Multiphoton Fluorescence Imaging Research Articles

Real-Time CARS Imaging Reveals a Calpain-Dependent Pathway for Paranodal Myelin Retraction during High-Frequency Stimulation

High-frequency electrical stimulation is becoming a promising therapy for neurological disorders, however the response of the central nervous system to stimulation remains poorly understood. The current work investigates the response of myelin to electrical stimulation by laser-scanning coherent anti-Stokes Raman scattering (CARS) imaging of myelin in live spinal tissues in real time. Paranodal myelin retraction at the nodes of Ranvier was observed during 200 Hz electrical stimulation. Retraction was seen to begin minutes after the onset of stimulation and continue for up to 10 min after stimulation was ceased, but was found to reverse after a 2 h recovery period. The myelin retraction resulted in exposure of Kv 1.2 potassium channels visualized by immunofluorescence. Accordingly, treating the stimulated tissue with a potassium channel blocker, 4-aminopyridine, led to the appearance of a shoulder peak in the compound action potential curve. Label-free CARS imaging of myelin coupled with multiphoton fluorescence imaging of immuno-labeled proteins at the nodes of Ranvier revealed that high-frequency stimulation induced paranodal myelin retraction via pathologic calcium influx into axons, calpain activation, and cytoskeleton degradation through spectrin break-down.

A Novel Assay of Mechano-Transduction in Single Muscle Cells

In striated muscle the tight integration of contractility and biomechanical properties with electrical, metabolic and hormonal signaling are defined as mechano-transduction signaling pathways. The great interest to define the molecular basis for mechano-transduction is underpinned by the growing number of loss-of-function disorders and diseases in which altered mechano-transduction has been identified. Intact, single cell preparations are attractive for studying mechano-transduction, however the mechanical loading of single cells requires technically challenging methods. We have recently developed MyoTak, a tenacious biological adhesive, which enables us to attach single dissociated striated myocytes (skeletal and cardiac) to force transducer and length controller units. The myocytes thus prepared can be readily imaged using an inverted microscope equipped with confocal, widefield and multiphoton fluorescence imaging systems. With this preparation we have been able to control myocyte length while assaying passive tension and electrically evoked contractility. In recent experiments we have explored the interaction of myocyte stretch and contractility with Ca2+ signaling, mitochondrial function and ROS production. We will present these new findings, the novel methods and additional experiments examining the role of microtubules as mechano-transducer elements that contribute to these interactions. Supported by RC2 NR011968 to CWW, and to WJL (NHLBI P01 HL67849 and R01-HL36974; Leducq North American-European Atrial Fibrillation Research Alliance; European Union Seventh Framework Program (FP7) “Identification and therapeutic targeting of common arrhythmia trigger mechanisms” and support from the Maryland Stem Cell Commission).

Glucose regulates fatty acid binding protein interaction with lipids and peroxisome proliferator-activated receptor α

Although the pathophysiology of diabetes is characterized by elevated levels of glucose and long-chain fatty acids (LCFA), nuclear mechanisms linking glucose and LCFA metabolism are poorly understood. As the liver fatty acid binding protein (L-FABP) shuttles LCFA to the nucleus, where L-FABP directly interacts with peroxisome proliferator-activated receptor-α (PPARα), the effect of glucose on these processes was examined. In vitro studies showed that L-FABP strongly bound glucose and glucose-1-phosphate (K(d) = 103 ± 19 nM and K(d) = 20 ± 3 nM, respectively), resulting in altered L-FABP conformation, increased affinity for lipid ligands, and enhanced interaction with PPARα. In living cells, glucose stimulated cellular uptake and nuclear localization of a nonmetabolizable fluorescent fatty acid analog (BODIPY C-16), particularly in the presence of L-FABP. These data suggest for the first time a direct role of glucose in facilitating L-FABP-mediated uptake and distribution of lipidic ligands to the nucleus for regulation of PPARα transcriptional activity.

Nanoparticles for highly efficient multiphoton fluorescence bioimaging

In this paper, we demonstrate for the first time that the new class of fluoride-based inorganic upconverting nanoparticles, NaYF4:Er3+, Yb3+, are the most efficient multiphoton excited fluorescent nanoparticles developed to date. The near-infrared-to-visible conversion efficiency of the aforementioned nanoparticles surpasses that of CdSe quantum dots and gold nanorods, which are the commercially available inorganic fluorescent nanoprobes presently used for multiphoton fluorescence bioimaging. The results presented here open new perspectives for the implementation of fluorescence tomography by multiphoton fluorescence imaging.

