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PubMed2002

Advantages and risks of multiphoton microscopy in physiology.

Multiphoton microscopy is based on the simultaneous absorption of two photons emitted by a pulsed infrared laser source. In this technique, the excitation is restricted to a very small focus and thus results in optical sectioning a priori without the need of a confocal aperture. Multiphoton microscopy was introduced in live cell imaging as an alternative to confocal microscopy due to its superior qualities, such as the deep penetration depth, the reduced photodamage and the lack of out-of-focus bleaching. However, during the past years, examinations revealed severe limitations to the initial expectations. In the focal plane, photodamage and photobleaching can be worse than in single photon microscopy. However, studies showed that with low excitation intensity and by special technical adaptations photodamage could be avoided successfully. For functional biological imaging multiphoton excitation provides an excellent tool such as the release of caged compounds in a diffraction-limited volume combined with multiphoton or confocal imaging.

Animals↗
PubMed2002

New modal wave-front sensor: application to adaptive confocal fluorescence microscopy and two-photon excitation fluorescence microscopy.

Confocal and multiphoton microscopes are particularly sensitive to specimen- or system-induced aberrations, which result in decreased resolution and signal-to-noise ratio. The inclusion of an adaptive optics correction system could help overcome this limitation and restore diffraction-limited performance, but such a system requires a suitable method of wave-front measurement. By extending the concept of a modal wave-front sensor previously described by Neil et al. [J. Opt. Soc. Am. A 17, 1098-1107 (2000)], we present a new sensor capable of measuring directly the Zernike aberration modes introduced by a specimen. This modal sensor is particularly suited to applications in three-dimensional microscopy because of its inherent axial selectivity; only those wave fronts originating in the focal region contribute to the measured signal. Four wave-front sensor configurations are presented and their input response is characterized. Sensitivity matrices and axial responses are presented.

Microscopy, Confocal↗
PubMed2006

Intraocular multiphoton microscopy with subcellular spatial resolution by infrared femtosecond lasers.

We reported on the in situ nonlinear optical sectioning of the corneal and retinal tissues based on the multiphoton microscopy (MPM) with different excitation wavelengths of infrared femtosecond (fs) lasers. The multiphoton nonlinear processing including two-photon fluorescence (2PF) and second harmonic generation (SHG) was induced under condition of high light intensities on an order of MW-GW/cm2. The laser beams emitted from the solid-state Ti: sapphire systems were focused in a 0.1 femtoliter focus volume of a high numerous aperture diffraction-limited objective (40 x 1.3 N.A., oil). The corneal layers have been visualized using nonlinear optical tomography. In particular, corneal Bowman's layer was optically determined in situ. The cellular and collagen components of tissues were selectively displayed with submicron spatial resolution and high efficiency without any assistance of staining or slicing. The preliminary study on retinal optical tomography is here also reported. MPM is a promising and convenient non-invasive technique by which the tissue layers can be visualized and the selective displaying of the tissue microstructures be realized. The optical biopsy based on intrinsic emission of MPM yields details that provide three-dimensional displaying of the tissue component and even have the potential to be used in clinical diagnostics.

Animals↗
PubMed2006

In situ three-dimensional characterization of membrane fouling by protein suspensions using multiphoton microscopy.

Fouling of microfiltration membranes leads to severe flux declines and the need to clean or replace the membrane. In situ 3D characterization of protein fouling both on the surface and within the pores of the membrane was achieved using multiphoton microscopy. Time-lapse images of the fouled membrane were obtained for single suspensions and mixtures of fluorescently labeled bovine serum albumin and ovalbumin. Deposited protein aggregates were visible on the membrane and evidently play an important role in fouling. A combination of 3D images and resistance versus time data was used to identify the dominant fouling mechanism. Fouling is initially internally dominated, but after 1 and 15 min for ovalbumin and bovine serum albumin, respectively, the fouling becomes externally dominated. This is in good agreement with two-stage protein fouling models.

Animals↗
PubMed2006

Moving toward the light: using new technology to answer old questions.

