Fluorescence Lifetime Data Research Articles

Mesoscopic fluorescence lifetime imaging: Fundamental principles, clinical applications and future directions.

Fluorescence lifetime imaging (FLIm) is an optical spectroscopic imaging technique capable of real-time assessments of tissue properties in clinical settings. Label-free FLIm is sensitive to changes in tissue structure and biochemistry resulting from pathological conditions, thus providing optical contrast to identify and monitor the progression of disease. Technical and methodological advances over the last two decades have enabled the development of FLIm instrumentation for real-time, in situ, mesoscopic imaging compatible with standard clinical workflows. Herein, we review the fundamental working principles of mesoscopic FLIm, discuss the technical characteristics of current clinical FLIm instrumentation, highlight the most commonly used analytical methods to interpret fluorescence lifetime data and discuss the recent applications of FLIm in surgical oncology and cardiovascular diagnostics. Finally, we conclude with an outlook on the future directions of clinical FLIm.

Insight into Sodium Pump Regulation by FXYD Proteins

The sodium pump (Na+/K+-ATPase, NKA) is a membrane transport ATPase responsible for establishing the vital gradients of sodium and potassium across the plasma membrane of all mammalian cells. NKA activity is regulated by FXYD proteins, a family of transmembrane proteins that serve as auxiliary subunits of the sodium pump. in the heart, NKA activity is modulated by phospholemman (FXYD1), but the structural determinants of this regulatory mechanism are incompletely understood. We used time-resolved and steady-state FRET techniques to study the stoichiometry and affinity of fluorescently labeled NKA and FXYD1. Progressive acceptor photobleaching revealed multiple acceptor-labeled FXYD1 bound to donor-labeled NKA. Complementary global analysis of time-correlated single photon counting experiments revealed presence of medium- and high-FRET species within the population of NKA-FXYD1 complexes. To explore the quaternary structure of the regulatory complex, we obtained fluorescence lifetime data from hundreds of cells spanning a wide range of protein expression level. Over this range, the high FRET species increased with increasing protein concentration at the expense of the medium-FRET population. We attribute these subpopulations to NKA-NKA dimers decorated with either one FXYD1 (at low concentration) or two (at high concentration). Moreover, acceptor photobleaching experiments revealed FRET between donor- and acceptor-labeled NKA α subunits consistent with NKA oligomerization. in addition, we quantified FRET between NKA α and β essential subunits to monitor changes in the dynamic equilibrium between open (E2) and closed (E1) states of NKA. Treatment of NKA with the specific inhibitor ouabain induced a shift in the dynamic equilibrium of NKA toward a higher FRET state, suggesting that the α and β n-termini moved closer together. The experiments reveal new insight into the regulation of NKA by FXYD proteins and by NKA oligomerization.

Spectroscopic Investigation into the Binding of Ferulic Acid with Sodium Deoxycholate: Hydrophobic Force Versus Hydrogen Bonding.

The binding of ferulic acid (FA) with sodium deoxycholate (NaDC) has been investigated using fluorescence and absorption measurements. The fluorescence probe technique of pyrene reveals that the presence of FA favors the micellization of NaDC, leading to the decreased critical micelle concentrations for the formation of NaDC micelles. As NaDC molecules change gradually from monomers via primary micelles into secondary micelles, the intensities of absorption and fluorescence spectra of FA increase at low NaDC concentrations, but decrease suddenly at intermediate NaDC concentrations, and finally increase again at high NaDC concentrations. These results corroborated well with FA fluorescence lifetime data suggesting that the aryl ring of FA hydrophobically binds to the convex surface of NaDC monomers, whereas the hydrogen bonding between FA and NaDC is significantly involved in NaDC primary micelles, which is gradually overcome by the hydrophobic interaction between FA and NaDC secondary micelles. The absorption and fluorescence spectra as well as the binding constant value of FA indicate the strong binding of FA in the large hydrophobic core of NaDC secondary micelles. At low FA concentrations, the measurement of FA anisotropy suggests that FA can increase the packing order of hydrophobic surfaces in NaDC secondary micelles, whereas the high amount of FA can greatly disrupt the packing structure of NaDC secondary micelles which is ascribed to the formation of FA dimers. The spectroscopic experiments outlined here present the binding events of FA with NaDC monomers and primary and secondary micelles, which are significantly related with the hydrophobic force and hydrogen bonding as well as the unique structural characteristics of bile salt.

