Here, we present a miniature endomicroscope that combines large field-of-view (FOV) (1.15 mm) reflectance modality and high-spatial resolution (~ 0.5 um) multiphoton imaging. The essential element of the endoscope is a 3 mm outside diameter (OD), catadioptric zoom lens based on the idea of separating the optical paths of excitation light with different wavelengths. The two imaging modes are switched by changing the wavelength of the excitation light and, therefore, the optical zoom operation is achieved without any mechanical adjustment at the endoscope distal end. We aligned in free space the zoom lens with a previously demonstrated miniaturized resonant/non-resonant fiber raster scanner. We tested the performance and confirmed the high resolution and large FOV of this miniature device by imaging a US Air Force test target in transmission. We acquired <i>ex vivo</i> images of unstained rodent tissues by using the large FOV mode to navigate to the site of interest and then using the high resolution modality to image with cellular details. The demonstrated endomicroscope with optical zoom capability is a significant step toward developing clinical optical tools for real time tissue diagnostics.
We use a compact and flexible multiphoton microendoscope (MPME) to acquire in vivo images of unstained liver, kidney, and colon from an anesthetized rat. The device delivers femtosecond pulsed 800 nm light from the core of a raster-scanned dual-clad fiber (DCF), which is focused by a miniaturized gradient-index lens assembly into tissue. Intrinsic fluorescence and second-harmonic generation signal from the tissue is epi-collected through the core and inner clad of the same DCF. The MPME has a rigid distal tip of 3 mm in outer diameter and 4 cm in length. The image field-of-view measures 115 μm by 115 μm and was acquired at 4.1 frames/s with 75 mW illumination power at the sample. Organs were imaged after anesthetizing Sprague-Dawley rats with isofluorane gas, accessing tissues via a ventral-midline abdominal incision, and isolating the organs with tongue depressors. In vivo multiphoton images acquired from liver, kidney, and colon using this device show features similar to that of conventional histology slides, without motion artifact, in ∼ 75% of imaged frames. To the best of our knowledge, this is the first demonstration of multiphoton imaging of unstained tissue from a live subject using a compact and flexible MPME device.
Limitations of current medical procedures for detecting early lung cancers inspire the need for new diagnostic imaging modalities for the direct microscopic visualization of lung nodules. Multiphoton microscopy (MPM) provides for subcellular resolution imaging of intrinsic fluorescence from unprocessed tissue with minimal optical attenuation and photodamage. We demonstrate that MPM detects morphological and spectral features of lung tissue and differentiates between normal, inflammatory and neoplastic lung. Ex vivo MPM imaging of intrinsic two-photon excited fluorescence was performed on mouse and canine neoplastic, inflammatory and tumor-free lung sites. Results showed that MPM detected microanatomical differences between tumor-free and neoplastic lung tissue similar to standard histopathology but without the need for tissue processing. Furthermore, inflammatory sites displayed a distinct red-shifted fluorescence compared to neoplasms in both mouse and canine lung, and adenocarcinomas displayed a less pronounced fluorescence emission in the 500 to 550 nm region compared to adenomas in mouse models of lung cancer. These spectral distinctions were also confirmed by two-photon excited fluorescence microspectroscopy. We demonstrate the feasibility of applying MPM imaging of intrinsic fluorescence for the differentiation of lung neoplasms, inflammatory and tumor-free lung, which motivates the application of multiphoton endoscopy for the in situ imaging of lung nodules.
Multiphoton microscopic endoscopy (MPM-E) is a promising medical in vivo diagnostic imaging technique because it
captures intrinsic fluorescence and second harmonic generation signals to reveal anatomical and histological information
about disease states in tissue. However, maximizing light collection from multiphoton endoscopes remains a challenge:
weak nonlinear emissions from endogenous structures, miniature optics, large imaging depths, and light scattering in
tissue all hamper light collection. The quantity of light that may be collected using a dual-clad fiber system from
scattering phantoms that mimic the properties of the in vivo environment is measured. In this experiment, 800nm
excitation light from a Ti:Sapphire laser is dispersion compensated and focused through a SM800 optical fiber and lens
system into the tissue phantom. Emission light from the phantom passes through the lens system, reflects off the dichroic
and is then collected by a second optical fiber actuated by a micromanipulator. The lateral position of the collection fiber
varies, measuring the distribution of emitted light 2000μm on either side of the focal point reimaged to the object plane.
This spatial collection measurement is performed at depths up to 200μm from the phantom surface. The tissue phantoms
are composed of a 15.8 μM fluorescein solution mixed with microspheres, approximating the scattering properties of
human bladder and dermis tissue. Results show that commercially available dual-clad optical fibers collect more than
47% of the total emission returning to the object plane from both phantoms. Based on these results, initial MPM-E
devices will image the surface of epithelial tissues.
