Lysosomal Probe Research Articles

NIR-Emitting Hemicyanines with Large Stokes’ Shifts for Live Cell Imaging: from Lysosome to Mitochondria Selectivity by Substituent Effect

Lysosome imaging without perturbing intracellular activity remains challenging, as the current commercial lysosome probes contain weakly basic amino groups that could perturb lysosome pH. Herein, we illustrate NIR-emitting dyes 2 and 3 (λem ≈ 700 nm) with very large Stokes' shifts (Δλ = 231-246 nm), attributing to the presence of a 2-hydroxyphenyl(benzo[d]oxazol) (HBO) unit that undergoes excited-state intramolecular proton transfer (ESIPT). The structures of 2 and 3 also contain a hemicyanine unit with benzothiazolium and indolium as a terminal group, respectively. Although the fluorescent probe 2 (Φfl ≈ 0.28-0.35 in CH2Cl2) does not contain any basic amino functional group, it exhibits excellent selectivity for staining intracellular lysosomes, showing the potential for long-term in vivo lysosome imaging without "alkalinizing effect." However, probe 3 (Φfl ≈ 0.27, in CH2Cl2) exhibits excellent selectivity toward mitochondria. The observation showed that the terminal group in the hemicyanine unit played an essential role in guiding the intracellular selectivity to different organelles. In addition, the probes also displayed a transparent optical window between 520 and 590 nm, which is useful to achieve multicolor co-staining study, without fluorescence crosstalk that is a common problem on fluorescence microscopes.

A pH-insensitive near-infrared fluorescent probe for wash-free lysosome-specific tracking with long time during physiological and pathological processes

A novel dicyanomethylene-4H-pyran based lysosome-specific near-infrared (NIR) probe, DCM-ML, is reported. It exhibits very weak fluorescence (Ф=0.007) in aqueous solution due to the energy relaxation from intramolecular rotation; but a large "off-on" NIR emission (20-folds) after specifically labeling lysosomes due to the local high viscosity instead of the local pH by both one- and two-photon excitation microscopy. Therefore, the imaging ability of DCM-ML will not confront the problems suffered by the currently used and reported pH-dependent lysosome probes (e.g., once lysosomal pH increases, their fluorescence gets quenched); The pH-independent feature of DCM-ML is further demonstrated to be a great advantage for tracing lysosomes’movements in a relatively long time, even during cellular processes when lysosomes are suffered from external stimulations that induce pH increase and apoptosis. In addition to the characteristics of pH-insensitivity and "off-on" NIR emission, DCM-ML also possesses characteristics of excellent membrane permeability, high selectivity, low cytotoxicity, good photostability and large Stokes shift (>200 nm), which qualify it as a superior wash-free NIR probe for tracking dynamic changes of lysosomes under both physiological and toxicological conditions. Hence, we believe DCM-ML may find a great potential of application in the future.

Epidermal growth factor receptor (EGFR) density may not be the only determinant for the efficacy of EGFR-targeted photoimmunotherapy in human head and neck cancer cell lines.