Photophysical Properties of Some Fluorescent Dyes in SDS Micellar Solutions.

Photophysical properties of fluorescent dyes such as Safranin T, Acridine Orange, Pyronin B and Pyronin Y in SDS micelles were examined by using spectroscopic techniques. Firstly, spherical micelles in deionized water were prepared with Sodium Dodecyl Sulfate (SDS) surfactants and they were transformed into their layered structures (lamellar micelles) by the aid of NaCl (sodium chloride). SEM studies confirmed the transformation of SDS micelles from the spherical structures to the lamellar structures. Secondly, absorption and fluorescence characteristics of the dyes in deionized water and the SDS micelles aqueous solutions were characterized in the presence of various NaCl concentrations at above the critical micelle concentration (CMC). Moreover, the photophysical properties of the dyes in various media were discussed by fluorescence quantum yield and fluorescence lifetime data. The micellar structures called a mimetic membrane system changed the photophysical properties of the dyes compared to those in deionized water.

Evaluation of cell metabolic adaptation in wound and tumour by Fluorescence Lifetime Imaging Microscopy

Acidic pH occurs in acute wounds progressing to healing as consequence of a cell metabolic adaptation in response to injury-induced tissue hypoperfusion. In tumours, high metabolic rate leads to acidosis affecting cancer progression. Acidic pH affects activities of remodelling cells in vitro. The pH measurement predicts healing in pathological wounds and success of surgical treatment of burns and chronic ulcers. However, current methods are limited to skin surface or based on detection of fluorescence intensity of specific sensitive probes that suffer of microenvironment factors. Herein, we ascertained relevance in vivo of cell metabolic adaptation in skin repair by interfering with anaerobic glycolysis. Moreover, a custom-designed skin imaging chamber, 2-Photon microscopy (2PM), fluorescence lifetime imaging (FLIM) and data mapping analyses were used to correlate maps of glycolytic activity in vivo as measurement of NADH intrinsic lifetime with areas of hypoxia and acidification in models of skin injury and cancer. The method was challenged by measuring the NADH profile by interfering with anaerobic glycolysis and oxidative phosphorylation in the mitochondrial respiratory chain. Therefore, intravital NADH FLIM represents a tool for investigating cell metabolic adaptation occurring in wounds, as well as the relationship between cell metabolism and cancer.

Simultaneous fluorescence lifetime and Raman fiber-based mapping of tissues.

We report the development of a novel, to the best of our knowledge, fiber-based system to realize coregistered simultaneous acquisition of fluorescence lifetime (FL) data and Raman spectra from the same area. FL measurements by means of time-correlated single photon counting are realized with periodic out-of-phase external illumination of the field of view, enabling acquisition of data under bright illumination of the specimen. Raman measurements in the near-infrared are realized asynchronously. We present a detailed characterization of this technique and validate its potential to report intrinsic contrast. Fiber-based FL and Raman maps report complementary structural, compositional, and molecular contrast in biological tissues with diverse compositional features.

