Two-photon Fluorescence Measurements Research Articles

Comparing different modalities of time-resolved two-photon fluorescence measurements for FRET analysis

Different modalities of time-resolved fluorescence lifetime techniques are typically used for Förster resonance energy transfer (FRET) studies in vivo and in solution and have been used to measure macromolecular processes and interactions. Here, we examine the sensitivity of the estimated FRET efficiency and donor-acceptor distance to different modalities of time-resolved two-photon (2P) fluorescence measurements, experimental design, and data analysis. As a model, we used the biosensor (mTurquoise-linker-mCitrine), where mTurquoise-mCitrine serves as the FRET pair tethered with a flexible linker.

Examining the donor effect on FRET efficiency in macromolecular crowding sensors

Macromolecular crowding influences many biological processes in living cells such as protein folding, protein-protein interactions, translational diffusion, and biochemical reaction kinetics. In order to investigate the correlation between the heterogenous, dynamic macromolecular crowding and cell physiology, site-specific biosensors and noninvasive, quantitative methodology are critically needed. Here, we investigate the effects of donor identity (mCerulean3, mTurqoise2.1, and mTurqoise2.0) on the environmental sensitivity of genetically encoded sensors (donor-linker-mCitrine) using time-resolved two-photon fluorescence measurements.

Unveiling Hg-binding protein within black deposit formed on Golgi-Cox-stained brain neuron

BackgroundGolgi-Cox staining has been conventionally used for investigating neuronal development. After the brain tissue is subject to Golgi-Cox staining, black deposits are formed on the surface of the stained neurons because of mercuric sulfide, which does not show a fluorescence response under two-photon excitation. However, we unexpectedly observed fluorescence emitted by these black deposits during two-photon fluorescence measurements. Further, the in-depth of physical and chemical methods analysis revealed that the black deposits on the stained neurons are composed of Hg-binding proteins. MethodsWe studied black deposits present in the Golgi-Cox-stained mouse brain neurons using techniques such as multiple-photon microscopy, scan electron microscopy, micro-Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. ResultsThe emitted fluorescence was because of the fluorescence groups of Hg-binding protein present within the Golgi-Cox deposits on the neuronal surface. ConclusionsThe presence of Hg-binding proteins within black deposits on the surface of Golgi-Cox-stained neurons was proven for the first time. The novel interaction between the neurons and Hg2+ ions during Golgi-Cox staining help to understand the mechanism of Golgi-Cox staining.

Second Harmonic Generation Spectroscopy of Membrane Probe Dynamics in Gram-Positive Bacteria

Bacterial membranes are complex mixtures with dispersity that is dynamic over scales of both space and time. To capture adsorption onto and transport within these mixtures, we conduct simultaneous second harmonic generation (SHG) and two-photon fluorescence measurements on two different gram-positive bacterial species as the cells uptake membrane-specific probe molecules. Our results show that SHG not only can monitor the movement of small molecules across membrane leaflets but also is sensitive to higher-level ordering of the molecules within the membrane. Further, we show that the membranes of Staphylococcus aureus remain more dynamic after longer times at room temperature in comparison to Enterococcus faecalis. Our findings provide insight into the variability of activities seen between structurally similar molecules in gram-positive bacteria while also demonstrating the power of SHG to examine these dynamics.

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy.

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-β). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Understanding the Dynamic Behavior of an Anticancer Drug, Doxorubicin, on a Lipid Membrane Using Multiple Spectroscopic Techniques.

The interaction, including the adsorption and embedding, of a widely applied anticancer drug, doxorubicin, with a lipid membrane was investigated. Second harmonic generation and two photon fluorescence were used as a powerful combination capable in revealing this dynamic process at the interface. The adsorption, association, deassociation and embedding of doxorubicin on the lipid membrane were clearly identified based on the consistency in the dynamic parameters revealed by the time dependent second harmonic generation and two-photon fluorescence measurements. This work also presents a new approach for in situ measurement of the adsorption density of doxorubicin on lipid membrane, benefiting from the two-photon fluorescence signal of doxorubicin being significantly altered by its chemical environment. The analysis of the location and molecular density based on the fluorescent efficiency of the chromophores makes the fluorescence measurement a "surface sensitive" technique as well. The analytical procedures used in this work are expected to aid in understanding the interaction between fluorescent molecules and lipid membranes in general.

