stimulated with PMA or COVID-19 sera was decreased in the presence of ASA, UFH, LMWH and cyclic nucleotide stimulators in a dose-dependent manner. In the first week of hospitalization, NET markers rose later than inflammatory markers in severe COVID-19 cases. Cyclic nucleotide stimulators, ASA and heparin may emerge as treatment approaches as they may modulate NETosis.

Chiral Self-Assembly of a Pyrene-Appended Glutamylalanine Dipeptide and Its Charge Transfer Complex: Fabrication of Magneto-Responsive Hydrogels and Human Cell Imaging.

The formation of a robust, self-healing hydrogel of a novel pyrene-appended dipeptide, Py-E-A (L-Glutamic acid short as E; L-Alanine short as A) is demonstrated. Detailed studies suggest that nanoscopic fibers with a length of several micrometers have formed by chiral self-organization of Py-E-A gelators. Additionally, live human PBMCs imaging is shown using the Py-E-A fluorophore. Interestingly, electron-rich Py-E-A couples with electron-deficient NDI-β-A (β-Alanine short as β-A) by charge transfer (CT) complexation and forms stable deep violet-colored CT super-hydrogel. X-ray diffraction, DFT, and 2D ROESY NMR studies suggest lamellar packing of both Py-E-A and the alternating CT stack in its hydrogel matrixes. Supramolecular chirality of the Py-E-A donor can be altered by adding an achiral acceptor NDI-β-A. Notably, the fibers of the CT hydrogel are found to be even thinner than the Py-E-A fibers, which, in turn, makes the CT hydrogel more tolerant to the applied strain. Further, the self-healing and injectable properties of the hydrogels are shown. Finally, the magneto-responsive behavior of the Py-E-A and CT hydrogels loaded with spin-canted Cu-ferrite (Cu0.6Zn0.4Fe2O4) nanoparticles (NPs) is demonstrated. The presence of magnetic NPs within the hydrogels has changed the fibrous morphology to rod-like nanoclusters.

Microwave-Assisted Synthesis of N, S Co-Doped Carbon Quantum Dots for Fluorescent Sensing of Fe(III) and Hydroquinone in Water and Cell Imaging

The detection of heavy metal ions and organic pollutants from water sources remains critical challenges due to their detrimental effects on human health and the environment. Herein, a nitrogen and sulfur co-doped carbon quantum dot (NS-CQDs) fluorescent sensor was developed using a microwave-assisted carbonization method for the detection of Fe3+ ions and hydroquinone (HQ) in aqueous solutions. NS-CQDs exhibit excellent optical properties, enabling sensitive detection of Fe3+ and HQ, with detection limits as low as 3.40 and 0.96 μM. Notably, with the alternating introduction of Fe3+ and HQ, NS-CQDs exhibit significant fluorescence (FL) quenching and recovery properties. Based on this property, a reliable “on-off-on” detection mechanism was established, enabling continuous and reversible detection of Fe3+ and HQ. Furthermore, the low cytotoxicity of NS-CQDs was confirmed through successful imaging of HeLa cells, indicating their potential for real-time intracellular detection of Fe3+ and HQ. This work not only provides a green and rapid synthesis strategy for CQDs but also highlights their versatility as fluorescent probes for environmental monitoring and bioimaging applications.

Abstract B022: Characterizing invasiveness in CCCR cells: Role of miR-100, miR-125b and EVs