Rapid modification of hemoglobin heme to form enhanced peroxidase-like activity for colorimetric assay

Modification of proteins is widespread throughout nature responsible for the vast biodiversity. In this study, we developed a facile method to rapid and selectively modify heme of hemoglobin (Hb) with tartaric acid to form complex (TA-Hb), which exhibits strong peroxidase-like activity comparable to the native horseradish peroxidase (HRP). The structural change of Hb accompanied by the formation of TA-Hb complex was validated by UV-vis absorption, fluorescence, fluorescence lifetime and circular dichroism data. The oxygen atom of the carboxyl group of tartaric acid can easily coordinate with heme and become the axial ligand of porphyrin, and this specific structure facilitates to form stable intermediate, significantly enhancing Hb peroxidase-like activity. This study is expected to provide important insight into the interactions of the physiologically important protein Hb with various ligands. In addition, the unique property of TA-Hb was employed to develop a rapid colorimetric assay for detecting concentration of Hb in human serum samples, regardless of whether the sample is in the form of fresh or long-term storage, liquid or dried blood. This colorimetric assay can detect Hb over the concentration range of 0.01 ~ 0.1 mg/mL and the limit of detection (LOD) is 0.1 μg/mL. Moreover, cascade reactions can be realized based on the peroxidase-like TA-Hb complex formed in situ from the serum samples by combining with the glucose oxidase, which enable this colorimetric assay to detect not only Hb but also blood glucose in the same sample.

Phasor analysis of fluorescence lifetime data and its application

Fluorescence lifetime imaging microscopy (FLIM) is widely used in biomedical, materials and other fields. It not only has strong specificity and high sensitivity, but also has the capability of quantitative measurement because the fluorescence lifetime is not affected by the intensity of excitation, the concentration of fluorophores and photobleaching, and consequently is able to monitor the changes of microenvironment and reflecting the interaction between molecules. However, its application is limited to some extent by the complexity of data analysis. In order to make FLIM technology more suitable for fast analysis of high-throughput data, a variety of new algorithms for fluorescence lifetime analysis have emerged in recent years, such as phasor analysis, maximum likelihood estimation, first-order moment, Bayesian analysis, and compressed sensing. Among them, the phasor analysis (PA) method obtains the fluorescence lifetime by converting the fitting in the time domain to the direct calculation in the frequency domain. Compared with traditional least-square fitting method, it is not only simpler and faster, but also more suitable for the case of low photon counts. In addition, in the PA approach to FLIM, the fluorescence decay is directly converted into a phasor diagram by simple mathematics, where the phasor points originating from different pixels in the image are represented by the positions in the phasor plot, and thus the graphical representation obtained by PA method is convenient for data visualization and cluster analysis. Therefore, it has become a simple and powerful analysis method for FLIM, and is increasingly favored by researchers. In this paper, the basic principle of PA method and how we can use it are described in detail. And on this basis, the latest application research progress of the method in cell metabolism state measurement, protein interaction study, cell microenvironment measurement, auxiliary pathological diagnosis, and resolution improvement in super-resolution imaging are introduced and summarized. The advantages of PA method in these FLIM applications are focused on, providing useful reference for the research in related fields. Finally, the phasor analysis method for FLIM data analysis and the development trend of its application are prospected.

Differential effects of simvastatin on membrane organization and dynamics in varying phases

Simvastatin belongs to the statin family of cholesterol lowering drugs which act as competitive inhibitors of HMG-CoA reductase, the rate-determining enzyme in cholesterol biosynthesis pathway. Simvastatin is a semi-synthetic, highly lipophilic statin, and has several side effects. Since HMG-CoA reductase is localized in the endoplasmic reticulum, orally administered simvastatin needs to cross the cellular plasma membrane to be able to act on HMG-CoA reductase. With an overall goal of exploring the interaction of simvastatin with membranes, we examined the effect of simvastatin on the organization and dynamics in membranes of varying phase, in a depth-dependent manner. For this, we employed DPH and TMA-DPH, which represent fluorescent membrane probes localized at two different locations (depths) in the membrane. Analysis of fluorescence anisotropy and lifetime data of these depth-specific probes in membranes of varying phase (gel/fluid/liquid-ordered) showed that the maximum membrane disordering was observed in gel phase, while moderate effects were observed in liquid-ordered phase, with no significant change in membrane order in fluid phase membranes. We conclude that simvastatin induces change in membrane order in a depth-dependent and phase-specific manner. These results provide novel insight in the membrane interaction of simvastatin and could be crucial for understanding its pharmacological effect.