Fluorescence microscopy has become a principle methodology in the field of developmental biology. Recent technological advances have led to the design of high-speed and high-resolution confocal and multiphoton microscopes that enable researchers to obtain three- and four-dimensional information in living cells and whole embryos. Paralleling this progress, the development of stable and bright vital fluorescent probes has revolutionized the ability to track individual cells in vitro and in vivo and to visualize intercellular and subcellular molecular interactions in real time. Combining imaging modalities and labeling techniques that are increasingly unobtrusive to cell and whole animal function, our understanding of how proteins interact, tissues take form, and organs synchronize to create a functioning animal is reaching a whole new level.

Animals↗
PubMed2006

Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure.

BACKGROUND: We characterize the application of multiphoton microscopy to the observation of the extracellular matrix of fresh unstained vessels. METHOD: Combined two-photon-excited fluorescence (2PEF) and second harmonic generation (SHG) imaging of large arteries reveals the architecture of elastin and collagen fibers in the vessel wall with remarkable specificity. RESULTS: We present elastin/collagen imaging in unstained rat vessels at both micrometer and whole vessel scales, and we characterize the optical properties of rat carotid artery and aorta walls. We apply this method to evidence deleterious effects of residual doses of a pesticide on the vessel wall. CONCLUSION: This study illustrates the potential of 2PEF/SHG microscopy for pharmacological studies in unlabeled arteries.

Animals↗
PubMed2003

Nonlinear magic: multiphoton microscopy in the biosciences.

Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.

Biological Science Disciplines↗
PubMed2006

Two-photon-excited fluorescence imaging of human RPE cells with a femtosecond Ti:Sapphire laser.

PURPOSE: To record the distribution and spectrum of human retinal pigment epithelial cell lipofuscin (LF) by two-photon-excited fluorescence (TPEF) and confocal laser scanning microscopy. METHODS: Ex vivo TPEF imaging of the human retinal pigment epithelium (RPE) of human donor eyes was conducted with a multiphoton laser scanning microscope that employs a femtosecond Ti:sapphire laser as an excitation laser source. The spectrum of autofluorescence of LF granules was analyzed with a confocal laser scanning microscope coupled to a UV argon laser. RESULTS: TPEF examination allowed for imaging of RPE cell morphology and intracellular distribution of LF granules with high-contrast and submicrometer resolution. Variations in cell size and shape as well as in autofluorescence spectra of individual LF granules were recorded. The typical diameter of LF granules was found to be below 1 mum, with some RPE cells possessing larger granules. Remarkably, enhanced blue-green autofluorescence was observed from these larger LF granules. CONCLUSIONS: TPEF imaging represents a novel tool for the investigation of morphologic and spectral characteristics of human RPE cells. Spectral variations of individual LF granules may indicate differences in the complex molecular composition. Compared to conventional single-photon excited autofluorescence, TPEF with a tunable laser source allows for reduced photo damage and deeper sensing depth. It may help to elucidate further the pathophysiological role of LF accumulation as a common downstream pathogenetic pathway in retinal diseases. With the proof of principle from this ex vivo study, further work is now planned to evaluate the safety of TPEF RPE imaging in RPE cultures and animal models.

Adult↗
PubMed2006

Fluid flow in the juxtaglomerular interstitium visualized in vivo.

Earlier electron microscopy studies demonstrated morphological signs of fluid flow in the juxtaglomerular apparatus (JGA), including fenestrations of the afferent arteriole (AA) endothelium facing renin granular cells. We aimed to directly visualize fluid flow in the JGA, the putative function of the fenestrated endothelium, using intravital multiphoton microscopy of Munich-Wistar rats and C57BL6 mice. Renin content of the AA correlated strongly with the length of the fenestrated, filtering AA segment. Fluorescence of the extracellular fluid marker lucifer yellow (LY) injected into the cannulated femoral vein in bolus was followed in the renal cortex by real-time imaging. LY was detected in the interstitium around the JG AA before the plasma LY filtered into Bowman's capsule and early proximal tubule. The fluorescence intensity of LY in the JGA interstitium was 17.9 +/- 3.5% of that in the AA plasma (n = 6). The JGA fluid flow was oscillatory, consisting of two components: a fast (one every 5-10 s) and a slow (one every 45-50 s) oscillation, most likely due to the rapid transmission of both the myogenic and tubuloglomerular feedback (TGF)-mediated hemodynamic changes. LY was also detected in the distal tubular lumen about 2-5 s later than in the AA, indicating the flow of JGA interstitial fluid through the macula densa. In the isolated microperfused JGA, blocking the early proximal tubule with a micropipette caused significant increases in MD cell volume by 62 +/- 4% (n = 4) and induced dilation of the intercellular lateral spaces. In summary, significant and dynamic fluid flow exists in the JGA which may help filter the released renin into the renal interstitium (endocrine function). It may also modulate TGF and renin signals in the JGA (hemodynamic function).

