A compact, prism-based spectrograph was designed for 2-photon light sheet microscopy based on broadband, ultrashort optical pulses that corrects for chromatic aberrations and distortion. Gaussian or Bessel beams of broadband optical pulses may be used to create narrow cylindrical, nonlinear excitation volumes from which fluorophores emit characteristic spectra. In practice, a slit aperture is often used to optically section the excitation volume before being imaged onto the camera chip. Optical dispersion of the fluorescence in the transverse direction of the imaging slit allows for hyperspectral image acquisition. Hyperspectral imaging systems may be used to simultaneously image and segment multiple fluorescent reporters in biological tissue. However, current systems have properties which are undesirable for low-light microscopy including chromatic aberrations, distortion, low optical transmission, and large footprint which consumes precious laboratory real estate. Here, we present a novel spectrograph that has sufficient optical transmission, achromaticity, and distortion correction for microscopy of fluorescent reporters spanning the visible spectrum (400 – 650 nm). Across the spatial dimension of the excitation volume, the spectrograph has uniform separation of the spectral bands while maintaining a compact size and profile. A second order deconvolution algorithm is used to spectrally deconvolve overlapping fluorophores.
During embryogenesis, presumptive brain compartments are patterned by dynamic networks of gene expression. The spatiotemporal dynamics of these networks, however, have not been characterized with sufficient resolution for us to understand the regulatory logic resulting in morphogenetic cellular behaviors that give the brain its shape. We have developed a new, integrated approach using ultrashort pulse microscopy [a high-resolution, two-photon fluorescence (2PF)-optical coherence microscopy (OCM) platform using 10-fs pulses] and image registration to study brain patterning and morphogenesis in zebrafish embryos. As a demonstration, we used time-lapse 2PF to capture midbrain-hindbrain boundary morphogenesis and a wnt1 lineage map from embryos during brain segmentation. We then performed in situ hybridization to deposit NBT/BCIP, where wnt1 remained actively expressed, and reimaged the embryos with combined 2PF-OCM. When we merged these datasets using morphological landmark registration, we found that the mechanism of boundary formation differs along the dorsoventral axis. Dorsally, boundary sharpening is dominated by changes in gene expression, while ventrally, sharpening may be accomplished by lineage sorting. We conclude that the integrated visualization of lineage reporter and gene expression domains simultaneously with brain morphology will be useful for understanding how changes in gene expression give rise to proper brain compartmentalization and structure.
Multicomponent tissue models are viable tools to better understand cell responses in complex environments, but present challenges when investigated with live cell microscopy noninvasively. In this study, integrated nonlinear optical microscopy-optical coherence microscopy (NLOM-OCM) was used to characterize cell interactions within three-dimensional (3-D), multicomponent extracellular matrices. In fibrin-collagen mixtures, 3T3 fibroblasts were observed to recruit both fibrin and collagen fibers while remodeling matrices. Also, NLOM-OCM was used to observe collagen deposition by neonatal human dermal fibroblasts within originally fibrin matrices over an extended time. It was observed that preferentially aligned collagen deposition could be achieved with aligned fibroblasts but that cell alignment could be achieved without aligning the extant extracellular matrix. In summary, this multimodel imaging system has potential for both real-time and longitudinal imaging of living 3-D cultures, which is particularly important for evaluating cell microenvironments in composite scaffolds or serial characterization of engineered tissue constructs during culture.
We report the application of ultrashort pulse microscopy (UPM) for integrated imaging of embryonic development at the tissue, cell, and molecular length scales. The UPM is a multimodal imaging platform that utilizes the broad-power spectrum and high-peak power of 10-fs pulses to render two-photon excited signals and the short coherence gate of such pulses to render optical coherence signals. We show that ultrashort pulses efficiently excite cellular autofluorescence in developing zebrafish embryos such that tissues are readily visualized and individual cells can be monitored, providing a potential method for label-free cell tracking. We also show the ability of ultrashort pulses, without tuning, to excite a broad spectrum of fluorescent protein variants for tracking genetically labeled cell lineages in live embryos, with no apparent damage to the embryos. Molecular information at the mRNA transcript level can also be obtained from embryos that have been stained to reveal the localization of the expression of a gene using NBT/BCIP, which we show can be detected with three-dimensional resolution using a combination of two-photon and optical coherence signals. From this demonstration, we conclude that UPM is an efficient and a powerful tool for elucidating the dynamic multiparameter and multiscale mechanisms of embryonic development.