The squared distance approach to frequency domain time-resolved fluorescence analysis.

A frequency-domain (FD) analysis of fluorescence lifetime (FLT) is a unique and rapid method for cellular and intracellular classifications that can serve for medical diagnostics purposes. Nevertheless, its data analysis process demands nonlinear fitting algorithms that may distort the resolution of the FLT data and hence diminish the classification ability of the method. This research suggests a sample classification technique that is unaffected by the analysis process as it is based on the squared distance (D2 ) between the raw frequency response data (FRD). In addition, it presents the theory behind this technique and its validation in two simulated data sets of six groups with similar widely and closely spaced FLT data as well as in experimental data of 43 samples from bacterial and viral infected and non-infected patients. In the two simulated tests, the classification accuracy was above 95% for all six groups. In the experimental data, the classification of 41 out of 43 samples matched earlier report and 29 out of 31 agreed with preliminary physician diagnosis. The D2 approach has the potential to promote FD-time resolved fluorescence measurements as a medical diagnostic technique with high specifity and high sensitivity for many of today's conventional diagnostic procedures.

Deciphering the interactions of substituted chromones possessing acetylcholinesterase inhibition activity in human serum albumin matrix

A combination of different biophysical techniques is applied in conjunction with molecular docking calculation to explore the binding interaction of two substituted chromone derivatives namely 7-amino-2-methylchromone (AMC) and 3-cyanochromone (CyC), both of which are screened as potential acetylcholinesterase inhibitors, with human serum albumin (HSA). Analysis of intrinsic tryptophan fluorescence quenching reveals the Stern-Volmer constant (KSV) for CyC (~1.2 × 105 M−1) to be one order in magnitude higher than AMC (~3.9 × 104 M−1). Analysis of fluorescence lifetime data indicates static quenching mechanism for AMC; while a significant contribution from the dynamic (collisional) quenching also exits for CyC. The difference in binding thermodynamics of the chromones into the protein binding domain is mainly due to the difference in the primary forces responsible for the binding. The fluorescence titration results are in nice agreement with the complementary circular dichroism (CD) experiments as well as with the results from molecular docking calculation. The results indicate that while CyC binds to Domain IIA (Sudlow's site 1) of HSA with a greater binding affinity of −28.84 kJ/mol, AMC binds at an alternate position, site IB with a lower binding affinity (−27.19 kJ/mol).

RETINAL FLECKS IN STARGARDT DISEASE REVEAL CHARACTERISTIC FLUORESCENCE LIFETIME TRANSITION OVER TIME.

Stargardt disease is the most common inherited juvenile macular dystrophy and is characterized by yellowish flecks across the posterior pole. The purpose of this study was to investigate fluorescence lifetime changes of retinal flecks over time using fluorescence lifetime imaging ophthalmoscopy. Longitudinal fluorescence lifetime data of 12 patients with Stargardt disease (mean age ± SEM, 42.25 ± 2.1 years; range, 28-58 years) were acquired using a fluorescence lifetime imaging ophthalmoscope based on a Heidelberg Engineering Spectralis system. Retinal autofluorescence was excited with a 470-nm laser. The emitted fluorescence was detected in two wavelength channels: a short spectral channel (498-560 nm) and a long spectral channel (560-720 nm). The mean retinal autofluorescence lifetimes were calculated and further analyzed with corresponding color fundus images, autofluorescence intensity images, and spectral domain optical coherence tomography. Patients were classified into three subtypes. All patients with Stargardt disease displayed characteristic autofluorescence lifetime patterns. Mean fluorescence lifetime values within areas of yellow flecks were significantly prolonged (long spectral channel 484 ps) compared with the surrounding tissue (long spectral channel 297 ps). In 91.6% of the eyes, flecks with short fluorescence lifetimes (long spectral channel 255 ps) were identified. Short lifetime flecks progressed to flecks with characteristic long lifetimes in 75.1% of eyes within a mean interval of 29.2 months (range 3-45 months). Between baseline and follow-up, the rate of newly developed short lifetime flecks (number/per year) based on subtypes was 2.62 in Group 1, 1.43 in Group 2, and 0.81 in Group 3. Recent onset flecks in Stargardt disease display short fluorescence lifetimes and convert into longer fluorescence lifetime flecks over time. This transition may represent a change in the composition of retinal deposits with accumulation of lipofuscin and retinoid by-products from the visual cycle. With emerging treatment options, these findings may prove useful to monitor disease progression and therapeutic effects.