The aim of this study was to investigate the effects of targeted photoimmunotherapy (PIT) in vitro on cell lines with various expression levels of epidermal growth factor receptor (EGFR) using an anti-EGFR targeted conjugate composed of Cetuximab and IR700DX, phthalocyanine dye. Relative EGFR density and cell binding assay was conducted in three human head & neck cancer cell lines (scc-U2, scc-U8, and OSC19) and one reference cell line A431. After incubation with the conjugate for 1 or 24 hours, cellular uptake and localization were investigated by confocal laser scanning microscopy and quantified by image analysis. Cell survival was determined using the MTS assay and alamarBlue assay after PIT with a 690 nm laser to a dose of 7 J.cm-2 (at 5 mW.cm-2 ). The mode of cell death was examined with flow cytometry using apoptosis/necrosis staining by Annexin V/propidium iodide, together with immunoblots of anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL. A431 cells had the highest EGFR density followed by OSC19, and then scc-U2 and scc-U8. The conjugates were localized both on the surface and in the cytosol of the cells after 1- and 24-hour incubation. After 24-hour incubation the granular pattern was more pronounced and in a similar pattern of a lysosomal probe, suggesting that the uptake of conjugates by cells was via receptor-mediated endocytosis. The results obtained from the quantitative imaging analysis correlate with the level of EGFR expression. Targeted PIT killed scc-U8 and A431 cells efficiently; while scc-U2 and OSC19 were less sensitive to this treatment, despite having similar EGFR density, uptake and localization pattern. Scc-U2 cells showed less apoptotic cell dealth than in A431 after 24-hour targeted PIT. Immunoblots showed significantly higher expression of anti-apoptotic Bcl-2 and Bcl-xL proteins in scc-U2 cell lines compared to scc-U8. Our study suggests that the effectiveness of EGFR targeted PIT is not only dependent upon EGFR density. Intrinsic biological properties of tumor cell lines also play a role in determining the efficacy of targeted PIT. We have shown that in scc-U2 cells this difference may be caused by differences in the apoptopic pathway. Lasers Surg. Med. 50:513-522, 2018. © 2018 Wiley Periodicals, Inc.

Real-Time Recording of the Cellular Effects of the Anion Transporter Prodigiosin

Summary The unraveling of the cellular effects of anion transporters is key to their potential development as apoptosis-inducing or autophagy-disrupting therapeutics. Here, we conducted a systematic study of the cellular responses to the anion transporter prodigiosin by using a pH on/off responsive near-infrared (NIR)-fluorescent probe in HeLa and LAMP1-GFP-transfected HeLa cell lines. The sequence of localized and global cellular acidity changes and the resulting outcomes induced by the anion transporter were visualized with high temporal and spatial resolution. The results show that prodigiosin causes the pH within the lysosomal lumen to rise, after which a non-organelle-specific increase in acidity of the cytosol takes place, which prompts cells to undergo apoptosis. This was confirmed by the quantification of NIR-emissive lysosomes, the intra-cellular fluorescence intensity, and fluorescence volume over time. This NIR probe overcame the limitations of acridine orange, which to date have severely restricted researchers in this field.

A long-lifetime iridium(iii) complex for lysosome tracking with high specificity and a large Stokes shift.

Investigating the role of lysosome dysfunction in cancer requires the development of efficient probes for lysosomes. We report herein a cyclometalated iridium(iii) complex (Ir-Ly) as a luminescent probe for visualizing lysosomes in cancer cells. The morpholine and hydroxy moieties within Ir-Ly provide suitable hydrophilicity and responsiveness to pH. Importantly, Ir-Ly exhibits a large Stokes shift, long lifetime and high photostability, which are important advantages for lysosome tracking in living cells.

Amino-Si-rhodamines: A new class of two-photon fluorescent dyes with intrinsic targeting ability for lysosomes

Noninvasive and specific visualization of lysosomes by fluorescence technology is critical for studying lysosomal trafficking in health and disease and for evaluating new cancer therapeutics that target tumor cell lysosomes. To date, there are two basic types of lysosomal probes whose lysosomal localization correlates with lysosomal acidity and endocytosis pathway, respectively. However, the former may suffer from pH-sensitive lysosomal localization and alkalization-induced lysosomal enzyme inactivation, and the latter need long incubation time to penetrate cell membrane due to the energy-dependency of endocytosis process. In this work, a new class of two-photon fluorescent dyes, termed amino-Si-rhodamines (ASiRs), were developed, which possess the intrinsic lysosome-targeted ability that is independent of lysosomal acidity and endocytosis pathway. As a result, ASiRs show not only the stable lysosomal localization against lysosomal pH changes and negligible interference to lysosomal function, but also excellent cell-membrane-permeability due to the energy-independent passive diffusion pathway. These merits, coupled with their excellent two-photon photophysical properties, long-term retention ability in lysosomes, and negligible cytotoxicity, make ASiRs very suitable for real-time and long-term tracking of lysosomes in living cells or tissues without interference to normal cellular processes. Moreover, the easy functionalization via amino linker further allows the construction of various fluorescent probes for biological targets of interest based on ASiR skeleton, as indicated by the cancer-targeted fluorescent probe ASiR6 as well as a fluorescent peroxynitrite probe ASiR-P.

