SignificanceFluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples.AimWe aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM.ApproachA single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells.ResultsAn NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a 30 × to 6 × acquisition speed advantage for the light sheet compared to the laser scanning geometry.ConclusionsFLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.
SPAD array sensors support higher-throughput fluorescence lifetime imaging microscopy (FLIM) by transitioning from laser-scanning to wide-field geometries. While a SPAD camera in epi-fluorescence geometry enables wide-field FLIM of fluorescently labeled samples, label-free imaging of single-cell autofluorescence is not feasible in an epi-fluorescence geometry because background fluorescence from out-of-focus culture medium masks cell autofluorescence and biases lifetime measurements. Here, we address this problem in a proof-of-concept implementation by integrating the SPAD camera in a light-sheet illumination geometry to achieve optical sectioning and limit out-of-focus contributions, enabling label-free wide-field FLIM of single-cell NAD(P)H autofluorescence.
Label-free methods provide image contrast without the need to introduce exogenous material that may alter the sample microstructure, perturb the microenvironment, or limit clinical translation. In addition to being label-free, it is increasingly important to consider modalities that work in reflection mode geometry to allow imaging intact tissue (in vivo or ex vivo) or 3D cell cultures too thick to image in transmission. However, reflection geometry precludes many useful contrast modalities, including transmitted phase and darkfield. Optical scattering in tissue limits contrast and depth of imaging, but scattering can also be exploited to quantify tissue structure and provide diagnostic markers. Here, we present another application of multiple scattering in tissue in which collection of scattered light is used to reconstruct images with contrast comparable to transmission imaging modalities.
A Scattered Light Contrast (SLC) microscope collects a descanned image of the scattered light surrounding the confocal illumination point. The illumination point is raster scanned and the scattered light distribution is collected for each image pixel, providing 4D data. By analyzing the scattered light, a range of image contrast modalities can be reconstructed including reflectance confocal microscopy, transmitted phase contrast, and dark field microscopy. Computational Monte Carlo simulations relate the SLC signal to the phase gradient in the image plane and can be used to reconstruct the phase gradient of transmitted light in the focal plane. This modality enables label-free imaging of cells and structures in the retina that will advance diagnosis and monitoring therapies at the cellular level.
Optical scattering properties are important diagnostic indicators with sensitivity to sub-resolution tissue structure as well as being necessary parameters for modeling light transport. Improved understanding of scattering in tissue is essential for optimizing and interpreting image contrast, modeling optogenetics, and developing next-generation optical imaging approaches. Despite the importance of optical scattering properties, most measurement methods rely on approximations or assumptions about the shape of the angular distribution of scattered light (phase function) or lack the spatial resolution to characterize heterogeneous tissue. There is a need for a spatially resolved method to quantify the optical scattering properties including the shape of the phase function.
This work presents a lens-based spectral goniometry system and spatially resolved measurement of 4pi optical scattering phase functions. Angle-space measurement of scattering is performed by imaging the Fourier plane of a high-NA microscope objective. By combining forward and backward images and varying the illumination beam angle, the entire 4pi phase function can be acquired. This method enables several capabilities: a) Spatially resolved measurement of properties combined with stage scanning provides mapping of layered or heterogeneous tissues with <100 micron sampling. b) By inverting the angular scattering measurements, this approach allows characterization of refractive index autocorrelation. c) As a camera and lens based measurement technique that collects large solid-angles of scattering in a single image, the non-axially symmetric scattering signature of fibrous or oriented tissue can be characterized. These applications as well as instrument design and analysis methodology will be presented.
Tissue optical properties have become an increasingly promising avenue of diagnosis and screening for cancers and may provide contrast for real time monitoring of tumor ablation therapy. Of particular interest, are methods that can quantify scattering properties while providing spatial context or a map of the tissue being measured. Optical Coherence Tomography (OCT) is a non-destructive imaging modality with high 3D resolution which can be miniaturized into a probe compatible with common endoscopes. OCT has recently been used to quantify optical scattering properties, and endoscopic access to luminal organs allows examination of the thin epithelial layer, wherein many cancers originate.
We present a fiber probe capable of quantifying optical properties with no distal optics providing low-cost disposable functionality. A reflection from the distal fiber face provides a common path reference through the fiber and eliminates the need for reference arm with dispersion compensation. A custom visible light OCT instrument was adapted to the self-reference fiber capable of a-scan imaging hundreds of microns into porcine esophagus tissue. B-scan images are produced by dragging the fiber along the tissue surface. Tissue was thermally ablated to create controllable scattering contrast with normal tissue. Image analysis with a custom MATLAB algorithm demonstrated significant increases in scattering coefficients which has been observed previously in a benchtop scanning OCT system.
