Application of nucleic acid-functionalized gold nanoprobes in live-cell fluorescence imaging
0
Citation
0
Reference
10
Related Paper
Abstract:
Combining nanomaterials and nucleic acid technology, fluorescent nucleic acid-functionalized gold nanoprobes possess many advantages, such as improved stability, good biocompatibility, unique optical properties and precise programmability, which open a new era for live-cell imaging. In particular, amplified nucleic acid-functionalized gold nanoprobes are ideal for
Keywords:
Live cell imaging
Fluorescence-lifetime imaging microscopy
Implementation of miniaturized modular-array fluorescence microscopy for long-term live-cell imaging
Fluorescence microscopy imaging of live cells has provided consistent monitoring of dynamic cellular activities and interactions. However, due to the limited adaptability of current live-cell imaging systems, portable cell imaging system has been adapted by broad strategies, including miniaturized fluorescence microscopy. Here, we provide a protocol for the construction and operational process of miniaturized modular-array fluorescence microscopy (MAM). The MAM system is built in a portable size (15 cm × 15 cm × 3 cm) and provides in situ cell imaging inside an incubator with a subcellular lateral resolution (~3 μm). We demonstrated the improved stability of the MAM system with fluorescent targets and live HeLa cells, enabling long-term imaging for 12 hours without the need for external support or post-processing. We believe the protocol could guide scientists to construct a compact portable fluorescence imaging system and perform time-lapse in situ single-cell imaging and analysis.
Fluorescence-lifetime imaging microscopy
Live cell imaging
Single-Cell Analysis
Cite
Citations (0)
Fluorescence imaging of green fluorescent protein (GFP) may be used to locate proteins in live cells and fluorescence lifetime imaging (FLIM) may be employed to probe the local microenvironment of proteins. Here we apply FLIM to GFP-tagged proteins at the cell surface and at an inhibitory natural killer (NK) cell immunological synapse (IS). We present a novel quantitative analysis of fluorescence lifetime images that we believe is useful to determine whether apparent FLIM heterogeneity is statistically significant. We observe that, although the variation of observed fluorescence lifetime of GFP-tagged proteins at the cell surface is close to the expected statistical range, the lifetime of GFP-tagged proteins in cells is shorter than recombinant GFP in solution. Furthermore the lifetime of GFP-tagged major histocompatibility complex class I protein is shortened at the inhibitory NK cell IS compared with the unconjugated membrane. Following our previous work demonstrating the ability of FLIM to report the local refractive index of GFP in solution, we speculate that these lifetime variations may indicate local refractive index changes. This application of our method for detecting small but significant differences in fluorescence lifetimes shows how FLIM could be broadly useful in imaging discrete membrane environments for a given protein.
Fluorescence-lifetime imaging microscopy
Immunological synapse
Live cell imaging
Cite
Citations (70)
We have performed multimodal imaging of live fibroblast cells infected by murine cytomegalovirus (mCMV). The infection process was monitored by imaging the two-photon fluorescence signal from a GFP-expressing strain of mCMV, whilst changes to lipid droplet configuration were observed by CARS imaging. This allowed us to identify three visually distinct stages of infection. Quantitative analysis of lipid droplet number and size distributions were obtained from live cells, which showed significant perturbations across the different stages of infection. The CARS and two-photon images were acquired simultaneously and the experimental design allowed incorporation of an environmental control chamber to maintain cell viability. Photodamage to the live cell population was also assessed.
Live cell imaging
Fluorescence-lifetime imaging microscopy
Lipid droplet
Two-photon excitation microscopy
Cite
Citations (11)
Ion concentrations in biological cells are widely studied with fluorescent probes. The probes have a high selectivity for specific ions and exhibit marked changes in their photophysical properties upon binding ions. The fluorescence decay behavior of the probes in the presence of ions can now be used as a contrast mechanism for imaging purposes. This technique can be further exploited for the quantitative determination of ion concentrations within living cells. Here we describe the fluorescence lifetime properties of the free calcium probe CalciumGreen and the pH probe carboxy SNAFL-1. The potential of fluorescence lifetime imaging is illustrated by the imaging of Ca2+ concentrations and pH in single cells. In the case of the emission ratio probe c.SNAFL-1, it was possible to determine the pH in the same cell using both the ratio and the fluorescence lifetime method. It turns out that no cumbersome in vivo calibration procedure is required when c.SNAFL-1 is used for quantitative fluorescence lifetime imaging of pH in single cells.