Cell-permeable organic fluorescent probes for live-cell long-term super-resolution imaging reveal lysosome-mitochondrion interactions

Characterizing the long-term nanometer-scale interactions between lysosomes and mitochondria in live cells is essential for understanding their functions but remains challenging due to limitations of the existing fluorescent probes. Here, we develop cell-permeable organic fluorescent probes for lysosomes with excellent specificity and high photostability. We also use an existing Atto 647N dye with high brightness and excellent photostability to achieve specific labeling of mitochondria in live cells. Using these probes, we obtain dual-color structured illumination microscopy (SIM) images of dynamic physical lysosome-mitochondrion interactions in live cells at an ~90-nm resolution over a long time course of ~13 min. We successfully record the consecutive dynamic processes of lysosomal fusion and fission, as well as four types of physical lysosome-mitochondrion interactions by super-resolution imaging. Our probes provide an avenue for understanding the functions and the dynamic interplay of lysosomes and mitochondria in live cells.

Morpholine Derivative-Functionalized Carbon Dots-Based Fluorescent Probe for Highly Selective Lysosomal Imaging in Living Cells.

The development of a suitable fluorescent probe for the specific labeling and imaging of lysosomes through the direct visual fluorescent signal is extremely important for understanding the dysfunction of lysosomes, which might induce various pathologies, including neurodegenerative diseases, cancer, and Alzheimer's disease. Herein, a new carbon dot-based fluorescent probe (CDs-PEI-ML) was designed and synthesized for highly selective imaging of lysosomes in live cells. In this probe, PEI (polyethylenimine) is introduced to improve water solubility and provide abundant amine groups for the as-prepared CDs-PEI, and the morpholine group (ML) serves as a targeting unit for lysosomes. More importantly, passivation with PEI could dramatically increase the fluorescence quantum yield of CDs-PEI-ML as well as their stability in fluorescence emission under different excitation wavelength. Consequently, experimental data demonstrated that the target probe CDs-PEI-ML has low cytotoxicity and excellent photostability. Additionally, further live cell imaging experiment indicated that CDs-PEI-ML is a highly selective fluorescent probe for lysosomes. We speculate the mechanism for selective staining of lysosomes that CDs-PEI-ML was initially taken up by lysosomes through the endocytic pathway and then accumulated in acidic lysosomes. It is notable that there was less diffusion of CDs-PEI-ML into cytoplasm, which could be ascribed to the presence of lysosome target group morpholine on surface of CDs-PEI-ML. The blue emission wavelength combined with the high photo stability and ability of long-lasting cell imaging makes CDs-PEI-ML become an alternative fluorescent probe for multicolor labeling and long-term tracking of lysosomes in live cells and the potential application in super-resolution imaging. To best of our knowledge, there are still limited carbon dots-based fluorescent probes that have been studied for specific lysosomal imaging in live cells. The concept of surface functionality of carbon dots will also pave a new avenue for developing carbon dots-based fluorescent probes for subcellular labeling.

An NIR-emitting lysosome-targeting probe with large Stokes shift via coupling cyanine and excited-state intramolecular proton transfer.

An NIR-emitting probe (λem ∼ 700 nm) with a large Stokes shift (Δλ ≈ 234 nm) is synthesized by using excited-state intramolecular proton transfer (ESIPT). The phenolic proton, which controls ESIPT, acts as a switch to give strong fluorescence at pH ≈ 5. The probe can selectively show lysosome organelles, therefore leading to a lysosome probe without exhibiting "an alkalinizing effect".