Quantum Dot Capped Magnetite Nanorings as High Performance Nanoprobe for Multiphoton Fluorescence and Magnetic Resonance Imaging

In the present study, quantum dot (QD) capped magnetite nanorings (NRs) with a high luminescence and magnetic vortex core have been successfully developed as a new class of magnetic-fluorescent nanoprobe. Through electrostatic interaction, cationic polyethylenimine (PEI) capped QD have been firmly graft into negatively charged magnetite NRs modified with citric acid on the surface. The obtained biocompatible multicolor QD capped magnetite NRs exhibit a much stronger magnetic resonance (MR) T2* effect where the r2* relaxivity and r2*/r1 ratio are 4 times and 110 times respectively larger than those of a commercial superparamagnetic iron oxide. The multiphoton fluorescence imaging and cell uptake of QD capped magnetite NRs are also demonstrated using MGH bladder cancer cells. In particular, these QD capped magnetite NRs can escape from endosomes and be released into the cytoplasm. The obtained results from these exploratory experiments suggest that the cell-penetrating QD capped magnetite NRs could be an excellent dual-modality nanoprobe for intracellular imaging and therapeutic applications. This work has shown great potential of the magnetic vortex core based multifunctional nanoparticle as a high performance nanoprobe for biomedical applications.

Single- and Multiphoton Turn-On Fluorescent Fe3+ Sensors Based on Bis(rhodamine)

Selective and sensitive turn-on fluorescent Fe(3+) sensors based on novel bis(rhodamine) dye molecules are reported. The compounds are synthesized with very high yields and characterized with NMR, ESI mass spectrometry, and elemental analysis. Single- and two-photon fluorescence enhancement is observed for both molecules in the presence of Fe(3+). High selectivity and sensitivity is observed over other metal ions and is shown to be due mainly to the spirolactam ring-opening power of Fe(3+). All measurements are made in buffer environments simulating biological conditions to facilitate single- and multiphoton fluorescence imaging of Fe(3+) in vivo and in vitro. Larger enhancement of fluorescence for both one- and two-photon excitation makes them suitable candidates for fluorescent labeling of biological systems. Two photon cross-section and time-resolved fluorescence measurements are utilized to understand the selectivity of the present sensors for Fe(3+)-sensing.

Peak Multiphoton Excitation of mCherry Using an Optical Parametric Oscillator (OPO)

mCherry is a red fluorescent protein which is bright, photostable, and has a low molecular weight. It is an attractive choice for multiphoton fluorescence imaging; however, the multiphoton excitation spectrum of mCherry is not known. In this paper we report the two photon excitation spectrum of mCherry measured up to 1190 nm in the near infrared (NIR) region. Skin tissues of transgenic mice that express mCherry were used in the experiments. mCherry in the tissues was excited with a Titanium:Sapphire laser and an optical parametric oscillator pumped by the Titanium:Sapphire laser. We found that the peak excitation of mCherry occurs at 1160 nm.

In vivo microcartography and subcellular imaging of tumor angiogenesis: A novel platform for translational angiogenesis research

Purpose To eliminate the variable of tumor heterogeneity from a novel in vivo model of tumor angiogenesis. Experimental design We developed a method to navigate tumor neovasculature in a living tissue microenvironment, enabling relocation of a cell- or microregion-of-interest, for serial in vivo imaging. Orthotopic melanoma was grown, in immunocompetent Tie2GFP mice. Intravital multiphoton fluorescence and confocal reflectance imaging was performed, on a custom microscope with motorized stage and coordinate navigation software. A point within a Tie2GFP+ microvessel was selected for relocation. Custom software predicted target coordinates based upon reference points (tissue-embedded polystyrene beads) and baseline target coordinates. Mice were removed from the stage to make previously-obtained target coordinates invalid in subsequent imaging. Results Coordinate predictions always relocated target points, in vivo, to within 10–200 μm (within a single 40× field-of-view). The model system provided a virtual living histology of tumor neovascularization and microenvironment, with subcellular spatial resolution and hemodynamic information. Conclusions The navigation procedure, termed in vivo microcartography, permits control of tissue heterogeneity, as a variable. Tie2 may be the best reporter gene identified, to-date, for intravital microscopy of tumor angiogenesis. This novel model system should strengthen intravital microscopy in its historical role as a vital tool in oncology, angiogenesis research, and angiotherapeutic drug development.

Third-Harmonic Generation and Multi-Photon Excitation Fluorescence Imaging Microscopy Techniques for Online Art Conservation Diagnosis

We present an appropriate methodology and results for using third-harmonic generation (THG) modality for nondestructive high resolution imaging measurements of varnished structures in model painted artifacts. Detection takes place in the reflection mode, demonstrating the ability of the technique to be applied to the evaluation of original artworks. Furthermore, multi-photon excitation fluorescence images were obtained, providing complementary information related to the identification of the chemical composition of the artifacts.

Coherent Control in Multiphoton Fluorescence Imaging.