Automated Fluorescence Lifetime Imaging High-Content Analysis of Förster Resonance Energy Transfer between Endogenously Labeled Kinetochore Proteins in Live Budding Yeast Cells.

We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) labeling endogenous kinetochore proteins (KPs) in live budding yeast cells. The low copy number of many KPs and their small spatial extent present significant challenges for the quantification of donor fluorescence lifetime in the presence of significant cellular autofluorescence and photobleaching. Automated FLIM data acquisition was controlled by µManager and incorporated wide-field time-gated imaging with optical sectioning to reduce background fluorescence. For data analysis, we used custom MATLAB-based software tools to perform kinetochore foci segmentation and local cellular background subtraction and fitted the fluorescence lifetime data using the open-source FLIMfit software. We validated the methodology using endogenous KPs labeled with mTurquoise2 FP and/or yellow FP and measured the donor fluorescence lifetimes for foci comprising 32 kinetochores with KP copy numbers as low as ~2 per kinetochore under an average labeling efficiency of 50%. We observed changes of median donor lifetime ≥250 ps for KPs known to form dimers. Thus, this FLIM high-content analysis platform enables the screening of relatively low-copy-number endogenous protein–protein interactions at spatially confined macromolecular complexes.

A readily usable two-photon fluorescence lifetime microendoscope.

A two-photon fluorescence lifetime (2P-FLIM) microendoscope, capable of energetic metabolism imaging through the intracellular nicotinamide adenine dinucleotide (NADH) autofluorescence, at sub-cellular resolution, is demonstrated. It exhibits readily usable characteristics such as convenient endoscope probe diameter (≈2 mm), fiber length (>5 m) and data accumulation rate (16 frames per second (fps)), leading to a FLIM refreshing rate of ≈0.1 to 1 fps depending on the sample. The spiral scanning image formation does not influence the instrument response function (IRF) characteristics of the system. Near table-top microscope performances are achieved through a comprehensive system including a home-designed spectro-temporal pulse shaper and a custom air-silica double-clad photonic crystal fiber, which enables to reach up to 40 mW of ≈100 fs pulses @ 760 nm with a 80 MHz repetition rate. A GRadient INdex (GRIN) lens provides a lateral resolution of 0.67 μm at the focus of the fiber probe. Intracellular NADH fluorescence lifetime data are finally acquired on cultured cells at 16 fps.

Semi-autonomous real-time programmable fluorescence lifetime segmentation with a digital micromirror device.