Ultrafast responses of two V-shaped compounds with a reverse conjugated structural configuration: an investigation of the reason for the enhanced two-photon absorption cross-section

To study the effect of the conjugated structural configuration on the two-photon absorption (TPA) properties of V-shaped compounds, two 1,3,5-triazine-based compounds with the same electron donor (D) and acceptor (A) connected in a reverse-conjugated structural configuration (T02: D- $$\pi$$ -A- $$\pi$$ -D; R02: A- $$\pi$$ -D- $$\pi$$ -A) were systematically investigated using steady-state and transient absorption spectroscopy, open-aperture Z-scan measurements, and two-photon fluorescence measurements. The TPA cross-section of compound R02 connected in a A- $$\pi$$ -D- $$\pi$$ -A-conjugated structural configuration with triphenylamine as the central core was 203 GM, which showed a 2.3-fold enhancement compared with compound T02 connected in a reverse D- $$\pi$$ -A- $$\pi$$ -D-conjugated structural configuration (90 GM, with 1,3,5-triazine as the central core). This result indicates that the conjugated structural configuration plays an important role in the TPA properties. A two-color pump–probe experiment was used to investigate the effect of the conjugated structural configuration on the excited state and intra-molecular charge transfer (ICT) properties of these V-shaped compounds. The formation and relaxation lifetimes of the ICT state were determined. The results indicate that the electron-donating/accepting strength of the central group, which serves as a communal group for two D- $$\pi$$ -A subunits, was confirmed to be a key role to the overall effect of the ICT for V-shaped compounds. These ultrafast dynamic results are in agreement with the TPA properties.

Ultrafast dynamics of a series multi-branched styryl derivatives with reverse conjugated structural configuration

To study the effect of conjugated structural configurations on the two-photon absorption properties, tri-branched compounds with the same electron-donating and electron-accepting groups connected in a reverse conjugated structural mode, are systematically investigated using steady-state and transient absorption spectroscopies, open-aperture Z-scan, and two-photon fluorescence measurements. The two-photon absorption cross-section of compound R03 with the strong electron donor triphenylamine as the central core of 766 GM is notably large and is approximately 1.7 times larger than that of the tri-branched compound T03-a with a reverse conjugated structural mode and with the 1,3,5-triazine electron acceptor as the central core. This finding indicates that the conjugated mode does have an important effect on the nonlinear optical properties. A two-color pump-probe experiment is used to investigate the ultrafast responses and intra-molecular charge transfer properties of these multi-branched compounds. The form...

SYNTHESIS, CHARACTERIZATION OF A NOVEL CONJUGATED STRUCTURAL OLIGOMER

An octupolar star-shaped ladder-type oligomer composed of electron deriving from 1,3,5-triazine core end-capped by either an electron donating carbazolemoiety (TA(TL)-Ph(3)-CBZ) have been designed and synthesized to investigate the structure-properties relationship of highly efficient multiphoton absorbing materials. The newly synthesized molecules were characterized by 1H NMR, 13C NMR and mass spectrometry. Linear optical properties of the oligomer were investigated by UV-vis and fluorescence spectrometries, as well as the nonlinear optical properties were characterized by two-photon excited fluorescence (2PEF) measurement.

Two-Photon Fluorescence Microscopy of Corneal Riboflavin Absorption

To correct for attenuation in two-photon fluorescence (TPF) measurements of riboflavin absorption in porcine corneas. Two-photon fluorescence imaging of riboflavin was performed using excitation at a wavelength of 890 nm, with fluorescence signal detected between 525 and 650 nm. TPF signal attenuation was demonstrated by imaging from either side of a uniformly soaked corneoscleral button. To overcome this attenuation, a reservoir of dextran-free 0.1% wt/vol riboflavin 5'-monophosphate in saline and hydroxypropyl methylcellulose (HPMC) was placed on top of porcine corneas (globe intact-epithelium removed). TPF imaging was performed through this reservoir with image stacks acquired at 10-μm steps through the cornea repeated at regular intervals for up to 60 minutes. A novel correction method was applied to achieve corneal riboflavin concentration measurements in whole eyes (n = 4). Significant attenuation of the TPF signal was observed in all eyes, with the signal decreasing approximately linearly with depth in uniformly soaked tissue. Cross-sectional TPF images taken of excised corneal strips confirmed the tissue was uniformly soaked so that the decrease in signal was not due to spatial variations in riboflavin concentration. After correcting for signal attenuation, we observed increased riboflavin concentrations with longer soak duration, with the mean (standard deviation) maximum tissue concentration recorded at 0.094% (± 0.001) wt/vol [1.36 mg/mL]. Uniform riboflavin absorption was achieved after a minimum 50 minutes. Following a standard corneal cross-linking soak of 30 minutes, a mean stromal concentration of 0.086% (± 0.001) wt/vol [1.25 mg/mL] was achieved at a depth of 300 μm. The accuracy of TPF measurements of corneal riboflavin absorption can be increased by applying a correction for depth-related signal attenuation.