In multiphoton fluorescence laser-scanning microscopy ultrafast laser pulses, i.e. light pulses having pulse-width ≤ 1picosecond (1 ps = 10-12s), are commonly used to circumvent the low multiphoton absorption cross-sections of common fluorophores. Starting with a discussion on how amplitude modulation of ultrashort pulse-train enhances the two-photon fluorescence providing deep insight into laser-induced photo-thermal damage, the effect of controlling time lag between phase-locked laser pulses on imaging is described. In addition, the prospects of laser pulse-shaping in signal enhancement (by temporal pulse-compression at the sample) and selective excitation of fluorophores (by manipulating the phase and/or amplitude of different frequency components within the pulse) are discussed with promising future applications lying ahead.

Ultrafast pulse-pair control in multiphoton fluorescence laser-scanning microscopy

In multiphoton fluorescence laser-scanning microscopy, ultrafast laser pulses [i.e., light pulses having pulse width <or=1 ps (1 ps=10(-12) s)] are commonly employed to circumvent the low-multiphoton absorption cross-sections of common fluorophores. Because of the broad overlapping two-photon absorption spectra of fluorophores and the large spectral bandwidth of a short pulse, simultaneous excitation of many fluorophores is common, which justifies a persistent demand for selective excitation of individual fluorophores. We describe the use of pulse-pair excitation with possibilities of controlling molecular fluorescence in laser-scanning microscopy and compare it with coherent control using pulse sequence [De and Goswami, "Coherent control in multiphoton fluorescence imaging," Proc. SPIE 7183, 71832B (2009)].

Photophysical Characterization of a Highly Conjugated Bipyridyl-Based Dye Synthesized by a Unique Two-Step Approach

In this article, we present a new and simple, yet efficient, two-step approach to synthesize 4,4'-bis(dibutylaminostyrylstyryl)-2,2'-bipyridine with high yield, as well as its linear and nonlinear optical characterizations in THF and toluene solutions. We show that its one- and two-photon absorption spectra are similar in both solvents. Nevertheless, the relaxation processes of this compound exhibit dependence on the solvent polarity. The one- and two-photon induced fluorescence signal of this molecule in solution reveals that its excited state is highly stabilized in THF solution rather than in toluene. Analysis of the fluorescence quantum yield, lifetime, and radiative and nonradiative decay rates are in agreement with Lippert's model for solute-solvent interactions. The optical measurements demonstrate that this dye is a promising candidate for multiphoton fluorescence imaging, optical limiting, and dye lasers.

Conjugated Polymer Dots for Multiphoton Fluorescence Imaging

We report on the two-photon excited fluorescence of conjugated polymer dots (CPdots). As a new class of two-photon fluorescent probes, CPdots exhibit two-photon action cross sections as high as 2.0 × 105 GM, to our knowledge, the largest reported thus far for a nanoparticle. The cross section values are 3−4 orders of magnitude higher than those of conventional fluorescent dyes and an order of magnitude higher than those of inorganic quantum dots. Single particle fluorescence imaging was achieved using relatively low laser power.

Multiphoton Fluorescence Imaging at Cryogenic Conditions

Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, September 2 – September 7, 2007

In vivo quantitation of rare circulating tumor cells by multiphoton intravital flow cytometry

Quantitation of circulating tumor cells (CTCs) constitutes an emerging tool for the diagnosis and staging of cancer, assessment of response to therapy, and evaluation of residual disease after surgery. Unfortunately, no existing technology has the sensitivity to measure the low numbers of tumor cells (<1 CTC per ml of whole blood) that characterize minimal levels of disease. We present a method, intravital flow cytometry, that noninvasively counts rare CTCs in vivo as they flow through the peripheral vasculature. The method involves i.v. injection of a tumor-specific fluorescent ligand followed by multiphoton fluorescence imaging of superficial blood vessels to quantitate the flowing CTCs. Studies in mice with metastatic tumors demonstrate that CTCs can be quantitated weeks before metastatic disease is detected by other means. Analysis of whole blood samples from cancer patients further establishes that human CTCs can be selectively labeled and quantitated when present at approximately 2 CTCs per ml, opening opportunities for earlier assessment of metastatic disease.

Spectral-domain optical coherence phase and multiphoton microscopy

We describe simultaneous quantitative phase contrast and multiphoton fluorescence imaging by combined spectral-domain optical coherence phase and multiphoton microscopy. The instrument employs two light sources for efficient optical coherence microscopic and multiphoton imaging and can generate structural and functional images of transparent specimens in the epidirection. Phase contrast imaging exhibits spatial and temporal phase stability in the subnanometer range. We also demonstrate the visualization of actin filaments in a fixed cell specimen, which is confirmed by simultaneous multiphoton fluorescence imaging.

Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging

The aim of this work is to demonstrate that multiphoton microscopy is a preferred technique to investigate intact cornea structure without slicing and staining. At the micron resolution, multiphoton imaging can provide both large morphological features and detailed structure of epithelium, corneal collagen fibril bundles and keratocytes. A large area multiphoton cross-section across an intact eye excised from a GFP mouse was obtained by a homebuilt multiphoton microscope. The broadband multiphoton fluorescence (435-700 nm) and second harmonic generation (SHG, 360-400 nm) signals were generated by the 760 nm output of a femtosecond titanium-sapphire laser. A water immersion objective (Fluor, 40X, NA 0.8; Nikon) was used to facilitate imaging the curve ocular surface. The multiphoton image over entire cornea provides morphological information of epithelial cells, keratocytes, and global collagen orientation. Specifically, our planar, large area multiphoton image reveals a concentric pattern of the stroma collagen, indicative of the laminar collagen organization throughout the stroma. In addition, the green fluorescence protein (GFP) labeling contributed to fluorescence contrast of cellular area and facilitated visualizing of inactive keratocytes. Our results show that multiphoton imaging of GFP labeled mouse cornea manifests both morphological significance and structural details. The second harmonic generation imaging reveals the collagen orientation, while the multiphoton fluorescence imaging indicates morphology and distribution of cells in cornea. Our results support that multiphoton microscopy is an appropriate technology for further in vivo investigation and diagnosis of cornea.

Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging

We performed multiphoton fluorescence (MF) and second-harmonic generation (SHG) imaging on human basal cell carcinoma samples. In the dermis, basal cell carcinomas can be identified by masses of autofluorescent cells with relatively large nuclei and marked peripheral palisading. In the normal dermis, SHG from dermal collagen contributes largely to the multiphoton signal. However, within the cancer stroma, SHG signals diminish and are replaced by autofluorescent signals, indicating that normal collagen structures responsible for SHG have been altered. To better delineate the cancer cells and cancer stroma from the normal dermis, a quantitative MF to SHG index is developed. We demonstrate that this index can be used to differentiate cancer cells and adjacent cancer stroma from the normal dermis. Our work shows that MF and SHG imaging can be an alternative for Mohs' surgery in the real-time guidance of the secure removal of basal cell carcinoma.

Quantitative imaging of basic functions in renal (patho)physiology

Multiphoton fluorescence microscopy offers the advantages of deep optical sectioning of living tissue with minimal phototoxicity and high optical resolution. More importantly, dynamic processes and multiple functions of an intact organ can be visualized in real time using noninvasive methods, and quantified. These studies aimed to extend existing methods of multiphoton fluorescence imaging to directly observe and quantify basic physiological parameters of the kidney including glomerular filtration rate (GFR) and permeability, blood flow, urinary concentration/dilution, renin content and release, as well as more integrated and complex functions like the tubuloglomerular feedback (TGF)-mediated oscillations in glomerular filtration and tubular flow. Streptozotocin-induced diabetes significantly increased single-nephron GFR (SNGFR) from 32.4 +/- 0.4 to 59.5 +/- 2.5 nl/min and glomerular permeability to a 70-kDa fluorophore approximately eightfold. The loop diuretic furosemide 2-fold diluted and increased approximately 10-fold the volume of distal tubular fluid, while also causing the release of 20% of juxtaglomerular renin content. Significantly higher speeds of individual red blood cells were measured in intraglomerular capillaries (16.7 +/- 0.4 mm/s) compared with peritubular vessels (4.7 +/- 0.2 mm/s). Regular periods of glomerular contraction-relaxation were observed, resulting in oscillations of filtration and tubular flow rate. Oscillations in proximal and distal tubular flow showed similar cycle times ( approximately 45 s) to glomerular filtration, with a delay of approximately 5-10 and 25-30 s, respectively. These innovative technologies provide the most complex, immediate, and dynamic portrayal of renal function, clearly depicting the components and mechanisms involved in normal physiology and pathophysiology.

Stochastic scanning multiphoton multifocal microscopy

Multiparticle tracking with scanning confocal and multiphoton fluorescence imaging is increasingly important for elucidating biological function, as in the transport of intracellular cargo-carrying vesicles. We demonstrate a simple rapid-sampling stochastic scanning multifocal multiphoton microscopy (SS-MMM) fluorescence imaging technique that enables multiparticle tracking without specialized hardware at rates 1,000 times greater than conventional single point raster scanning. Stochastic scanning of a diffractive optic generated 10x10 hexagonal array of foci with a white noise driven galvanometer yields a scan pattern that is random yet space-filling. SS-MMM creates a more uniformly sampled image with fewer spatio-temporal artifacts than obtained by conventional or multibeam raster scanning. SS-MMM is verified by simulation and experimentally demonstrated by tracking microsphere diffusion in solution.