Time-correlated single-photon counting (TCSPC) is the gold standard for performing lifetime spectroscopy in biological assays. Traditional fluorescence lifetime imaging (FLIM) using laser scanning microscopes are inherently slow due to point scanning all pixels in the field-of-view. Wide-field implementations of TCSPC spectroscopy using microchannel plates benefit from particularly fast acquisition times at the expense of temporal resolution, and are fundamentally limited by photon counting rates. Here, we introduce programmable lifetime imaging (PLI), combining the advantages of wide-field imaging using total internal reflection excitation with state-of-the-art TCSPC detector technology for accurate lifetime determination in an object-oriented manner using a digital micromirror device (DMD). The fluorescent emission is projected onto the DMD to facilitate the sequential segmentation of fluorescence from individual objects in the field-of-view, allowing for both image acquisition and fluorescence lifetime determination of the assay. The sensitivity of PLI is demonstrated by manually segmenting fluorescence from fixed cell assays. We also demonstrate an automated implementation of PLI, using a camera as a feedback mechanism to segment fluorescence produced by emitting objects of interest in the imaging field-of-view, highlighting the advantages of measurement only in areas where valuable information exists. As a result, PLI is able to reduce acquisition time of fluorescence lifetime data by at least an order of magnitude compared to laser scanning implementations.

Two-Photon Active Boron Nitride Quantum Dots for Multiplexed Imaging, Intracellular Ferric Ion Biosensing, and pH Tracking in Living Cells.

Nanoparticles are key vehicles for targeted therapies because they can pass through biological barriers, enter into cells, and distribute within cell structures. We investigated the synthesis of blue and green emissive hexagonal boron nitride quantum dots (hBNQDs) using a liquid-exfoliation technique followed by hydrothermal treatment. A distinct shift from blue to bright-green emission was observed upon surface passivating the dots using poly(ethylene glycol) or PEG200 under the same UV irradiation. The quantum yield of the hBNQDs increased with the surface passivation. Multiplexed imaging was accomplished using the hBNQDs in conjunction with organic dyes. The hBNQDs provided images with distinctive emission wavelengths and fluorescence lifetimes. Although the fluorescence signals of blue- and green-emissive hBNQDs overlap spectrally with those of the emission wavelengths of the organic dyes, the fluorescence lifetime data were resolved temporally using software-based time gates. The blue-emissive hBNQD-b quantum dots were validated as sensitive platforms for detecting intracellular ferric ions with a low limit of detection (20.6 nM). The green-emissive hBNQD-g quantum dots successfully identified intracellular variations in pH, and the localization in human breast cancer cells was determined during their life cycles via fluorescence lifetime imaging.

Detail Photophysical Studies of Sulfonated Polyaniline in Aqueous Medium.

Sulfonated polyaniline (SPANI) has emerged as a promising polymer in the past few decades due to its solubility in water and relatively moderate conductivity. However, to date, literature data on the optical characterization of SPANI are very limited and preliminary in nature. In the present work, SPANI is synthesized by direct sulfonation of emeraldine salt form of polyaniline with chlorosulfonic acid in an inert solvent. Detail photophysical properties of SPANI are investigated in aqueous medium by using steady state (concentration, temperature, pH, and excitation wavelength dependence) and time-resolved spectroscopic techniques. The steady state fluorescence emission measurements are carried out carefully to avoid inner filter effect (especially secondary inner filter effect or reabsorption effect) as well as scattering. Two ground state conformations of SPANI are suggested to exist in aqueous medium. Excitation wavelength dependence of the fluorescence emission spectra is attributed to red-edge effect. All these observations are nicely corroborated by the fluorescence lifetime data of SPANI obtained from time-resolved measurements. All these new findings are extremely important in view of the potential applications of SPANI in polymer optoelectronics.

Synthesis and luminescence properties of LiBaPO4:Bi3+ yellow-emitting phosphor for solid-state lighting

Novel LiBaPO4:Bi3+ yellow-emitting phosphor is synthesized by high temperature solid-state reaction method in air. With excitation 260 nm, LiBaPO4:Bi3+ phosphor emits yellow light with the chromaticity coordinate (0.4272, 0.4657) and color rendering index 77.7. Emission band peaking at ~ 588 nm of LiBaPO4:Bi3+ phosphor in the range of 400–790 nm is attributed to the 3P1 → 1S0 electron transition of Bi3+ ion. Excitation band monitored at 588 nm in the range of 220–300 nm is assigned to the 1S0 → 3P1 electron transition of Bi3+ ion. The optimal Bi3+ ion concentration in LiBaPO4:Bi3+ phosphor is ~ 1.0 mol%. Time resolved spectra and fluorescence lifetime data confirm that there is only Bi3+ ion luminous center in LiBaPO4:Bi3+ phosphor. The luminous mechanism is analyzed by configurational coordinate diagram of Bi3+ ion. The experiment results are helpful to develop other new Bi3+-doped optical materials for solid-state lighting.