PEGYLATED CONJUGATED OLIGOMERS FOR TARGETED TWO-PHOTON FLUORESCENCE IMAGING OF CANCER CELLS

A water-dispersible PEGylated conjugated oligomer (PEGTVFVBN) is synthesized for two-photon fluorescence imaging applications. PEGTVFVBN could aggregate into nanoparticles of ~ 110 nm size in H2O , which show a fluorescence quantum yield of 0.12, 20 times higher than that for its cationic counterpart (TVFVBC). Two-photon fluorescence measurement reveals a maximum two-photon absorption (TPA) cross-section of 263 GM at 780 nm based on molecules for PEGTVFVBN nanoparticles in H2O , while the TPA spectra of TVFVBC are barely detectable under the same experimental conditions. The N , N' -diacetylchitobiotic acid conjugated derivative (ChPEGTVFVBN) could also aggregate into nanoparticles of ~ 90 nm sizes, showing a slightly smaller fluorescence quantum yield of 0.08 and a maximum TPA cross-section of 213 GM at 780 nm. ChPEGTVFVBN could be specifically internalized by vimentin over expressing Hela cells through a receptor mediated endocytosis, giving two-photon fluorescence images in a bright and high contrast manner. In addition, both PEGTVFVBN and ChPEGTVFVBN show low cytotoxicity, which is essential for cell imaging applications. This study thus highlights the molecular engineering strategy of PEGylation to construct robust TPA materials for two-photon fluorescence imaging applications.

Solvent effect on two-photon absorption (TPA) of three novel dyes with large TPA cross-section and red emission

Abstract Three novel organic dyes containing 2,6-dicarbonyl-pyridine as the central electron acceptor with different end-caped amino groups as donors in the D-π-A-π-D arrangement are designed and synthesized. All three molecules exhibit large TPA cross-sections (δ > 1000 GM). The cooperative effects of donor strength, conjugation length and solvent polarity on the TPA activity are studied. The conjugated system plays an important role on the enhancement of δ in the highly polar solvent, while donor strength has more impact on TPA properties. The influence of the solvent polarity on TPA properties is studied by two-photon excited fluorescence (TPEF) measurement. The result shows a nonmonotonic relationship between the TPA cross-section and the solvent, and the maximum of δ is found in solvents with moderate polarity. The δ of molecules with stronger donor reaches the maximum in more polar solvents. The highest δ is found to be 5250 GM in CHCl3. All compounds exhibit large Stokes shifts due to the strong intramolecular charge transfer (ICT) effect.

Glycosylated Star‐Shaped Conjugated Oligomers for Targeted Two‐Photon Fluorescence Imaging

A glucopyranose functionalized star-shaped oligomer, N-tris{4,4',4''-[(1E)-2-(2-{(E)-2-[4-(benzo[d]thiazol-2-yl)phenyl]vinyl}-9,9-bis(6-2-amido-2-deoxy-1-thio-β-D-glucopyranose-hexyl)-9H-fluoren-7-yl)vinyl]phenyl}phenylamine (TVFVBN-S-NH(2)), is synthesized for two-photon fluorescence imaging. In water, TVFVBN-S-NH(2) self-assembles into nanoparticles with an average diameter of ∼49 nm and shows a fluorescence quantum yield of 0.21. Two-photon fluorescence measurements reveal that TVFVBN-S-NH(2) has a two-photon absorption cross-section of ∼1100 GM at 780 nm in water. The active amine group on the glucopyranose moiety allows further functionalization of TVFVBN-S-NH(2) with folic acid to yield TVFVBN-S-NH(2) FA with similar optical and physical properties as those for TVFVBN-S-NH(2). Cellular imaging studies reveal that TVFVBN-S-NH(2) FA has increased uptake by MCF-7 cells relative to that for TVFVBN-S-NH(2), due to specific interactions between folic acid and folate receptors on the MCF-7 cell membrane. This study demonstrates the effectiveness of glycosylation as a molecular engineering strategy to yield water-soluble materials with a large two-photon absorption (TPA) cross-section for targeted cancer-cell imaging.