Visible light maximizes scatter contrast making Vis-OCT an ideal tool for cancer screening. Additionally, the lack of a distal optics and scanning mechanism offers a cost-effective, disposable functionality.
Optical scattering properties in tissue are diagnostic markers, ingredients to increasingly sophisticated models, and undergird many optical imaging modalities. Advances in all these areas hinge on obtaining high fidelity scattering measurements. A new optical scattering goniometry method is discussed which measures scattering into 4π sr from small (~100um) tissue regions in flat-mounted samples.
This novel tissue scattering gonoimeter images the back focal plane of two opposing microscope objectives to collect light in the forward and backward direction and has several key advantages: (1) scanning the incident angle allows measurement of scattering over 4π steradians to determine the complete scattering phase function of tissue, (2) specificity of measuring scattering from small ~50um regions combined with obliquely sectioned tissue allows mapping of layered tissue, (3) spectral characterization through tuning illumination wavelength, (4) concurrent measurement of scattering coefficient. This opens up the prospect of a new level of detail in the characterization of optical scattering from tissue, including distinguishing properties of thin layers.
A tissue system of particular interest and an excellent candidate on which to apply this new goniometry method is the retina. Existing measurements are limited to bulk retina properties or inferred from methodologies that do not have access to transmitted scattering. Scattering coefficient and anisotropy measurements are presented for the various retinal layers. These novel measurements may be used to model light transport and scattering in the retina. Examples of modeling imaging modalities based on scattered light are discussed.
Numerous methods have been developed to quantify the light scattering properties of tissue. These properties are of interest in diagnostic and screening applications due to sensitivity to changes in tissue ultrastructure and changes associated with disease such as cancer. Tissue is considered a weak scatterer because that the mean free path is much larger than the correlation length. When this is the case, all scattering properties can be calculated from the refractive index correlation function Bn(r).
Direct measurement of Bn(r) is challenging because it requires refractive index measurement at high resolution over a large tissue volume. Instead, a model is usually assumed. One particularly useful model, the Whittle-Matern function includes several realistic function types such as mass fractal and exponential. Optical scattering properties for weakly scattering media can be determined analytically from Bn(r) by applying the Rayleigh-Gans-Debye (RGD) or Born Approximation, and so measured scattering properties are used to fit parameters of the model function. Direct measurement of Bn(r) would provide confirmation that the function is a good representation of tissue or help in identifying the length scale at which changes occur.
The RGD approximation relates the scattering phase function to the refractive index correlation function through a Fourier transform. This can be inverted without approximation, so goniometric measurement of the scattering can be converted to Bn(r). However, geometric constraints of the measurement of the phase function, angular resolution, and wavelength result in a band limited measurement of Bn(r). These limits are discussed and example measurements are described.
Optical characterization of biological tissue in field carcinogenesis offers a method with which to study the mechanisms behind early cancer development and the potential to perform clinical diagnosis. Previously, low-coherence enhanced backscattering spectroscopy (LEBS) has demonstrated the ability to discriminate between normal and diseased organs based on measurements of histologically normal-appearing tissue in the field of colorectal (CRC) and pancreatic (PC) cancers. Here, we implement the more comprehensive enhanced backscattering (EBS) spectroscopy to better understand the structural and optical changes which lead to the previous findings. EBS provides high-resolution measurement of the spatial reflectance profile P(r s ) between 30 microns and 2.7 mm, where information about nanoscale mass density fluctuations in the mucosa can be quantified. A demonstration of the length-scales at which P(r s ) is optimally altered in CRC and PC field carcinogenesis is given and subsequently these changes are related to the tissue’s structural composition. Three main conclusions are made. First, the most significant changes in P(r s ) occur at short length-scales corresponding to the superficial mucosal layer. Second, these changes are predominantly attributable to a reduction in the presence of subdiffractional structures. Third, similar trends are seen for both cancer types, suggesting a common progression of structural alterations in each.
We present an open source electric field tracking Monte Carlo program to model backscattering in biological media containing birefringence, with computation of the coherent backscattering phenomenon as an example. These simulations enable the modeling of tissue scattering as a statistically homogeneous continuous random media under the Whittle-Matérn model, which includes the Henyey-Greenstein phase function as a special case, or as a composition of discrete spherical scatterers under Mie theory. The calculation of the amplitude scattering matrix for the above two cases as well as the implementation of birefringence using the Jones N-matrix formalism is presented. For ease of operator use and data processing, our simulation incorporates a graphical user interface written in MATLAB to interact with the underlying C code. Additionally, an increase in computational speed is achieved through implementation of message passing interface and the semi-analytical approach. Finally, we provide demonstrations of the results of our simulation for purely scattering media and scattering media containing linear birefringence.