Fluorescence-lifetime imaging microscopy
Live cell imaging
Cite
Citations (3)
Implementation of miniaturized modular-array fluorescence microscopy for long-term live-cell imaging
Fluorescence microscopy imaging of live cells has provided consistent monitoring of dynamic cellular activities and interactions. However, because current live-cell imaging systems are limited in their adaptability, portable cell imaging systems have been adapted by a variety of strategies, including miniaturized fluorescence microscopy. Here, we provide a protocol for the construction and operational process of miniaturized modular-array fluorescence microscopy (MAM). The MAM system is built in a portable size (15
Live cell imaging
Fluorescence-lifetime imaging microscopy
Photoactivated localization microscopy
Cite
Citations (0)
Autofluorescence
Fluorescence-lifetime imaging microscopy
Cite
Citations (0)
Fluorescence is a very promising radioactive-free technique for functional imaging in small animals and, in the future, in humans. However, most commercial near-infrared dyes display poor optical properties, such as low fluorescence quantum yields and short fluorescence lifetimes. In this paper, we explore whether the encapsulation of infrared cyanine dyes within the core of lipid nanoparticles (LNPs) could improve their optical properties. Lipophilic dialkylcarbocyanines DiD and DiR are loaded very efficiently in 30-35-nm-diam lipid droplets stabilized in water by surfactants. No significant fluorescence autoquenching is observed up to 53 dyes per particle. Encapsulated in LNP, which are stable for more than one year at room temperature in HBS buffer (HEPES 0.02 M, EDTA 0.01 M, pH 5.5), DiD and DiR display far improved fluorescence quantum yields (respectively, 0.38 and 0.25) and longer fluorescence lifetimes (respectively, 1.8 and 1.1 ns) in comparison to their hydrophilic counterparts Cy5 (=0.28, =1.0 ns) and Cy7 (=0.13, =0.57 ns). Moreover, dye-loaded LNPs are able to accumulate passively in various subcutaneous tumors in mice, thanks to the enhanced permeability and retention effect. These new fluorescent nanoparticles therefore appear as very promising labels for in vivo fluorescence imaging.
Cyanine
Nile red
Fluorescence-lifetime imaging microscopy
Indocyanine Green
Cite
Citations (142)
Because of the tremendously sensitive emission profiles and high specificity of fluorescence signals, as well as the selectivity offered by antibody labeling, fluorescence imaging has become one of the most powerful techniques in biomedical research and clinical applications in recent years. Long after the phenomenon of fluorescence was discovered by Stokes in 1852, fluorescence in the biomedical field was first reported in 1911 when fluorescence was observed coming from an animal subjected to UV illumination. Fluorescence imaging was proposed as a method for cancer diagnostics in 1948. Winkelman and Rasmussen-Taxdal performed the first quantitative measurements of in vivo fluorescence using exogenous fluorophores in 1960. Since then, fluorescence technology has attracted extensive research for biomedical applications in both autofluorescence and fluorescence imaging using contrast agents. In this chapter, the principle of fluorescence imaging and fluorescence imaging techniques will be discussed in Secs. 5.1 and 5.2. Sections 5.3 and 5.4 will focus on the key components of fluorescence imaging systems and fluorescence filters. The configurations and optical design of fluorescence imaging systems will be discussed in Secs. 5.5 and 5.6. 5.1 Introduction to Fluorescence 5.1.1 Fluorescence process When illuminated with light possessing a suitable spectrum, some specimens, living or nonliving, organic or inorganic, absorb the illumination light and then radiate light with a different wavelength. This phenomenon, called the fluorescence process, was discovered by British scientist Sir George G. Stokes in the middle of the nineteenth century. The fluorescence process is commonly illustrated by the simple electronic-state diagram called the Jablonski energy diagram, as shown in Fig. 5.1. When molecules absorb light of a suitable wavelength λex, electrons may be raised from the ground state S0 to a higher-energy and vibrationally excited state S2. This process may only take 10-15 s. Within 10-14 to 10-11 s, the excited electrons may lose some vibrational energy to the surrounding environment in the form of heat and then relax to the lowest vibrational energy level S1 within the electronically excited state from which the fluorescence emission originates.
Fluorescence-lifetime imaging microscopy
Autofluorescence
Cite
Citations (0)
Implementation of miniaturized modular-array fluorescence microscopy for long-term live-cell imaging
Fluorescence microscopy imaging of live cells has provided consistent monitoring of dynamic cellular activities and interactions. However, due to the limited adaptability of current live-cell imaging systems, portable cell imaging system has been adapted by broad strategies, including miniaturized fluorescence microscopy. Here, we provide a protocol for the construction and operational process of miniaturized modular-array fluorescence microscopy (MAM). The MAM system is built in a portable size (15 cm × 15 cm × 3 cm) and provides in situ cell imaging inside an incubator with a subcellular lateral resolution (~3 μm). We demonstrated the improved stability of the MAM system with fluorescent targets and live HeLa cells, enabling long-term imaging for 12 hours without the need for external support or post-processing. We believe the protocol could guide scientists to construct a compact portable fluorescence imaging system and perform time-lapse in situ single-cell imaging and analysis.
Fluorescence-lifetime imaging microscopy
Live cell imaging
Single-Cell Analysis
Cite
Citations (0)
We present a time domain optically sectioned fluorescence lifetime imaging (FLIM) microscope developed for high-speed live cell imaging. This single photon excited system combines wide field parallel pixel detection with confocal sectioning utilizing spinning Nipkow disc microscopy. It can acquire fluorescence lifetime images of live cells at up to 10 frames per second (fps), permitting high-speed FLIM of cell dynamics and protein interactions with potential for high throughput cell imaging and screening applications. We demonstrate the application of this FLIM microscope to real-time monitoring of changes in lipid order in cell membranes following cholesterol depletion using cyclodextrin and to the activation of the small GTP-ase Ras in live cells using FRET.
Live cell imaging
Fluorescence-lifetime imaging microscopy
Biological Imaging
Cite
Citations (80)