Lung cancer is the leading killer among all cancers for both men and women in the US, and is associated with one of the
lowest 5-year survival rates. Current diagnostic techniques, such as histopathological assessment of tissue obtained by
computed tomography guided biopsies, have limited accuracy, especially for small lesions. Early diagnosis of lung
cancer can be improved by introducing a real-time, optical guidance method based on the in vivo application of
multiphoton microscopy (MPM). In particular, we hypothesize that MPM imaging of living lung tissue based on twophoton
excited intrinsic fluorescence and second harmonic generation can provide sufficient morphologic and
spectroscopic information to distinguish between normal and diseased lung tissue. Here, we used an experimental
approach based on MPM with multichannel fluorescence detection for initial discovery that MPM spectral imaging
could differentiate between normal and neoplastic lung in ex vivo samples from a murine model of lung cancer. Current
results indicate that MPM imaging can directly distinguish normal and neoplastic lung tissues based on their distinct
morphologies and fluorescence emission properties in non-processed lung tissue. Moreover, we found initial indication
that MPM imaging differentiates between normal alveolar tissue, inflammatory foci, and lung neoplasms. Our long-term
goal is to apply results from ex vivo lung specimens to aid in the development of multiphoton endoscopy for in vivo
imaging of lung abnormalities in various animal models, and ultimately for the diagnosis of human lung cancer.
At the time of diagnosis, approximately 75% of bladder cancers are non-muscle invasive. Appropriate diagnosis and
surgical resection at this stage improves prognosis dramatically. However, these lesions, being small and/or flat, are
often missed by conventional white-light cystoscopes. Furthermore, it is difficult to assess the surgical margin for
negativity using conventional cystoscopes. Resultantly, the recurrence rates in patients with early bladder cancer are very
high. This is currently addressed by repeat cystoscopies and biopsies, which can last throughout the life of a patient,
increasing cost and patient morbidity. Multiphoton endoscopes offer a potential solution, allowing real time, noninvasive
biopsies of the human bladder, as well as an up-close assessment of the resection margin. While miniaturization
of the Multiphoton microscope into an endoscopic format is currently in progress, we present results here indicating that
Multiphoton imaging (using a bench-top Multiphoton microscope) can indeed identify cancers in fresh, unfixed human
bladder biopsies. Multiphoton images are acquired in two channels: (1) broadband autofluorescence from cells, and (2)
second harmonic generation (SHG), mostly by tissue collagen. These images are then compared with gold standard
hematoxylin/eosin (H&E) stained histopathology slides from the same specimen. Based on a "training set" and a very
small "blinded set" of samples, we have found excellent correlation between the Multiphoton and histopathological
diagnoses. A larger blinded analysis by two independent uropathologists is currently in progress. We expect that the
conclusion of this phase will provide us with diagnostic accuracy estimates, as well as the degree of inter-observer
Water-soluble quantum dots (qdots) have been introduced as bright fluorophores into life sciences research. Although various photophysical pathologies of qdots have been found, how their biological applications will be affected --- particularly in the native biological environment --- has not been evaluated. By fluorescence coincidence analysis and fluorescence cross-correlation spectroscopy, we studied the dark fraction of free-diffusing qdots in aqueous solution. We were able to detect individual qdots and found significant heterogeneity --- well-distinguished dark qdots and bright qdots. We estimated the bright fraction of Qdot525-Streptavidin to be about 55%. Blinking events were also noticed in fluorescence coincidence analysis, with “on/off” timescale from submilliseconds to tens of milliseconds.
Gradient index lenses enable multiphoton microscopy of deep tissues in the intact animal. In order to assess their applicability to clinical research, we present in vivo multiphoton microscopy with gradient index lenses in brain regions associated with Alzheimer's disease and Parkinson's disease in both transgenic and wild-type mice. We also demonstrate microscopy of ovary in wild type mouse using only intrinsic fluorescence and second harmonic generation, signal sources which may prove useful for both the study and diagnosis of cancer.