We have developed a combined NLOM-OCM method using ultrashort sub-10-fs pulses to study cell lineages and their
gene expression profiles in zebrafish. First, time-lapse NLOM is used to capture embryo morphology (broadly excited
autofluorescence) and cell lineage dynamics (eGFP reporter). The embryo is then fixed and an in situ hybridization performed,
depositing NBT/BCIP precipitate where a gene of interest is actively expressed. Combined NLOM-OCM is then
used to capture the gene expression pattern with 3-D resolution and these two data sets acquired from the same embryo
are merged using morphological landmarks. We have used this approach to study the dynamics of the wnt1 lineage at the
midbrain-hindbrain boundary (MHB) in normal and in fgf8a(ace) morphant embryos. We show that with fgf8a knock-down,
the MHB constriction begins to form but subsequent failure of the constriction causes the incorporation of a transient
cerebellar structure into caudal tectum. Concomitantly, this morphological distortion in the dorsal MHB causes
anterior displacement in a ventral subpopulation of the wnt1 lineage at the MHB. NLOM-OCM confirms the displaced
wnt1 MHB lineage stops expressing the wnt1 reporter, and with further experiments we can investigate markers such as
wnt4 or ascl1a, which have been shown to be expanded caudally in ace mutants, to understand the transformed molecular
fate of this displaced tissue. We conclude this approach of co-registering dynamic lineage tracing and in situ hybridization
data sets using morphological context will help shed light on developmental mechanisms by integrating established
analysis techniques at the morphological, cellular, and molecular levels.
KEYWORDS: Microscopy, Collagen, Ultrafast phenomena, 3D image processing, Time lapse microscopy, 3D modeling, Environmental sensing, Tissues, Binary data, Animal model studies
The mechanisms that enable viruses to harness cellular machinery for their own survival are primarily studied in cell lines cultured in two-dimensional (2-D) environments. However, there are increasing reports of biological differences between cells cultured in 2-D versus three-dimensional (3-D) environments. Here we report differences in host-virus interactions based on differences in culture environment. Using ultrashort pulse microscopy (UPM), a form of two-photon microscopy that utilizes sub-10-fs pulses to efficiently excite fluorophores, we have shown that de novo development of extra-chromosomal virus replication compartments (VRCs) upon murine cytomegalovirus (mCMV) infection is markedly enhanced when host cells are cultured in 3-D collagen gels versus 2-D monolayers. In addition, time-lapse imaging revealed that mCMV-induced VRCs have the capacity to grow by coalescence. This work supports the future potential of 3-D culture as a useful bridge between traditional monolayer cultures and animal models to study host-virus interactions in a more physiologically relevant environment for the development of effective anti-viral therapeutics. These advances will require broader adoption of modalities, such as UPM, to image deep within scattering tissues.
The inverse relationship between two-photon excited fluorescence (TPEF) and laser pulse duration suggests that two-photon microscopy (TPM) performance may be improved by decreasing pulse duration. However, for ultrashort pulses of sub-10 femtosecond (fs) in duration, its spectrum contains the effective gain bandwidth of Ti:Sapphire and its central wavelength is no longer tunable. An experimental study was performed to explore this apparent tradeoff between untuned sub-10 fs transform-limited pulse (TLP) and tunable 140 fs pulse for TPEF. Enhancement factors of 1.6, 6.7, and 5.2 are measured for Indo-1, FITC, and TRITC excited by sub-10 fs TLP compared with 140 fs pulse tuned to the two-photon excitation (TPE) maxima at 730 nm, 800 nm, and 840 nm, respectively. Both degenerate (v1 = v2) and nondegenerate (v1 ≠ v2) mixing of sub-10 fs TLP spectral components result in its broad second-harmonic (SH) power spectrum and high spectral density, which can effectively compensate for the lack of central wavelength tuning and lead to large overlap with dye TPE spectra for TPEF enhancements. These pulse properties were also exploited for demonstrating its potential applications in multicolor imaging with TPM.