Abstract Introduction: Extracellular vesicles have been found to be important regulators of intercellular communication between cancer cells. EV cargo varies greatly between different cell types, the composition of which provides important insights into the role of donor and recipient cells. Cetuximab Resistant Colorectal Cancer (CCCR) cells upregulate miR-100 and miR-125b and select them as cargo for their respective secreted EVs. These miRNAs are responsible for altered gene expression in the CCCR cells and can also be functionally transferred as part of EVs between donor and recipient cells. Bioinformatic analysis identified Cingulin (CGN) mRNA as one of the possible targets of miR-100 and miR-125b. The action of miRNAs leads to degradation of CGN mRNA ultimately decreasing cellular concentration of the functional protein. Absence of CGN has been associated with increased invasiveness and metastatic potential in the cancer cells. We carried out immunofluorescent imaging of CCCR cells which revealed downregulation of CGN in CCCR cells compared to the parental Cetuximab sensitive Colorectal Cancer (CC) cells. Similar effects were observed in recipient cells that obtained the miRNAs as part of EVs originating from CCCR donor cells. This project helps elucidate the roles of miR-100 and miR-125b in CCCR cells and how EVs facilitate their functional transfer between nearby cancer cells. Methods: CCCR cell lines were derived from parental CC cells in the lab of Dr. Robert Coffey by iterative selection of cetuximab resistance after growth in 3D. CRISPR/Cas9 technology was used to knockout miR-100 and miR-125b in CCCR cells. Transwell co-culture experiments were carried out to analyze EV transfer of miRNAs. Transfer was monitored by immunofluorescence using antibodies against CGN. Cells were stained and imaged as Z stacks under the 63x Immersion setting. ImageJ was used to analyze the stacks and CGN concentrations between the cells. For quantification of immunofluorescence, additional Z stack images were obtained from an inverted microscope and fluorescence digital camera. Images were analyzed using Fiji software to obtain specific CGN concentrations between the cells. Results and Discussion: Immunofluorescent imaging revealed statistically significant downregulation of CGN in CCCR cells. The protein was also downregulated in the recipient knockout cells as part of the Transwell co-culture experiment with CCCR donor cells. CGN is a tight junction protein and its absence confers increased invasiveness in 3D growth with enhanced metastasis. Decreased CGN concentration in the miR-100 and miR-125b enriched EV recipient knockout cells is indicative of functional transfer of the two miRNAs between cancer cells. These distinctive cellular dynamics could be harnessed to develop potential cancer therapies that provide a better prognosis and limit the metastatic potential of targeted cancer cells. Citation Format: Muhammad Shameer, Hannah M Nelson, Shimian Qu, Liyu Huang, Kevin C Corn, Sydney N Chapman, Nicole L Luthcke, Sara A Schuster, Tellie D Stamaris, Lauren A Turnbull, Lucas L Guy, Xiao Liu, Danielle L Michell, Elizabeth M Semler, Kasey C Vickers, Qi Liu, Jeffrey L Franklin, Alissa M Weaver, Marjan Rafat, Robert J Coffey, James G Patton. Characterizing invasiveness in CCCR cells: Role of miR-100, miR-125b and EVs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B022.

Live-Cell Imaging of miRNAs with the Combination of CHA and HCR Techniques.

The abnormal expression of miRNA-21 is closely related to many cancers, and its sensitive detection plays an important role in the diagnosis and treatment of cancers. In the report, we designed a non-enzymatic amplification sensing system based on the catalytic hairpin assembly (CHA) and palindrome-based hybridization chain reaction (PHCR) technique for the detection and imaging of miRNA in living cells. Based on PHCR technology, two ingeniously designed palindrome-contained fluorescently labeled hairpin probes are sequentially opened upon the stimulation of short oligonucleotide triggers, promoting the autonomous assembly of cross-linked network-like structures (CNSs) and generating fluorescence resonance energy transfer (FRET) signal. Moreover, the PHCR technique can be combined with CHA technology to sufficiently improve amplification efficiency and signal output. By utilizing the CHA-PHCR sensing system, one miRNA-21 activates many triggers through CHA process and in turn initiates the assembly of CNSs by PHCR reaction, efficiently amplifying the FRET signal output. As such, the sensing system can detect target miRNAs down to 10pm with high detection specificity. Moreover, the CNS exhibits the enhanced intracellular stability, enabling the living cell imaging of miRNA-21. The CHA-PHCR sensing system can also screen the expression level of miRNA-21 in different living cells and differentiate cancer cells from healthy cells, which is consistent with the gold-standard PCR method.

Benzo[c,d]Indole-Quinoline-Based Deep-Red Emissive Probes for Live-Cell Imaging of Nucleolar RNA.