High-Resolution, Noninvasive, Two-Photon Fluorescence Measurement of Molecular Concentrations in Corneal Tissue

To perform high-resolution, noninvasive, calibrated measurements of the concentrations and diffusion profiles of fluorescent molecules in the live cornea after topical application to the ocular surface. An 800-nm femtosecond laser was used to perform two-photon fluorescence (TPF) axial scanning measurements. Calibration solutions consisting of sodium fluorescein (Na-Fl; concentration range, 0.01%-2.5%) and riboflavin (concentration range, 0.0125%-0.1%) were tested in well slides, and TPF signals were assessed. Excised feline eyeballs preserved in corneal storage medium and with either intact or removed corneal epithelia were then treated with Na-Fl, riboflavin, or fluorescein dextran (Fl-d) of different molecular weight (MW) for 30 minutes. Calibrated TPF was then used immediately to measure the concentration of these molecules across the central corneal depth. The axial resolution of our TPF system was 6 μm, and a linear relationship was observed between TPF signal and low concentrations of most fluorophores. Intact corneas treated with Na-Fl or riboflavin exhibited a detectable penetration depth of only approximately 20 μm, compared with approximately 400 to 600 μm when the epithelium was removed before fluorophore application. Peak concentrations for intact corneas were half those attained with epithelial removal. Debrided corneas treated with 2,000,000 MW Fl-d showed a half-maximum penetration depth of 156.7 μm compared with 384 μm for the 3,000 MW dextran. The peak concentration of the high MW dextran was one quarter that of the lower MW dextran. TPF is an effective, high-resolution, noninvasive method of quantifying the diffusion and concentration of fluorescent molecules across the cornea.

Pulse characterization during femtosecond laser filamentation in air by two-photon fluorescence measurement

This work reports a temporal characteristic of a laser pulse undergoing filamentation in air. The diagnostic method is based on two-photon fluorescence measurement. The results show that the pulse duration, chirp rate and beam radius could be simultaneously quantitatively retrieved. This simple technique would be useful in practice to trace the underlying dynamics of filamentation in air.

基于双光子荧光原理的空气中飞秒激光成丝区域脉冲特性的测量

基于双光子荧光原理的空气中飞秒激光成丝区域脉冲特性的测量

Single-snapshot and intensity-resolved two-photon fluorescence measurements

We present a single-snapshot (SSS) method for obtaining intensity-resolved two-photon fluorescence (TPF). This simple method uses a digital camera to image the TPF spot on a liquid dye jet. By making a comparison between the local laser and TPF intensities, TPF probabilities are reconstructed. We compare our intensity-resolved TPF results with those obtained by the more common intensity scanning (IS) and z-scan methods. The dependence of the TPF probability on intensity obtained by the SSS method for coumarin-30 exhibits a clear maximum around I approximately 4 x 10(12) W/cm(2) and a postsaturation decrease, while no such effects were found in the data obtained by the other methods. Additionally, theoretical models are presented to extract the overall probability from within the volume integral. To our knowledge, we present the first reported measurements of such intensity-resolved TPF.

In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy

In this study, we use combined two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG) and third-harmonic generation (THG) measurements to image cellular structures of the nematode Caenorhabditis elegans, in vivo. To our knowledge, this is the first time that a THG modality is employed to image live C. elegans specimens. Femtosecond laser pulses (1028 nm) were utilized for excitation. Detailed and specific structural and anatomical features can be visualized, by recording THG signals. Thus, the combination of three image-contrast modes (TPEF-SHG-THG) in a single instrument has the potential to provide unique and complementary information about the structure and function of tissues and individual cells of live biological specimens.

Two-photon fluorescence measurements of reversible photodegradation in a dye-doped polymer

We report on the dynamics of photodegradation and subsequent recovery of two-photon fluorescence in a dye-doped polymer. The energy dependence suggests that photodegradation is a linear process, while recovery is entropic. Such recovery could be useful to high-intensity devices such as two-photon absorbers, which can be used in many applications.

Biosensing based on two-photon fluorescence measurements through optical fibers

We have performed two-photon fluorescence detection in a new scheme in which femtosecond laser pulses were delivered thorugh an optical fiber for nonlinear excitation and the emitted fluorescence was collected through the same fiber. Single-mode fibers were determined to give higher detection efficiency than multimode fibers, consistent with theoretical considerations. The utility of fiber-optic sensing based on two-photon fluorescence detection was proved by an experiment that measured the uptake of a targeted drug delivery agent into cultured cancer cells.