Low-coherence enhanced backscattering (LEBS) is a depth selective technique that allows noninvasive characterization of turbid media such as biological tissue. LEBS provides a spectral measurement of the tissue reflectance distribution as a function of distance between incident and reflected ray pairs through the use of partial spatial coherence broadband illumination. We present LEBS as a new depth-selective technique to measure optical properties of tissue in situ. Because LEBS enables measurements of reflectance due to initial scattering events, LEBS is sensitive to the shape of the phase function in addition to the reduced scattering coefficient (μs*). We introduce a simulation of LEBS that implements a two parameter phase function based on the Whittle-Matérn refractive index correlation function model. We show that the LEBS enhancement factor (E) primarily depends on μs*, the normalized spectral dependence of E (Sn) depends on one of the two parameters of the phase function that also defines the functional type of the refractive index correlation function (m), and the LEBS peak width depends on both the anisotropy factor (g) and m. Three inverse models for calculating these optical properties are described and the calculations are validated with an experimental measurement from a tissue phantom.
A comprehensive three-parameter statistical model is presented for the refractive index fluctuations in continuous
homogeneous random media, and the light-scattering properties of these media are investigated in the Born
(or single-scattering) approximation. Because biological media are usually weakly scattering, the results are
applicable to many biomedical light-scattering problems. A rigorous error analysis is presented for the scattering
coefficient under the Born approximation in a biologically-relevant, albeit more simplified geometry. The finitedifference-
time-domain (FDTD) computational electromagnetic analysis is used to obtain the exact solutions for this error analysis. The ranges for the correlation length and the refractive index fluctuation strength under which Born approximation is valid are clearly identified.
There is always a tradeoff between resolution and Field of View (FOV) in an imaging system. This limit can be
due to the number of pixels in the detector, however a fundamental limit also exists in any optical system called
the Space Bandwidth Product (SBP) which scales as the FOV area divided by the area of the diffraction limited
spot. The SBP can only be increased by increasing the size of the optical system. In applications where the size
of the optical system is constrained such as endoscopes, the SBC will ultimately limit the resolution or FOV.
However, there is a way to provide both high resolution and a wide FOV without changing the total number of
pixels in the image. The technique is called foveated imaging because is mimics this characteristic of the human
eye in which the fovea has a higher resolution at the center of the FOV than the surrounding retina. A similar
effect can be achieved optically by introducing a large amount of barrel distortion in the lens design. The result
is an effective increase in the magnification at the center of the FOV, and reduced resolution but larger angular
sampling at the edge. The stretching effect of the distortion can be compensated for computationally to provide
an onscreen display that is not distorted, but merely appears blurred at the edges. Such an objective will enable
for endomicroscopy while still providing "peripheral vision" to allow endoscopists to navigate and locate regions
of interest.
Noninvasive and real-time analysis of tissue properties, in particular, the quantitative assessment of blood content and
light scattering properties of mucosa is useful in a wide variety of applications. However, the nature of interactions
between contact fiber-optic probes and the tissue surface presents a challenging problem with respect to the variability of
in vivo measurements, for example affects due to variations in the pressure and angle of the probe tip on the tissue
surface. Previously, pressure and angle effects have been investigated for other modalities (i.e. diffuse reflectance and
Raman spectroscopy). We present an evaluation of this variability, as well as the length of time in contact with tissue for
polarization-gated spectroscopy. The evaluation is based on the quantification of mucosal blood content at superficial
depths (within 100 to 200 microns of tissue surface) for in vivo measurements of oral mucosa. Measurements are
presented for different pressures, angles and time scales and the variability due to these factors is assessed.
An integrated miniature multi-modal microscope (4M device) for microendoscopy was built and tested. Imaging performance is evaluated and imaging results are presented for both fluorescence and reflectance samples. Images of biological samples show successful imaging of both thin layers of fixed cells prepared on a slide as well as thick samples of excised fixed porcine epithelial tissue, thus demonstrating the potential for in vivo use.
The multi-modal miniature microscope (4M) device for early cancer detection is based on micro-optical table (MOT) platform which accommodates on a chip: optical, micro-mechanical, and electronic components. The MOT is a zeroalignment optical-system concept developed for a wide variety of opto-mechanical instruments. In practical terms this concept translates into assembly errors that are smaller than the tolerances on the performance of the optical system. This paper discusses all major system elements: optical system, custom high speed CMOS detector and comb drive actuator. It also points to mutual relations between different technologies. The hybrid sol-gel lenses, their fabrication and assembling techniques, optical system parameters, and various operation modes are also discussed. A particularly interesting mode is a structured illumination technique that delivers confocal-imaging capabilities and may be used for optical sectioning. Structured illumination is produced with LIGA fabricated actuator scanning in resonance and reconstructed using sine approximation algorithm.