Molecular interactions of optical clearing agents were investigated using a combination of molecular dynamics (MD) simulations and optical spectroscopy. For a series of sugar alcohols with low to high optical clearing potential, Raman spectroscopy and integrating sphere measurements were used to quantitatively characterize tissue water loss and reduction in light scattering following agent exposures. The rate of tissue water loss was found to correlate with agent optical clearing potential, but equivalent tissue optical clearing was measured in native and fixed tissue in vitro, given long-enough exposure times to the polyol series. MD simulations showed that the rate of tissue optical clearing correlated with the preferential formation of hydrogen bond bridges between agent and collagen. Hydrogen bond bridge formation disrupts the collagen hydration layer and facilitates replacement by a chemical agent to homogenize tissue refractive index. However, the reduction in tissue light scattering did not correlate with the agent index of refraction. Our results suggest that a necessary property of optical clearing agents is hyperosmolarity to tissue, but that the most effective agents with the highest rates of optical clearing are a subset with the highest collagen solubilities.
Two-photon-excited fluorescence and second-harmonic generation are characterized as a function of laser pulse duration as short as sub-10-fs. A comparative study is performed where pulse duration is varied by introducing dispersion, as reported previously, and by tailoring pulse spectral width and minimizing its time-bandwidth product (transform-limited pulses). Experimental data and calculations show that characterizing a two-photon signal with the two schemes to vary pulse duration measures different phenomena. Two-photon signal characterization using dispersion-broadened pulses measures only the effect of chirp on the pulse two-photon-excitation spectrum and is independent of molecular response. Transform-limited pulses are used to measure the dependence of two-photon signal generation on pulse duration. Calculations show that deviation from the 1/Tp relationship would be expected as the transform-limited pulse spectral width approaches the molecular two-photon absorption linewidth and exhibits asymptotic behavior for pulse spectral widths 10 times greater than the absorption linewidth. Experimental measurements are consistent with the predicted behavior. The impact of using ultrashort laser pulses on the performance characteristics of nonlinear optical microscopy is discussed.
Broadband, sub-10-fs pulses, can be propagated through polarization maintaining
silica core single mode fibers for use in NLOM. In a manner similar to all fiber chirped
pulse amplifiers, we stretch the pulse sufficiently, ~-4000 fs2 post fiber residual chirp,
such that nonlinearity is minimized owing to the significant impact of dispersion on pulse
width. The optics of the imaging system provide the remaining positive dispersion
delivering a near transform limited, 12.7 fs pulse, to the specimen plane. We are able to
achieve average powers up to 75 mW from the fiber with minimal changes in spectra at a
fiber length of 400 mm. Image intensity analysis of identical images taken with and
without the fiber indicates that the fiber based system is capable of generating signals that
are within a factor of two of our traditional NLOM. Autocorrelations and pulse spectra
are also presented following propagation through the fiber and imaging system.
Biomedical optics and photomedicine applications are challenged by the turbidity of most biological tissue systems. Nonreactive, biocompatible chemical agents can induce a reversible reduction in optical scattering of collagenous tissues such as human skin. Herein we show that a chemical agent's tissue optical clearing potential is directly related to its collagen solubility, providing a rational design basis for effective, percutaneous formulations.