Animals↗
PubMed2006

[Second- and third-harmonic generation microscopies for the structural imaging of intact tissues].

One principal advantage of multiphoton excitation microscopy is that it preserves its three-dimensional micrometer resolution when imaging inside light-scattering samples. For that reason two-photon-excited fluorescence microscopy has become an invaluable tool for cellular imaging in intact tissue, with applications in many fields of physiology. This success has driven increasing interest in other forms of nonlinear microscopy that can provide additional information on cells and tissues, such as second- (SHG) and third- (THG) harmonic generation microscopies. In recent years, significant progress has been made in understanding the contrast mechanisms of these recent methodologies, and high-resolution imaging based on intrinsic sources of signal has been demonstrated in cells and tissues. Harmonic generation exhibits structural rather than chemical specificity and can be obtained from a variety of non-fluorescent samples. SHG is observed specifically in dense, non-centrosymmetric arrangements of polarizable molecules, such as collagen fibrils, myofilaments, and polarized microtubule bundles. SHG imaging is therefore emerging as a novel approach for studying processes such as the physiopathological remodelling of the collagen matrix and myofibrillogenesis in intact tissue. THG does not require a non-centrosymmetric system ; however no signal can be obtained from a homogeneous medium. THG imaging therefore provides maps of sub-micrometer heterogeneities (interfaces, inclusions) in unstained samples, and can be used as a general purpose structural imaging tool. Recent studies showed that this technique can be used to image embryo development in small organisms and to characterize the accumulation of large lipid bodies in specialized cells. SHG and THG microscopy both rely on femtosecond laser technology and are easily combined with two-photon microscopy.

Histological Techniques↗
PubMed2006

In vivo delivery of fluoresceinated dextrans to the murine growth plate: imaging of three vascular routes by multiphoton microscopy.

Bone elongation by endochondral ossification occurs through the differentiation cascade of chondrocytes of cartilaginous growth plates. Molecules from the systemic vasculature reach the growth plate from three different directions: epiphyseal, metaphyseal, and a ring vessel and plexus associated with the perichondrium. This study is an analysis of the real-time dynamics of entrance of fluoresceinated tracers of different molecular weights into the growth plate from the systemic vasculature and tests the hypothesis that molecular weight is a key variable in the determination of both the directionality and the extent of tracer movement into the growth plate. Multiphoton microscopy was used for direct in vivo imaging of the murine proximal tibial growth plate in anesthetized 4- to 5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter. Mice were given an intracardiac injection of either fluorescein (332.3 Da) or fluoresceinated dextrans of 3, 10, 40, 70 kDa, singly or sequentially. For each tracer, directionality and rate of arrival, together with extent of movement within the growth plate, were imaged in real time. For small molecules (up to 10 kDa), vascular access from all three directions was observed and entrance was equally permissive from the metaphyseal and the epiphyseal sides. Within our detection limit (a few percent of vascular concentration), 40 kDa and larger dextrans did not enter. These results have implications both for understanding systemic and paracrine regulation of growth plate chondrocytic differentiation, as well as variables associated with effective drug delivery to growth plate chondrocytes.

Animals↗
PubMed2004

FRET multiphoton spectral imaging microscopy of 7-ketocholesterol and Nile Red in U937 monocytic cells loaded with 7-ketocholesterol.