Small molecular weight fluorescent probes capable of binding to RNAs have been powerful tools for understanding the intracellular behaviors of RNAs. In this chapter, we describe the fluorescence imaging of nucleolar RNA in living cells using deep-red emissive probes with benzo[c,d]indole-quinoline (BIQ) monomethine cyanine scaffolds. These probes feature a significant fluorescence "off-on" ability upon binding to RNAs (>100-fold) in the deep-red spectral region (λem>650nm). In addition, they have many advantages for fluorescence sensing of RNAs and nucleolar RNA imaging, such as longer emission wavelength, higher photostability, and better counterstaining compatibility for live cell imaging, compared to a commercially available RNA-binding probe, SYTO RNA select.

Rosuvastatin activates autophagy via inhibition of the Akt/mTOR axis in vascular smooth muscle cells.

The proliferation and migration of vascular smooth muscle cells (VSMCs) are key contributors to the development of atherosclerosis and restenosis. We investigated the impact of rosuvastatin (RSV) on platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of VSMCs, with a focus on the Akt/mTOR-autophagy signaling pathways. The cytotoxicity of RSV was assessed using MTT and annexin V staining, while the proliferation and migration capabilities of PDGF-BB-induced VSMCs were evaluated using MTT and cell migration assays. Confocal microscopy was employed to examine autophagic cell images, and protein expressions were analyzed via Western blotting. Our key findings revealed that RSV inhibited PDGF-BB-induced proliferation and migration of VSMCs, significantly reducing the expression of proliferating cell nuclear antigen and matrix metalloproteinase-2, which are crucial for these processes. RSV also enhanced autophagy in PDGF-BB-stimulated cells by inducing the maturation of microtubule-associated protein light chain 3 and increasing the expression of Beclin-1, autophagy related (Atg)3, Atg5, and Atg7. The regulatory effects of RSV on PDGF-BB-induced autophagy, proliferation, and migration were associated with the suppression of the Akt/mTOR signaling pathway. These findings suggest that RSV may have potential therapeutic benefits in preventing and treating vascular diseases by targeting the Akt/mTOR pathway and inducing autophagy.

Rational Design of Biocompatible Ir(III) Photosensitizer to Overcome Drug-Resistant Cancer via Oxidative Autophagy Inhibition.

Autophagy is a crucial quality control mechanism that degrades damaged cellular components through lysosomal fusion with autophagosomes. However, elevated autophagy levels can promote drug resistance in cancer cells, enhancing their survival. Downregulation of autophagy through oxidative stress is a clinically promising strategy to counteract drug resistance, yet precise control of oxidative stress in autophagic proteins remains challenging. Here, a molecular design strategy of biocompatible neutral Ir(III) photosensitizers is demonstrated, B2 and B4, for precise reactive oxygen species (ROS) control at lysosomes to inhibit autophagy. The underlying molecular mechanisms for the biocompatibility and lysosome selectivity of Ir(III) complexes are explored by comparing B2 with the cationic or the non-lysosome-targeting analogs. Also, the biological mechanisms for autophagy inhibition via lysosomal oxidation are explored. Proteome analyses reveal significant oxidation of proteins essential for autophagy, including lysosomal and fusion-mediator proteins. These findings are verified in vitro, using mass spectrometry, live cell imaging, and a model SNARE complex. The anti-tumor efficacy of the precise lysosomal oxidation strategy is further validated in vivo with B4, engineered for red light absorbance. This study is expected to inspire thetherapeutic use of spatiotemporal ROS control for sophisticated modulation of autophagy.

Live-Cell Imaging of RNA G-Quadruplex with a Dual-Color Fluorescence Switch-on Probe.

In-cell imaging of the G-quadruplex (G4) formation of nucleic acids remains challenging for revealing G4's functions in cells. This study describes the cell imaging method of G4 nucleic acids using our unique tripodal quinone-cyanine fluorescent dye, QCy(MeBT)3, whose 600nm and 700nm fluorescence is enhanced independently upon the binding with double-stranded DNA and G4-DNA/RNA, respectively. The use of QCy(MeBT)3 enables us to visualize cytosolic G4 RNAs in fixed and living HeLa cells with near-infrared 700nm fluorescence.

Green synthesis of fluorescent N-doped carbon quantum dots from castor seeds and their applications in cell imaging and pH sensing.

Water-soluble fluorescent N-doped carbon quantum dots (N-CQDs) were hydrothermally prepared through a green synthesis route using castor seeds as a single precursor and a hydrothermal method. Several experimental techniques have been used to characterize synthesized N-CQDs to confirm their structure and to verify their applicability in cell imaging and pH sensing. The synthesized N-CQDs were found to have are characterized by amorphous nature with a spherical shape with an average particle size of 6.57nm as revealed from XRD and TEM measurements. The FTIR results reveal the presence of carboxylic and hydroxyl functional groups on the surface of the CQDs, which was also confirmed by XPS analysis. The fluorescence characterization of the synthesized N-CQDs showed blue emission and excitation dependence with good photostability. It was found that the optimal excitation and emission wavelengths were (λEx = 360) and (λEm = 432) nm, respectively. The fluorescence quantum yield (QY) of about 9.6% at the optimum excitation wavelength 360nm. Moreover, the fluorescence intensity of N-CQDs showed good linear dependence with the pH values in ranges of 3.5 - 7.5 and 8 - 12 as well as high sensitivity for slight changes of pH values. According to these results, two fluorescent pH sensors were created based on acidic and basic media. The obtained N-CQDs have zeta potential of -21.86 mV and thus have excellent stability in water. Moreover, N-CQDs derived from the castor seeds have antimicrobial activity and exhibits low cytotoxicity to WI-13 cells with IC50 = 394.4 ± 13.8µg/mL. The results of this study demonstrated that the synthesized N-CQDs derived from castor seeds can be used as pH sensing and antimicrobial materials. On the other hand, they are also promising in applications in cell imaging, thermo-sensing and optoelectronics.

A Ratiometric Fluorescence Probe for Visualized Detection of Heavy Metal Cadmium and Application in Water Samples and Living Cells

Heavy metal cadmium (II) residuals have inflicted severe damage to human health and ecosystems. It has become imperative to devise straightforward and highly selective sensing methods for the detection of Cd2+. In this work, a ratiometric benzothiazole-based fluorescence probe (BQFA) was effortlessly synthesized and characterized using standard optical techniques for the visual detection of Cd2+ with a change in color from blue to green, exhibiting a significant Stokes shift. Moreover, the binding ratio of BQFA to Cd2+ was established as 1:1 by the Job’s plot and was further confirmed by FT-IR and 1HNMR titrations. The ratiometric fluorescence response via the ICT mechanism was confirmed by DFT calculations. Furthermore, the limit of detection for detecting Cd2+ was determined to be 68 nM. Furthermore, it is noteworthy that BQFA showed good performance in real water samples, paper strips, smartphone colorimetric identification, and cell imaging.

Size dependent properties of Gd3+-free versus Gd3+-doped carbon dots for bioimaging application.

Gd3+- free carbon dots (CDs) were synthesized by one-step solvothermal method using urea, citric acid and 3-(trifluoromethyl)aniline as precursors. Additionally, Gd3+-doped CDs were prepared by incorporating gadolinium chloride (Gd3+ ions) into the synthesis. Size selection of the purified CDs was achieved through filter membranes ranging from 3kDa to 100kDa. The chemical composition and optical properties of the obtained samples were characterized by Energy dispersive X-ray (EDX), Fourier-transform infrared spectroscopy (FTIR), Dynamic Light Scattering (DLS), proton relaxation time measurements, Ultraviolet-visible (UV-vis) and fluorescence spectroscopies. A comparative analysis revealed a strong size-dependent behavior in the optical properties of both Gd3+-doped and Gd3+-free CDs. Furthermore, in vitro tests confirmed the non-cytotoxicity of Gd3+-doped CDs, indicating their potential applicability in biomedicine for magnetic resonance imaging (MRI) and red fluorescence-based cell and tissue imaging.

Dual-Locked Chemiluminescent Probe Enables Precise Imaging and Timely Diagnosis of Colitis via Chymotrypsin/Vanin-1 Cascade Activation.

The development of precise diagnosis and the discovery of individualized drugs go together to provide effective therapy against inflammatory bowel disease (IBD). The exploitation of the unique imaging advantages of chemiluminescent probes represents a pivotal strategy for achieving this goal. Nevertheless, the dual-locked strategy, which is believed to enhance precision, is rarely employed in the design of chemiluminescent probes. A novel dual-locked chemiluminescent probe, BPan-CL, was designed based on IBD candidate biomarkers chymotrypsin (CHT) and vanin-1. BPan-CL exhibited specific reactivity and chemiluminescence response when subjected to simultaneous stimulation of CHT and vanin-1, with a signal-to-noise ratio superior to that of the fluorescent probe with the same dual-locked mode. In both live cell and IBD mice imaging, BPan-CL demonstrated superior sensitivity compared to its single-locked counterpart, Pan-CL. In contrast to Pan-CL, BPan-CL was able to more accurately identify IBD and healthy mice by in vivo imaging and allowed for early prediction of IBD using a noninvasive fecal test. BPan-CL has identified CHT and vanin-1 as valuable combinatorial biomarkers for accurate and early IBD diagnosis. This strategy has significant potential for use in biomedical imaging and future individualized therapies.

Pyrene Based Schiff Base for Cascade Fluorescent "On-Off" Detection of Aluminium(III) and Fluoride Ions and its Applications in Live Cells Imaging.

An easy-to-prepare pyrene-based Schiff base PNZ was synthesized by condensing equimolar amount of 1-pyrenebutyric hydrazide with 2-hydroxy-naphthaldehyde, and employed as a fluorescent chemosensor for in-situ cascade detection of aluminium (Al3+) and fluoride (F-) ions. In DMSO:H2O (1 : 1, v/v), the weakly emissive PNZ showed a significant fluorescence enhancement at 455 nm selectively upon the addition of Al3+ due to the complexation-induced formation of a pyrene excimer. Schiff base PNZ and Al3+ formed a complex in 2 : 1 binding ratio with the estimated binding constants of K1:1=9826.01 M-1 and K2:1=3188.49 M-1. The sensing mechanism was explored by performing quantum mechanical calculations and 1H-NMR titration of PNZ with Al3+. The in-situ formed PNZ-Al3+ complex species enabled the fluorescent turn-off detection of F-. Using PNZ and PNZ-Al3+, the concentrations of Al3+ and F- ions can be detected down to 2.89×10-7 M and 1.88×10-7 M, respectively. The cytotoxicity of the PNZ and its ability to bioimage Al3+ and F- ions was examined in the human cervical cancer cell line. Finally, the receptor PNZ was applied for the quantification of Al3+ and F- ions in various real samples, such as tap water, river water, rainwater, mouthwash, and toothpaste.

Environment-sensitive turn-on fluorescent probe enables live cell imaging of myeloperoxidase activity during NETosis.

Myeloperoxidase (MPO) plays an important role in the immune response of human neutrophils and has been implicated in autoimmune conditions, cardiovascular disorders, and neurodegeneration. Current methods to detect MPO activity rely on the detection of HOCl using activatable probes or require challenging experimental procedures. Therefore, these tools provide limited information about the dynamics and localization of MPO in complex molecular processes such as NETosis in real time. In this study, we report a ''turn-on" activity-based probe that fluoresces exclusively upon binding to MPO, exhibits minimal background fluorescence in buffered aqueous media, and is blocked by MPO inhibitors. Our probe facilitates real-time imaging of direct MPO activity in human neutrophils and HL-60-derived granulocytes during NETosis under wash-free conditions. Furthermore, it allows for the discrimination between different triggers of NETosis in human neutrophils. These findings hold promise for advancing our understanding of the role of MPO in immune responses and inflammatory conditions.

Metal-Responsive Fluorophore and Amikacin-Conjugated Heparin for Bacterial Cell Imaging and Antibacterial Applications.