This study reports on current work involving the use of Surface Enhanced Raman Spectroscopy (SERS) for the intracellular detection of cell constituents in mouse fibroblast cells using gold nanoshells. Gold nanoshells were acquired from Nanospectra Biosciences that are based on a silica dielectric core and an outer gold shell layer. They
have the unique property of a tunable surface plasmon resonance wavelength from the visible through the near infrared which allows control of the electromagnetic field strength on its surface. Hence gold nanoshells can serve as SERS substrates with plasmonic properties that are not aggregation dependent and thus can be expected to overcome the reproducibility problem that is generally associated with aggregation based colloidal metal nanoparticles. These results represent the first steps in the development of a nanoshell-based SERS probe to detect cell organelles and/or intracellular biochemicals with the goal of ultimately improving the ability to monitor intracellular biological processes in real time.
Reduction of optical scattering in turbid biological tissues using nonreactive chemical agents has potential applications for light-based diagnostics and therapeutics. Optical clearing effects by exogenous chemical agents, in particular sugars and sugar alcohols, have been found to be temporary with tissue rehydration. Applications with dermatologic laser therapies are now being investigated, but suffer from the inability of studied agents to penetrate the superficial layers of human skin. Selection, design, and refinement of topically effective chemical agents are hindered by a lack of fundamental understanding of tissue clearing mechanisms. We present recent work, particularly from the biochemistry community, detailing molecular interactions between chemical agents and collagen. This body of work demonstrates the perturbative effects of sugars and sugar alcohols on collagen high-order structures at micro- and nanometer length scales by screening noncovalent bonding forces. In addition, these studies emphasize the nonreactive nature of agent-collagen interactions and the ability of noncovalent bonding forces to recover with agent removal and drive reassembly of destabilized collagen structures. A mechanism of tissue optical clearing is proposed based on agent destabilization of high-order collagen structures.
Wound healing is a physiologic process that acts to repair disruptions in the continuity of tissue caused by injury or surgical incision. Keloids and hypertrophic scars are forms of aberrant wound healing, which are characterized by the overproduction of collagen, resulting in an excessive amount of scar tissue. Keloid tumors, by definition, grow outside the boundary of the original tissue damage. Multiphoton microscopy (MPM) is an imaging technique which allows imaging of living specimens, without the use of fixation or stains. Images of collagen fibers are produced by the second harmonic signal intensity generated by endogenous fluorescence through excitation by infrared laser light. A postauricular keloid tumor was excised from a patient. The tissue was dissected, and a portion was imaged using MPM. Normal skin tissue was isolated from a patient undergoing a facelift. A portion of this tissue was also dissected and imaged using MPM. MPM images were taken using a 63X water immersion objective lens on a two-photon microscope and a titanium-sapphire laser. Images were taken beginning at the surface of the tissue and moving in at intervals of 200 nm to a final depth of 30 μm. The two-photon images were used to reconstruct three-dimensional representations of the collagen matrix within the tissues, which are readily contrasted. Density of the collagen within each tissue was also ascertained using depth dependant decay of the image intensity. Multiphoton imaging was successfully used to image the collagen matrix of normal skin and a keloid scar, demonstrating differences in their microstructures.
A broad range of excitation wavelengths (730-880nm) was used to demonstrate the co-registration of two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) in unstained turbid tissues in reflection geometry. The composite TPEF/SHG microscopic technique was applied to imaging an organotypic tissue model (RAFT). The origin of the image-forming signal from the various RAFT constituents was determined by spectral measurements. It was shown that at shorter excitation wavelengths the signal emitted from the extracellular matrix (ECM) is a combination of SHG and TPEF from collagen, whereas at longer excitation wavelengths the ECM signal is exclusively due to SHG. The cellular signal is due to TPEF at all excitation wavelengths. The reflected SHG intensity followed a quadratic dependence on the excitation power and exhibited a spectral dependence in accordance with previous theoretical studies. Understanding the structural origin of signal provided a stratagem for enhancing contrast between cellular structures, and components of the extracellular matrix. The use of SHG and TPEF in combination provides complementary information that allows non-invasive, spatially localized in vivo characterization of cell-ECM interactions and pathology.
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