Fluorescence lifetimes of drusen in age‐related macular degeneration

PurposeFluorescence Lifetime Imaging Ophthalmoscopy (FLIO) allows non‐invasive in vivo measurement of fluorescence lifetimes of natural fluorophores of the retina upon excitation with a laser light. FLIO in different retinal diseases showed that the technique is useful to detect early metabolic changes and can differentiate between retinal deposits.The aim of this study was to characterize fluorescence lifetimes in retinal drusen due to age‐related macular degeneration (AMD).MethodsFLIO imaging was performed using a Fluorescence Lifetime Imaging Ophthalmoscope (Heidelberg Engineering, Germany). Autofluorescence was elicited with a 473 nm laser and decay times were measured in a short (498–560 nm) and in a long (560–720 nm) spectral channel. Fluorescence lifetime data was compared with autofluorescence intensity images, optical coherence tomography (OCT) and color fundus images. Soft drusen and reticular pseudodrusen were analysed and compared to an age matched control group.Results64 eyes from 64 patients with AMD and retinal drusen (age: mean ± SD 78 ± 8.5 years) were investigated and compared to 20 age matched healthy controls. Mean fluorescence lifetime in patients with AMD was significantly prolonged compared to the healthy control eyes (mean ± SEM; SSC 486 ± 18 ps vs 332 ± 11 ps, p > 0.0001; LSC: 493 ± 9 ps vs 382 ± 17 ps, p > 0.0001). Areas of drusen featured a broader range of fluorescence lifetime values. Long lifetimes were identified in areas of atrophy and intraretinal hyperreflective deposits. Areas of short lifetime corresponded to deposits within the photoreceptor outer segment band.ConclusionsMean retinal autofluorescence lifetimes in AMD were significantly prolonged compared to healthy control eyes. Intraretinal deposits caused prolonged lifetimes whereas deposits in the area of the outer photoreceptor segments lead to short fluorescence lifetimes.

Low-cost, fast and easy production of germanium nanostructures and interfacial electron transfer dynamics of BODIPY–germanium nanostructure system

Germanium nanostructures are prepared by electrochemical etching of n-type Sb-doped (100) oriented germanium (Ge) substrates with resistivity of 0.01 Ω cm. Ge substrates are etched in an electrochemical double cell containing hydrofluoric acid and ethanol solution at room temperature. Although the use of illumination source is essential for etching of an n-type semiconductor material, the influence of illumination source type on the germanium surface morphology has not yet been investigated. In this work, the illumination effect is studied by halogen lamp, white LED, 470- and 405-nm pulsed diode laser. It is demonstrated that different Ge surface morphologies such as nanocone, nanorod, nanoplate and nanopyramid are obtained using different illumination source. The current density, anodization time and pulsed laser power density effects on Ge nanopyramid are investigated in order to optimize anodization conditions. The most uniform and continuous Ge nanopyramid array is obtained at the current density of 30 mA/cm2 for 45 min under cathode side illumination with 470-nm pulsed diode laser. It is observed that the nanostructured Ge surfaces exhibit a broad photoluminescence band between 400 and 650 nm. Time-resolved fluorescence spectroscopy studies of electron transfer process between BODIPY dye and Ge nanostructures are reported. The obtained fluorescence lifetime data are analyzed in the light of the Marcus electron transfer theory to determine the conduction band energy level of nanostructured germanium substrates.