OBJECTIVE: To show the effect of 7-ketocholesterol (7KC) on cellular lipid content by means of flow cytometry and the interaction of 7KC with Nile Red (NR) via ultraviolet fluorescence resonance energy transfer (FRET) excitation of NR on U937 monocytic cells by means of 2-photon excitation confocal laser scanning microscopy (CLSM). STUDY DESIGN: Untreated and 7KC-treated U937 cells were stained with NR and analyzed by flow cytometry and CLSM. 3D sequences of images were obtained by spectral analysis in a 2-photon excitation CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, which provides factor curves and images. Factor images are the result of the FAMIS image processing method, which handles emission spectra. In FRET analysis, preparations are screened at selected UV wavelengths to avoid emission of NR in the absence of 7KC. RESULTS: During 7KC-induced cell death,flow cytometry and CLSM revealed a modification of the cellular lipid content. Factor images show FRET occurrence and subsequent colocalization of 7KC and NR. CONCLUSION: This investigation established the utility of 2-photon excitation CLSM to assess colocalization of 7KC with NR by FRET and to identify and distinguish polar and neutral lipids stained by NR that accumulate from the effect of 7KC.

Cell Death↗
PubMedDate not supplied

Fast three-dimensional laser scanning scheme using acousto-optic deflectors.

A scheme for fast 3-D laser scanning using acousto-optic deflectors is proposed and demonstrated. By employing counterpropagating acoustic waves that are both chirped and offset in their frequencies, we show that it is possible to simultaneously scan both axially and laterally with frame rates on the order of tens of kilohertz. This scheme was specifically designed for application with multiphoton imaging, particularly of neurons, where it will enable the concurrent monitoring of physiological signals at multiple locations within a microscopic 3-D volume (350 x 350 x 200 microm). When used for this purpose, we demonstrate how this scheme would also inherently compensate for spatial dispersion when ultrafast laser pulses are used in acousto-optic multiphoton microscopy.

Acoustics↗
PubMed2006

Multiphoton imaging of ultrasound/Optison mediated cerebrovascular effects in vivo.

Ultrasound (US) enhanced with microbubble contrast agents may transiently disrupt the blood-brain barrier (BBB) with minimal damage, providing a technique for noninvasive, localized drug-delivery deep within the brain. The mechanism and temporal profile of disruption are not understood, owing to the limitations of imaging modalities used previously. In this study, we monitored US-induced BBB disruption with multiphoton microscopy, providing high-resolution temporal and spatial information about the permeabilization mechanism and immediate effects of US exposure. Anesthetized C57 mice were prepared with a craniotomy and injected intravenously with fluorescent dyes to permit visualization of the vasculature and BBB integrity. The animals were imaged through a cranial window while exposed to low-intensity US (f=1.029 MHz, power=0.2 W) with a coincident intravenous injection of Optison (a microbubble contrast agent). We observed arteriolar vasoconstriction on US exposure that disrupted blood flow and lasted up to 5 mins; BBB disruption occurred via two characteristically distinct processes-perivascular fluorescence gradually increased (over minutes) along the length of the affected vessel without apparent rupture of the vessel wall or rapidly (seconds) increased in select, focal regions. These data corroborated previous studies suggesting increased endothelial transcytosis and breached tight junctions and demonstrated vasoconstriction, which might alter BBB permeability by modifying cerebral blood flow.

Albumins↗
PubMed2006

Hybrid reflecting objectives for functional multiphoton microscopy in turbid media.

Most multiphoton imaging of biological specimens is performed using microscope objectives optimized for high image quality under wide-field illumination. We present a class of objectives designed de novo without regard for these traditional constraints, driven exclusively by the needs of fast multiphoton imaging in turbid media: the delivery of femtosecond pulses without dispersion and the efficient collection of fluorescence. We model the performance of one such design optimized for a typical brain-imaging setup and show that it can greatly outperform objectives commonly used for this task.

Computer-Aided Design↗
PubMed2004

Coherence-gated wave-front sensing in strongly scattering samples.

We show that, by coherence-gate rejection of out-of-focus light, wave-front distortions can be measured in the presence of a scattering background that is dominant by several orders of magnitude. Applications are expected for multiphoton and confocal laser-scanning microscopy.

Algorithms↗