The inherent advantages of nonlinear excitation make multiphoton fluorescence microscopy (MPFM) awell-suited imaging technique for extracting valuable information from turbid and thick biological samples. These advantages include high three-dimensional spatial resolution, large penetration depth, minimum out-of-focus cellular photodamage, and high signal-to-noise contrast. We have investigated the nonlinear spectroscopy of biologically important molecules such as NADH, flavins, and intrinsically fluorescent proteins. Fundamental understanding of the molecular spectroscopy and dynamics of these biomolecules is essential for advancing their applications in biological research. MPFM has been utilized for monitoring a large spectrum of biological processes including metabolic activity and exocytosis. We will discuss two-photon (2P) redox fluorescence microscopy of NADH, which gives a quantitative measure of the respiratory chain activity, thus allowing functional imaging of energy metabolism in neurons and native brain tissue. Finally, a rational design strategy, based on donor-acceptor-donor configuration, will be elucidated for fluorescent probes with large 2P-excitation cross-section. These dyes are water-soluble, yet possess a high affinity to organic phases with site-specific labeling and Ca<sup>+2</sup> sensitivity (K<sub>d</sub> ~ 350 nM). A brief account on the biological application of nanocrystals and second harmonic imaging will be reviewed.
Transgenic mice expressing the human Amyloid Precursor Protein (APP) develop amyloid plaques as they age. These plaques resemble those found in the human disease. Multiphoton laser scanning microscopy combined with a novel surgical approach was used to measure amyloid plaque dynamics chronically in the cortex of living transgenic mice. Thioflavine S (thioS) was used as a fluorescent marker of amyloid deposits. Multiphoton excitation allowed visualization of amyloid plaques up to 200 micrometers deep into the brain. The surgical site could be imaged repeatedly without overt damage to the tissue, and individual plaques within this volume could be reliably identified over periods of several days to several months. On average, plaque sizes remained constant over time, supporting a model of rapid deposition, followed by relative stability. Alternative reporters for in vivo histology include thiazine red, and FITC-labeled amyloid-(Beta) peptide. We also present examples of multi-color imaging using Hoechst dyes and FITC-labeled tomato lectin. These approaches allow us to observe cell nuclei or microglia simultaneously with amyloid-(Beta) deposits in vivo. Chronic imaging of a variety of reporters in these transgenic mice should provide insight into the dynamics of amyloid-(Beta) activity in the brain.
Nonlinear excitation of fluorophores through molecular absorption of two or three near infrared photons from the tightly focused femtosecond pulses of a mode-locked laser offers the cellular biologist an unprecedented panoply of biomolecular indicators for microscopic imaging and cellular analysis. Measurements of the two-photon excitation spectra of more than twenty ultra-violet and visible absorbing fluorophores from 690 to 1050 nm reveal useful cross sections for near infrared excitation, providing an artist's palette of emission markers and chemical indicators for living biological preparations. Measurements of three-photon fluorophore excitation spectra now define alternative windows of relatively benign wavelength to excite deeper UV fluorophores. The three-photon excitation spectrum of the amino acid tryptophan, measured 700-900 nm, delivers native fluorescence for imaging and assay of proteins and neurotransmitter sin living tissues. The inherent optical sectioning capabilities of focused nonlinear excitation provides 3D resolution for imaging and avoids out of focus background. Here, we describe the characteristics of the measured nonlinear excitation spectra and define the resulting features of nonlinear microscopy for biological imaging.
The development of the single beam gradient force optical trap has improved the experimental capabilities available to cell biologists for noninvasive micromanipulation and mechanical measurement on living cells. Laser traps can be used not only to optically manipulate particles including bacteria, yeast cells, and intracellular organelles ranging in size from 25 nm to 25 micrometers with fine control of position (10 nm) but also to measure small (0.1 pN) forces in biological systems. For a given particle, trapping forces are linearly related to the laser power so that a relatively simple way of measuring force is to trap a particle at high power and gradually reduce it until the particle just escapes from the trap. The `escape' power, which is usually calibrated against the viscous drag of the aqueous medium at varying laser power levels, is a measure of the force.
Multilayered refractive data storage allows serial optical recording at data densities exceeding 10<SUP>12</SUP>bits/cm<SUP>3</SUP>. Data may be recorded in a write once mode using two-photon excitation of a photopolymer film. Data is read with minimal cross-talk between layers by wavefront shearing interferometry, for example by differential interference contrast microscopy. The readout signal from such a multilayered refractive memory initially increases with the refractive index contrast. However, high index contrast may eventually lead to excessive scattering which could reduce signal quality. We report here an empirical method for quantitatively assessing the readout performance of multilayer refractive memory media using laser scanning differential interference contrast microscopy and simple image processing. Signal level distributions for a multilayered refractive write once data storage structure are presented.