The multi-modal miniature microscope (4M) device to image morphology and cytochemistry in vivo is a microscope on a chip including optical, micro-mechanical, and electronic components. This paper describes all major system components: optical system, custom high speed CMOS detector and comb drive actuator. The hybrid sol-gel lenses, their fabrication and assembling technology, optical system parameters, and various operation modes (fluorescence, reflectance, structured illumination) are also discussed. A particularly interesting method is a structured illumination technique that delivers confocal-imaging capabilities and may be used for optical sectioning. For reconstruction of the sectioned layer a sine approximation algorithm is applied. Structured illumination is produced with LIGA fabricated actuator scanning in resonance. The spatial resolution of the system is 1 μm, and was magnified by 4x matching the CMOS pixel size of 4 μm (a lateral magnification is 4:1), and the extent of field of the system is 250μm. An overview of the 4M device is combined with the presentation of imaging results for epithelial cell phantoms with optical properties characteristic of normal and cancerous tissue labeled with nanoparticles.
This paper discusses the selection of parameters and the design of a CMOS detector for use in a structured illumination technique implemented with a 4M device (Miniaturized Multi-Modal Microscope) for precancer detection. To obtain real time sectioning the framing rate was set on the order of 500 frames / sec. 500 images allow us to obtain 8 to 16 sections / sec reconstructed from 16 to 64 images. The reconstruction technique is a sine approximation algorithm. To obtain a 1 μm spatial resolution, the required pixel size is 4 μm with a magnification 4:1. The field of extent of 250um required approximately 350 x 350 array size.
We are developing a multi-modal miniature microscope (4M device) for imaging morphology and cytochemistry in vivo and providing better delineation of tumors. The 4M device is designed to be a complete microscope on a chip, including optical, micro-mechanical, and electronic components. It has advantages such as compact size and capability for microscopic-scale imaging. This paper presents the recent imaging experiment of 4M device including trans-illumination imaging, TIR illumination imaging and fluorescent imsging. We built a multi-modal imaging test-bed to demonstrate multi-modality of 4M device. In this paper, we present imaging experiment results by implementing various imaging modality with cervical cancer cells. In order to enhance image contrast, some imaging modality uses cells attached with contrast agency such as silver nano-particles. Imaging results indicate that the 4M prototype can resolve cellular detail necessary for detection of precancer.
Most conventional imaging systems suffer from unwanted and unexpected stray light that is often caused by reflections and scattering from optics and opto-mechanical features. This problem is easily missed during a design procedure that concentrates on improvement of imaging performance. The problem becomes apparent at the final step of production in most cases. If an imaging system consists of micro-optics, a stray light problem may become more difficult to solve due to the system's micro-scale size.
The purpose of this stray light analysis is to improve imaging performance of the multi-modal miniature microscope (4M). The 4M device is a complete microscope on a chip, including optical, micro-mechanical, and electronic components. The 4M device is potentially a useful tool for early detection of pre-cancer due to its very compact size and capability for microscopic-scale imaging. Before actual fabrication of this device, however, we built the same geometry as the real 4M device in a commercial non-sequential ray tracing code and implemented stray light analysis of 4M device.
Our findings indicate that most of the stray light in a 4M device is created by reflection from optics that are nominally supposed to be transparent. Due to a low signal level associated with the object, it is required to add high quality anti-reflection coatings on optics to achieve reasonable SNR.
Hybrid glass materials are used in the photolithographic fabrication of optical and opto-mechanical structures. Two different methods are introduced. The first one is referred as photolithographic patterning and the other as direct photolithographic deforming of hybrid glass materials. Fabrication of isolated lenslets, lens arrays, gratings and other binary structures is presented. The hybrid glass material used in the photolithographic patterning features a maximum spectral extinction coefficient of 2.0 X 10-4 micrometers -1 between 450 nm and 1,600 nm and a refractive index of 1.53 at 632.8 nm. The fabricated structures feature large convex lens sags (up to 100 microns) with rms surface roughness values ranging from 10 to 45 nm, when the photolithographic patterning is applied. The hybrid glass material used in the direct photolithographic deforming exhibits a maximum spectral extinction coefficient of 1.6 X 10-3 micrometers -1 at wavelengths ranging from 450 nm to 2200 nm and a refractive index of 1.52 at 632.8 nm. The fabricated structures exhibit rms surface roughness between 1 and 5 nm, when direct photolithographic deforming is applied. These materials and methods are highly promising for micro- optics fabrication.
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