The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Luminescence imaging of biological specimens using non-invasive probes is a basic technique in life and biomedical sciences for studying the morphologic characteristics of tissue at high resolution  — .
Since the cell is the primary structural and functional unit of all known living organisms, the morphological aberration of certain cell types can lead to various diseases such as sickle cell anemia  , . Consequently, a great deal of attention has been invested into the development of luminescent probes for live cell imaging in recent years. Currently, organic dyes constitute the majority of the most commonly-used fluorescent probes . However, organic dyes can be subject to various drawbacks, including small Stokes shift values and short luminescence lifetimes  — .
In eukaryotes, the cytoplasm is an aqueous fluid that primarily consists of a transparent substance termed hyaloplasm or cytosol. Numerous life processes take place within the cytoplasm, including protein synthesis, metabolic reactions, and cellular signaling. However, only a few phosphorescent metal complexes have been developed for cytoplasmic staining. Barton and co-workers investigated a series of phosphorescent ruthenium II complexes with different ancillary ligands that selectively stain the cytoplasm .
The groups of Li and Lo have developed a series of cationic iridium III complexes as phosphorescent probes for luminescence staining of the cytoplasm of living cells  ,  — . Iridium III complexes with d 6 electronic structures often possess excellent photophysical properties such as tunable excitation and emission wavelengths from blue to red , high luminescent quantum yields, and relatively long phosphorescence lifetimes  , .
Iridium complexes have received considerable attention in inorganic photochemistry  —  , phosphorescent materials for optoelectronics  —  , chemosensors  —  , biolabeling  —  , live cell imaging  ,  —  , and in vivo tumor imaging .
We demonstrate that the complex is successfully taken up by both living and dead cells and can function as a selective luminescent probe for cytoplasmic staining. The luminescence response of complex 1 to various natural amino acids was investigated Figure 3. Complex 1 is non-emissive in aqueous buffered solution in the absence of analyte.
No significant change in the emission of the complex 1 was observed upon the addition of other natural amino acids Figure 3. This result indicates that complex 1 displays a high degree of selectivity for histidine over other amino acids. This suggests that complex 1 may be potentially developed for in vivo imaging applications. Complex 1 displayed an intense luminescence upon interaction with the histidine-rich BSA, but was only weakly emissive in the presence of ct DNA Figure 4.
Furthermore, the change in luminescence intensity of complex 1 upon the addition of various amounts The results showed that BSA or histidine were able to induce significant luminescence enhancements in complex 1 Figure 5. In combination with previously published reports  ,  , we propose that the labile solvato co-ligands of complex 1 are displaced by the imidazole N-donor moieties of histidine residues via coordinative bond formation.
This shelters the metal center within the hydrophobic environment of the protein, reducing solvent-mediated non-radiative decay of the excited state and thereby enhancing the phosphorescence of complex 1. The cyclometalated iridium III solvent complexes 1—3 Figure 1 were synthesized according to previously reported methods see Materials and Methods. Interestingly, complex 1 emits an intense orange luminescence in DMSO under UV-transillumination and was thus considered as a promising candidate for further cell imaging studies. On the other hand, luminescence of 1 was significantly suppressed in Tris buffer Figure 2B.
We rationalize that the reduced luminescence intensity of 1 in aqueous solution is due to non-radiative decay of the excited state of complex 1 by complex-solvent interactions. Presumably, this effect is less pronounced in DMSO, leading to a higher luminescence signal. We also investigated the application of iridium III complex 1 for staining fixed cells.
Similar to the results with live cells, only weak luminescence was observed in the nucleus of the fixed cells Figure 8c,d. These results suggest that complex 1 is an effective luminescent cytoplasmic stain for both living and dead cells. The practical application of complex 1 as a luminescent probe in living cells was investigated using confocal laser scanning microscopy Figure 6.
HeLa cells showed negligible background fluorescence. No cell death was observed under the staining and imaging conditions used Figure 7. Overlay images revealed that the luminescence pattern of complex 1 differed considerably from that of DNA-binding dye Hoechst Figure 6d. Furthermore, a large signal ratio was observed between the nuclei and cytoplasm, indicating that complex 1 prefers to stain the cytoplasmic regions of the cells. We presume that the observed luminescence enhancement of complex 1 is due to its interactions with histidine or histidine-rich proteins in the cellular cytoplasm.
These results indicate that complex 1 acts as a luminescent imaging agent for live cells without requiring prior membrane permeabilization.
Automated single-molecule imaging in living cells
Overlay of images in b and c bottom right. In conclusion, we have presented the cytoplasmic permeable iridium III complex 1 as a phosphorescent dye for live and fixed cell imaging. Complex 1 shows a bright phosphorescence in living cells, and effectively enters and stains the cytoplasm. Given that the emission properties of metal complexes can be fine-tuned through modifications of auxiliary ligands, we envision that further improvements can be achieved in the application of luminescent iridium III complexes as cellular imaging probes.
Iridium chloride hydrate IrCl 3. Louis, MO. Hoechst and cell culture reagents were purchased from Invitrogen Carlsbad, CA. For colocalization imaging of living cells. For colocalization imaging of fixed cells. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract A cell permeable cyclometalated iridium III complex has been developed as a phosphorescent probe for cell imaging.
Introduction Luminescence imaging of biological specimens using non-invasive probes is a basic technique in life and biomedical sciences for studying the morphologic characteristics of tissue at high resolution  — . Results and Discussion The cyclometalated iridium III solvent complexes 1—3 Figure 1 were synthesized according to previously reported methods see Materials and Methods.
Download: PPT. Figure 1. Figure 2. Live cell imaging is the study of living cells using images acquired from imaging systems such as microscopes and high content screening systems. It is used by scientists to give a better view of biological function through the study of cellular dynamics.
Adding Efficiency to Your Fluorescence Imaging
In recent years, this technology has become widely accessible and there is an increasing number of leading research biologists using live-cell imaging techniques to produce pivotal publications in a wide range of research areas. Live cell imaging is becoming a requisite technique for cell biology, developmental biology, cancer biology, and many other related biomedical research laboratories. A major challenge of live cell imaging is keeping cells alive and functioning as naturally as possible for the duration of the experiment.
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Fluorescence illumination, especially in the UV range, is harmful for cells and causes photobleaching and phototoxicity. The use of high power lasers as the excitation source adds to this challenge.
Fluorescence live cell imaging.
Successful experiments must be designed to minimize specimen illumination whilst maintaining the environment. In addition, some cell types such as cultured cells require incubation during the experiment. Careful control of temperature and CO 2 concentration within the incubation chamber allow cells to be cultured in HCS instruments with minimum disruption.
With well-designed systems, live cell assay allows research biologists and drug discovery scientists to discover more, achieve greater understanding and provides the confidence and reassurance that their results are true to life. Brightfield and digital phase imaging also enable gentle and label-free live cell analysis. Live cell imaging of HeLa cells expressing a GFP-tagged cameloid small antibody fragment Chromobody against a cell cycle clock protein. The protein of interest was followed in live cells for over 22 hrs, with images acquired every 30 min.
To analyze live cells without any fluorescent dye labels, you can choose to use the digital-phase contrast imaging mode. High performance sCMOS cameras offer high resolution and high sensitivity and allow the fast acquisition speeds so are the optimum choice for your high speed, 3D live cell experiments.