Two-photon excitation in laser scanning photolithography allows exposure of patterns not possible with conventional one-photon direct writing. In our experiments two red photons from a highly focused subpicosecond colliding pulse mode-locked dye laser are simultaneously absorbed by initiator molecules to affect a photochemical reaction that is normally driven by single-photon absorption using ultraviolet light. The quadratic dependence of the two-photon absorption rate on the incident intensity confines excitation to a submicron focal volume. By scanning this volume in a 3-d pattern through a thick layer of photoresist it is possible to expose arbitrary three dimensionally defined regions. Preliminary results showing half micron wide trenches of very high aspect ratio, and resist structures with undercutting edges, all produced with only a single development step, demonstrate. the potential utility of two-photon excitation in microfabrication.
A new dimension in quantitative fluorescence microscopy may be accessed by imaging of fluorescence decay times. To obtain spatially resolved information from microscopic sample locations, one must not only have sufficient optical resolution and detection sensitivity but also the ability to exclude fluorescence photons originating from outside the focal volume of interest. This background rejection is measured by the signal-to-background ratio, which must be large if three-dimensional information is to be obtained from a thick fluorescence sample. Two-photon excitation in laser scanning microscopy has an unparalleled ability to meet these demands. The two-photon excitation of a transition normally in the ultraviolet arises from the simultaneous non-linear absorption of two red photons. Because two-photon excitation depends on the square of the incident intensity, the resulting fluorescence is limited to the focal volume where the photon density of the focused laser illumination is high. This localization limits photobleaching and any photodamage to the focal plane of the image. This property is a major advantage over widefield or confocal microscopy. Two-photon excitation provides the depth discrimination associated with confocal microscopy without a confocal spatial filter, an advantage which allows for major simplifications of the apparatus. The resolution and background rejection properties of two-photon excitation have been calculated and measured, and have been shown to be identical to an ideal confocal microscope with the same optical wavelengths. Combined, these properties provide the ideal conditions for time-resolved imaging of fluorescence decay dynamics in order to characterize the submicroscopic environment of the fluorophore molecules within the specimen. A preliminary apparatus was designed and built to test these concepts, and it was found that fluorescence decay time images of living cells can be conveniently recorded with diffraction limited resolution in a few seconds of image acquisition time.
Two-photon excitation in scanning laser fluorescence microscopy provides axial resolution and rejection of out of focus background similar to that provided by confocal microscopy. Two- photon excitation is allowed by the extremely high peak intensity (> 1010 W/cm2) at the waist of a highly focused beam of subpicosecond pulses from a modelocked laser. Chromophore excitation and photobleaching are restricted to the focal plane by quadratic dependence of the excitation rate on incident intensity. Sectional imaging is therefore possible without the need of a confocal aperture allowing the use of UV excited chromophores, including quantitative fluorescent indicators of divalent cations, which are problematic for confocal microscopy because of chromatic aberration of objective lenses. Furthermore, sectional imaging becomes possible using widefield viewing detectors such as CCD arrays or video camera. The confinement of two-photon excitation to the focal volume allows point localized photochemistry. Two-photon excited release of caged biological probes makes possible the observation of the living cell's response to chemical stimuli with the spatial resolution of optical microscopy. Two-photon excited laser scanning photolithography allows the generation of complicated 3-D forms with a single patterned exposure and one development step. Two-photon activation of fluorescent dyes allows 3-D optical data storage at extremely high density. Two-photon excited release of caged fluorescent probes allows micromobility measurements in 3-D bulk media. By photolytic release of fluorescent dye and subsequent observation of its redistribution it is possible to quantitatively study diffusional transport properties in polymers and living biological specimens.
Simultaneous absorption of two red photons from a strongly focused subpicosecond colliding pulse mode4ocked dye laser stimulates visible fluorescence emission from fluorophores having their normal absorption in the ultraviolet1. The quadratic increase of the two-photon excitation rate with excitation intensity restricts fluorescence emission to the focal volume thus providing the same depth resolution as does confocal microscopy. Image degradation due to out of focus backround is thus avoided. Photobleaching and most cellular photodamage are similarly confined to the focus thereby minimizing sample degredation during acquisition of the multiple sections required for 3-d image reconstruction. Fluorescence images of living cells and other thick photolabile fluorescence labled assemblies illustrate the depth discrimination of both two-photon fluorescence excitation and photobleaching. The quadratic intensity dependence of two-photon excitation allows 3-d spatially resolved photochemistry in particular the photolytic release of caged compounds such as neurotransmitters nucleotides fluorescent dyes and second messengers such as 1P3 and Ca. The two-photon release of cased ATP has been measured and release of a caged fluorescent dye has been shown. Point photobleaching and a 3-d " write once read many" optical memory have been demonstrated. Two-photon excitation of photo-initiated polymerization with a sharply focused single beam allows microfabrication of complex structures of arbitrary form. By scanning the focused beam through a liquid polymer with a UV excited initiator it is possible to harden the polymer only at the focus thereby creating