Project description:We report what we believe to be the first near-infrared pH-sensitive fluorescence lifetime molecular probe suitable for biological applications in physiological range. Specifically, we modified a known fluorophore skeleton, hexamethylindotricarbocyanine, with a tertiary amine functionality that was electronically coupled to the fluorophore, to generate a pH-sensitive probe. The pK(a) of the probe depended critically on the location of the amine. Peripheral substitution at the 5-position of the indole ring resulted in a compound with pK(a) ? 4.9 as determined by emission spectroscopy. In contrast, substitution at the meso-position shifted the pK(a) to 5.5. The resulting compound, LS482, demonstrated steady-state and fluorescence-lifetime pH-sensitivity. This sensitivity stemmed from distinct lifetimes for protonated (?1.16 ns in acidic DMSO) and deprotonated (?1.4 ns in basic DMSO) components. The suitability of the fluorescent dyes for biological applications was demonstrated with a fluorescence-lifetime tomography system. The ability to interrogate cellular processes and subsequently translate the findings in living organisms further augments the potential of these lifetime-based pH probes.
Project description:The layered silicates Egyptian Blue (CaCuSi4O10, EB), Han Blue (BaCuSi4O10, HB) and Han Purple (BaCuSi2O6, HP) emit as bulk materials bright and stable fluorescence in the near-infrared (NIR), which is of high interest for (bio)photonics due to minimal scattering, absorption and phototoxicity in this spectral range. So far the optical properties of nanosheets (NS) of these silicates are poorly understood. Here, we exfoliate them into monodisperse nanosheets, report their physicochemical properties and use them for (bio)photonics. The approach uses ball milling followed by tip sonication and centrifugation steps to exfoliate the silicates into NS with lateral size and thickness down to ≈ 16-27 nm and 1-4 nm, respectively. They emit at ≈ 927 nm (EB-NS), 953 nm (HB-NS) and 924 nm (HP-NS), and single NS can be imaged in the NIR. The fluorescence lifetimes decrease from ≈ 30-100 μs (bulk) to 17 μs (EB-NS), 8 μs (HB-NS) and 7 μs (HP-NS), thus enabling lifetime-encoded multicolor imaging both on the microscopic and the macroscopic scale. Finally, remote imaging through tissue phantoms reveals the potential for bioimaging. In summary, we report a procedure to gain monodisperse NIR fluorescent silicate nanosheets, determine their size-dependent photophysical properties and showcase the potential for NIR photonics.
Project description:Sensing and imaging methods based on the dynamic scattering of coherent light (including laser speckle, laser Doppler, diffuse correlation spectroscopy, dynamic light scattering, and diffusing wave spectroscopy) quantify scatterer motion using light intensity fluctuations. The underlying optical field autocorrelation, rather than being measured directly, is typically inferred from the intensity autocorrelation through the Siegert relationship, assuming that the scattered field obeys Gaussian statistics. Here, we demonstrate interferometric near-infrared spectroscopy for measuring the time-of-flight (TOF) resolved field and intensity autocorrelations in turbid media. We find that the Siegert relationship breaks down for short TOFs due to static paths whose optical field does not decorrelate over experimental time scales. We also show that eliminating such paths by polarization gating restores the validity of the Siegert relationship. The unique capability of measuring optical field autocorrelations, as demonstrated here, enables the study of non-Gaussian and non-ergodic light scattering processes. Moreover, direct measurements of field autocorrelations are more efficient than indirect measurements based on intensity autocorrelations. Thus, optical field measurements may improve the quantiffcation of scatterer dynamics with coherent light.
Project description:BackgroundLight sheet microscopy became a popular tool allowing fast imaging with reduced out of focus light. However, when light penetrates turbid media such as biological tissues, multiple scattering scrambles the illumination into a speckle pattern and severely challenges conventional fluorescence imaging with focused light or with a light sheet. In this article, we present generation of light sheet type illumination patterns despite scattering.MethodsWe optimize the wave-front of the incoming light to transform the speckle pattern behind the scattering layer into a light sheet within the region of interest. We utilize a fast spatial light modulator for phase modulation and a genetic optimization algorithm. The light pattern behind the scattering layer is detected via a clear detection path and acts as a feedback signal for the algorithm.ResultsWe enabled homogenous light sheet illumination behind turbid media and enhanced the signal of fluorescent beads selectively at the desired focal plane up to eight times on average. The technique is capable to compensate the dynamic changes of the speckle pattern as well, as shown on samples consisting of living drosophila pupae.ConclusionOur technique shows that not only single foci, but also a homogenous light sheet illumination can directly be created and maintained behind static and dynamic scattering media. To make the technique suitable for common biological settings, where the detection path is turbid as well, a fluorescent probe can be used to provide the feedback signal.
Project description:The near-infrared (NIR) range of the electromagnetic (EM) spectrum offers a nearly transparent window for imaging tissue. Despite the significant potential of NIR fluorescence-based imaging, its establishment in basic research and clinical applications remains limited due to the scarcity of fluorescent molecules with absorption and emission properties in the NIR region, especially those suitable for biological applications. In this study, we present a novel approach by combining the widely used IRdye 800NHS fluorophore with gold nanospheres (GNSs) and gold nanorods (GNRs) to create Au nanodyes, with improved quantum yield (QY) and distinct lifetimes. These nanodyes exhibit varying photophysical properties due to the differences in the separation distance between the dye and the gold nanoparticles (GNP). Leveraging a rapid and highly sensitive wide-field fluorescence lifetime imaging (FLI) macroscopic set up, along with phasor based analysis, we introduce multiplexing capabilities for the Au nanodyes. Our approach showcases the ability to differentiate between NIR dyes with very similar, short lifetimes within a single image, using the combination of Au nanodyes and wide-field FLI. Furthermore, we demonstrate the uptake of Au nanodyes by mineral-oil induced plasmacytomas (MOPC315.bm) cells, indicating their potential for in vitro and in vivo applications.