A lysosomal probe for monitoring of pH in living cells and ovarian tumour

Intracellular pH is important in regulating various cellular behaviour and pathological conditions. We synthesised a rhodamine-based lysosomal pH probe (hereafter referred to as RD) with a pKa of 4.10. The probe exhibited favourable “on-off” reversibility between pH 3.25 and 7.20 for at least five cycles. RD can be employed to distinguish cancer cells from normal cells on the basis of different fluorescence response. RD performed well in the detection of lysosomal pH induced by artesunate, chloroquine and dexamethasone in MCF-7 cells. The intense fluorescence of RD in ovarian tumour indicated the higher acidity in tumour tissues than that in normal tissues. Furthermore, RD worked well in the imaging of excised mouse stomach and living mice under acidic stimulation. These data demonstrate the applicability of RD in monitoring pH in complex biological systems.

A small-structure two-photon platform equipped with weak electron donor: Development of permeable two-photon probes for lysosomes and mitochondria in living cells

Small-structure two-photon dyes displaying intense and long-wavelength emission in aqueous solutions are extremely attractive, because they are powerful platforms for design of fluorescent probes targeting various targets in cytoplasma. However, design of such dyes is difficult, because the photo-induced twisted intramolecular charge transfer (TICT) process often leads to low quantum yields. An efficient strategy is to introduce covalent bonds to constrain the intramolecular rotation, such as fluorecein and rhodamine dyes. But this strategy would bring great difficulty in molecular design and synthesis, and therefore cannot be always used. In this work, we provide a novel strategy by linking weak electron donor and strong electron acceptor to a simple conjugated system, and a small-molecule two-photon dye 1-(2-hydroxyethyl)-4-(4-(methylthio)styryl) pyridin-1-ium iodide (HMTP) was synthesized. Experimental results demonstrated that HMTP exhibited suppressed TICT process and strong green fluorescence in buffer solutions with modest two-photon performances (δ=320 GM). In consequence, HMTP has been modified as two probes, 4-(4-(methylthio)styryl)-1-propylpyridin-1-ium iodide (HMTP-MT) and 4-(4-(methylthio)styryl)pyridine (HMTP-LY), to bioimage mitochondria and lysosomes with intense one- and two-photon fluorescence. These results demonstrate the ability of HMTP as novel platform for the design of various one- and two-photon fluorescent probes, and also the validity of the strategy to construct fluorophores with weak electron donor and strong electron acceptor.

Cresyl violet: a superior fluorescent lysosomal marker.

We have characterized cresyl violet as a membrane-permeant fluorophore that localizes to lysosomes and acidic vacuoles of budding yeast, Drosophila, human, murine and canine cells. An acidotropic weak base, cresyl violet is shown to be virtually insensitive to physiological alkali and divalent cations. Because of its unique spectral properties, it can be used in combination with green, red and far-red fluorophores, is less susceptible to photobleaching than alternative acidotropic probes, and does not undergo photoconversion. At concentrations that yield bright labeling of acidic compartments, cresyl violet does not alter the organellar pH nor does it affect the buffering capacity. Its affordability, together with its chemical and spectral properties, make cresyl violet a superior lysosomal marker devoid of many of the negative characteristics associated with other lysosomal probes.

Fluorogenic Probes for Multicolor Imaging in Living Cells.

Here we present a far-red, silicon-rhodamine-based fluorophore (SiR700) for live-cell multicolor imaging. SiR700 has excitation and emission maxima at 690 and 715 nm, respectively. SiR700-based probes for F-actin, microtubules, lysosomes, and SNAP-tag are fluorogenic, cell-permeable, and compatible with superresolution microscopy. In conjunction with probes based on the previously introduced carboxy-SiR650, SiR700-based probes permit multicolor live-cell superresolution microscopy in the far-red, thus significantly expanding our capacity for imaging living cells.