The escalating prevalence of bacterial infections presents a formidable challenge to current global healthcare systems. Rapid identification and quantification of bacterial pathogens with anticipated sensitivity and selectivity are crucial for targeted therapeutic interventions to mitigate disease burden, drug resistance, and further transmission. Concurrently, there is a pressing need to innovate novel approaches to combat infections and counter antibiotic resistance. Herein, we demonstrated the development of heparin (HP) conjugates modified with a Zn2+-induced "turn-on" fluorophore, 2-(pyridin-2-yl)-1H-benzo[d]imidazole (PBI), that interacts with bacterial cells via specific binding with the surface-exposed heparin-binding proteins (HPBs), thereby inducing fluorescence signals for rapid and selective sensing of whole bacterial cells. Additionally, amikacin (Amk) antibiotic was integrated into the modified heparin polymer (HP-PBI-Amk) to augment its antibacterial efficacy via reactive oxygen species generation. Despite the nephrotoxicity of only amikacin, its inclusion in the biopolymer retains its antibacterial properties while providing biocompatibility. The outcome of this study demonstrates the development of HP-PBI and HP-PBI-Amk as promising strategies for bacterial detection and eradication, respectively, offering potential avenues for future research and clinical applications.

A Spectroscopic Study on the Amyloid-β Interaction with Clicked Peptide-Porphyrin Conjugates: a Vision Toward the Detection of Aβ Peptides in Aqueous Solution.

Alzheimer's disease (AD) is a multifactorial form of dementia mainly affecting people in the elderly, but no effective cure is available. According to the amyloid hypothesis the aggregation of Amyloid-β (Aβ) into oligomeric toxic species is believed to concur with the onset and progression of the disease heavily. By using a click chemistry approach, we conjugated a suitable designed peptide sequence to a metalloporphyrin moiety to obtain three hybrid peptide systems to be studied for their interaction with Amyloid-β peptides. The aim is to get new tools for the diagnosis and therapy in AD. The results described in this study, which were obtained through spectroscopic techniques (UV-Vis, CD, bis-Ans and intrinsic porphyrin Fluorescence), Microfluidics (GCI) and cell biology (MTT, Live cell imaging and flow cytometry), reveal interesting features about the structure-activity relationships connecting these conjugates with the interaction with Aβ, as well as on their potential use as sensing systems. In our opinion the data reported in this paper make the porphyrin-peptide conjugates highly compelling for further exploration as spectroscopic probes to detect Aβ biomarkers in biological fluids.

Clickable HaloTag ligands for live cell labeling and imaging

Self-labeling protein (SLP) tags, such as HaloTag, have gained considerable interest as advanced tools for live cell labeling. However, the chloroalkane-based substrates that can be directly used for protein labeling are limited. Here, we report two bioorthogonal small molecule linkers, chloroalkane-tetrazine (CA-Tz) and chloroalkane-azide (CA-N3), which can penetrate cell membranes and facilitate click chemistry-based labeling in live cells. We compare their labeling capability using two clickable silicon rhodamine dyes (SiR-PEG3-TCO and SiR-PEG4-DBCO). Confocal imaging results demonstrate that using CA-Tz and SiR-PEG3-TCO dye exhibits superior intracellular labeling with low nonspecific signals. We subsequently compared the photostability of SiR dyes with that of green fluorescent proteins (mEmerald). Total internal reflection fluorescence (TIRF) imaging indicates that SiR dyes exhibit superior photostability under identical excitation conditions, making them suitable for long-term cell imaging. Furthermore, SiR dyes labeling also shows high structure retention for the fourth-order super-resolution optical fluctuation imaging (SOFI) compared to fluorescent proteins. This study presents clickable HaloTag linkers as effective tools for live cell labeling and imaging, highlighting the high-quality labeling of chloroalkane linkers and clickable dyes for live cell imaging.

LASCA-Based Monitoring of Drug Impact and Classification using Machine Learning for Biospeckle Images of Melanoma Cells

LASCA-Based Monitoring of Drug Impact and Classification using Machine Learning for Biospeckle Images of Melanoma Cells