Project description:Enzymatic activity sensing in fluorescence lifetime (FLT) mode with "self-quenched" macromolecular near-infrared (NIR) sensors is a highly promising strategy for in vivo imaging of proteolysis. However, the mechanisms of FLT changes in such substrate-based NIR sensors have not yet been studied. We synthesized two types of sensors by linking the near-infrared fluorophore IRDye 800CW to macromolecular graft copolymers of methoxy polyethylene glycol and polylysine (MPEG-gPLL) with varying degrees of MPEGylation and studied their fragmentation induced by trypsin, elastase, plasmin and cathepsins (B,S,L,K). We determined that the efficiency of such NIR sensors in FLT mode depends on sensor composition. While MPEG-gPLL with a high degree of MPEGylation showed rapid (τ1/2=0.1-0.2 min) FLT increase (Δτ=0.25 ns) upon model proteinase-mediated hydrolysis in vivo, lower MPEGylation density resulted in no such FLT increase. Temperature-dependence of fluorescence de-quenching of NIR sensors pointed to a mixed dynamic/static-quenching mode of MPEG-gPLL-linked fluorophores. We further demonstrated that although the bulk of sensor-linked fluorophores were de-quenched due to the elimination of static quenching, proteolysis-mediated deletion of a fraction of short (8-10kD) negatively charged fragments of highly MPEGylated NIR sensor is the most likely event leading to a rapid FLT increase phenomenon in quenched NIR sensors. Therefore, the optimization of "built-in" dynamic quenching elements of macromolecular NIR sensors is a potential avenue for improving their response in FLT mode.
Project description:Accurate detection of tumor margins is essential for successful cancer surgery. While indocyanine green (ICG)-based near-infrared (NIR) fluorescence (FL) surgical navigation enhances the visual identification of tumor margins, its accuracy remains subjective, underscoring the need for quantitative approaches. In this study, a high spatiotemporal fluorescence lifetime (FLT) imaging technology is developed in the second near-infrared window (NIR-II, 1000-1700 nm) for quantitative tumor margin detection, utilizing folate receptor-targeted ICG nanoprobes (FPH-ICG). FPH-ICG exhibits a significantly prolonged NIR-II FLT (750 ± 7 ps vs 260 ± 3 ps) and increased NIR-II FL brightness (FPH-ICG/ICG = 3.8). In vitro and in vivo studies confirm that FPH-ICG specifically targets folate receptor-α (FRα) on SK-OV-3 ovarian cancer cells, achieving high-contrast NIR-II FL imaging with a signal-to-background ratio of 10.8. Notably, NIR-II FLT imaging demonstrates superior accuracy (90%) and consistency in defining tumor margins compared to NIR-II FL imaging (58%) in both SK-OV-3 tumor-bearing mice and clinical tumor samples. These findings underscore the potential of NIR-II FLT imaging as a quantitative tool for guiding surgical tumor margin detection.
Project description:Near-infrared (NIR) light possesses many suitable optophysical properties for medical imaging including low autofluorescence, deep tissue penetration, and minimal light scattering, which together allow for high-resolution imaging of biological tissue. NIR imaging has proven to be a noninvasive and effective real-time imaging methodology that provides a high signal-to-background ratio compared to other potential optical imaging modalities. In response to this, the use of NIR imaging has been extensively explored in the field of immunotherapy. To date, NIR fluorescence imaging has successfully offered reliable monitoring of the localization, dynamics, and function of immune responses, which are vital in assessing not only the efficacy but also the safety of treatments to design immunotherapies optimally. This review aims to provide an overview of the current research on NIR imaging of the immune response. We expect that the use of NIR imaging will expand further in response to the recent success in cancer immunotherapy. We will also offer our insights on how this technology will meet rapidly growing expectations in the future.
Project description:Fluorescence lifetime imaging microscopy (FLIM) opens new dimensions for highly multiplexed imaging in live cells and organisms using differences in fluorescence lifetime to distinguish spectrally identical fluorescent probes. Here, a set of fluorescence-activating and absorption-shifting tags (FASTs) capable of modulating the fluorescence lifetime of embedded fluorogenic 4-hydroxybenzylidene rhodanine (HBR) derivatives is described. It is shown that changes in the FAST protein sequence can vary the local environment of the chromophore and lead to significant changes in fluorescence lifetime. These fluorescence lifetime-modulating tags enable multiplexed imaging of up to three targets in one spectral channel using a single HBR derivative in live cells and live zebrafish larvae. The combination of fluorescence lifetime multiplexing with spectral multiplexing allows to successfully image six targets in live cells, opening great prospects for multicolor fluorescence lifetime multiplexing.
Project description:We report a platform combining multicomponent reaction synthesis and automated cell-based screening to develop biocompatible NIR-BODIPY fluorophores. From a library of over 60 fluorophores, we optimised compound NIRBD-62c as a multimodal probe with suitable properties for STED super-resolution and fluorescence lifetime imaging. Furthermore, we employed NIRBD-62c for imaging trafficking inside cells and to examine how pharmacological inhibitors can alter the vesicular traffic between intracellular compartments and the plasma membrane.