An efficient two-photon ratiometric fluorescent probe platform for dual-channel imaging of lysosomes in living cells and tissues

In contrast to one-photon microscopy, two-photon probe-based fluorescence imaging can provide improved three-dimensional spatial localization and increased imaging depth. Therefore, the development of new functional two-photon fluorescent dye has attracted great attention. Herein, we have adopted the fluorescence resonance energy transfer (FRET) strategy to design a unique type of two-photon ratiometric fluorescent probe platform. Specifically, a two-photon fluorophore (D-π-A-structured naphthalimide derivative) and a rhodamine B fluorophore are directly connected by a flexible piperidine linker. We further used this platform to develop a new type of two-photon ratiometric fluorescent probe, NRLys, for lysosomal pH detection and bioimaging. The experiments demonstrated that the NRLys is a reliable and specific probe for labeling lysosomes in living cells and tissues with two well-resolved emission peaks separated by 60nm. The probe showed high ratiometric imaging resolution and deep-tissue imaging depth of over 180μm. Based on the above results, we expect that the new platform may prompt the development of a wide variety of two-photon ratiometric fluorescent probes application in biological systems.

A new ratiometric two-photon fluorescent probe for imaging of lysosomes in living cells and tissues

Intracellular pH plays a pivotal role in various biological processes. Herein, we adopted the through bond energy transfer (TBET) strategy to design a unique type of ratiometric two-photon fluorescent probe for imaging of lysosomal pH in live cells and tissues, termed Np-Rh-Lys. Specifically, a two-photon fluorophore (D-π-A-structured naphthalimide derivative) and a rhodamine B fluorophore were directly connected, and dimethylamino moiety served as a lysosomal targeting-group. The experiments demonstrate new probe Np-Rh-Lys was a reliable and specific probe for labeling lysosomes in living cells with two well-resolved emission peaks separated by 80 nm, and which showed high ratiometric imaging resolution and deep-tissue imaging depth over 180 μm, we thus may expect the new platform prompt the development of a wide variety of ratiometric two-photon fluorescent probes application in biological systems.

Two-Photon Probes for Lysosomes and Mitochondria: Simultaneous Detection of Lysosomes and Mitochondria in Live Tissues by Dual-Color Two-Photon Microscopy Imaging.

Novel two-photon (TP) probes were developed for lysosomes (PLT-yellow) and mitochondria (BMT-blue and PMT-yellow). These probes emitted strong TP-excited fluorescence in cells at widely separated wavelength regions and displayed high organelle selectivity, good cell permeability, low cytotoxicity, and pH insensitivity. The BMT-blue and PLT-yellow probes could be utilized to detect lysosomes and mitochondria simultaneously in live tissues by using dual-color two-photon microscopy, with minimum interference from each other.

Spiroboronate Si-rhodamine as a near-infrared probe for imaging lysosomes based on the reversible ring-opening process.

A cyclic boronate structure was incorporated into Si-rhodamine to design a pH-activatable near-infrared (NIR) probe based on the reversible ring-opening process triggered by H(+). The probe showed general suitability of specific labelling of acidic lysosomes and tracking their pH changes in living cells.

Thiophene-based fluorescent probes with low cytotoxicity and high photostability for lysosomes in living cells

BackgroundSelective imaging of lysosomes by fluorescence microscopy using specific fluorescent probes allows the study of biological processes and it is potentially useful also for diagnosis. Lysosomes are involved in numerous physiological processes, such as bone and tissue remodeling, plasma membrane repair, and cholesterol homeostasis, along with cell death and cell signaling. Despite the great number of dyes available today on the market, the search for new fluorescent dyes easily up-taken by cells, biocompatible and bearing bright and long-lasting fluorescence is still a priority. MethodsTwo thiophene-based fluorescent dyes, TC1 and TC2, were synthetized as lysosome-specific probes. ResultsThe new dyes showed high selectivity for fluorescent staining and imaging of lysosomes and disclosed high photostability, low toxicity and pH insensitivity in the range 2–10. ConclusionsThe TC dyes exhibited high co-localization coefficients (>95%) and moderate quantum yields. They showed high biocompatibility and long-term retention, important features for biological applications. General significanceThe results of the present work disclose a new class of organic dyes with potential wide applications as specific and efficient lysosome probes in the study of various biological processes.

High-content screening using metabolic lysosomal enzyme probes

High-content screening using metabolic lysosomal enzyme probes

A new fluorescent pH probe for imaging lysosomes in living cells

A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5–4.1 with the pKa value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no ‘alkalizing effect’ on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells.