SignificanceThe reprojection setup typical of oblique plane microscopy (OPM) limits the effective aperture of the imaging system, and therefore its efficiency and resolution. Large aperture system is only possible through the use of custom specialized optics. A full-aperture OPM made with off the shelf components would both improve the performance of the method and encourage its widespread adoption.AimTo prove the feasibility of an OPM without a conventional reprojection setup, retaining the full aperture of the primary objective employed.ApproachA deformable lens based remote focusing setup synchronized with the rolling shutter of a complementary metal-oxide semiconductor detector is used instead of a traditional reprojection system.ResultsThe system was tested on microbeads, prepared slides, and zebrafish embryos. Resolution and pixel throughput were superior to conventional OPM with cropped apertures, and comparable with OPM implementations with custom made optical components.ConclusionsAn easily reproducible approach to OPM imaging is presented, eliminating the conventional reprojection setup and exploiting the full aperture of the employed objective.
Multispectral fluorescence lifetime imaging microscopy (λFLIM) is a powerful optical technique to investigate biological processes, which generally requires long acquisition time. Single Pixel Camera (SPC) is an imaging architecture base on Compressive Sensing (CS) techniques which allows to strongly reduce the acquisition time while preserving the information content at the cost of an increased computational time. In this work we present a λFLIM microscope based on CS-SPC architecture. We have tested the multiscale capability of the system by merging SPC zooming with data fusion and proposed a fast fitting framework, which runs in parallel with the acquisition, allowing a fast visualization.
In this novel multimodal wide-field Raman microscope, spectra are obtained by the time-domain Fourier-transform method. The wide-field approach enables faster collection of Raman maps, while the time-domain method disentangles fluorescence and Raman signals. This is obtained by choosing a proper sampling of the interferogram, thanks to the use of an ultrastable common-path birefringent interferometer. Validation of the system is performed on plastic microbeads; multimodality is demonstrated by fluorescence and Raman maps of a few-layers transition metal dichalcogenide sample.
We present a novel wide-field Raman microscope, based on the time-domain Fourier-transform method. This enables parallel acquisition of Raman spectra on all the pixels of the 2D detector; the resulting wide-field approach allows faster collection of Raman maps with respect to standard raster-scanning methods. In addition, the time-domain method disentangles fluorescence and Raman signals. The system is robust and stable, thanks to the use of an ultrastable common-path birefringent interferometer. Validation of the system is performed on plastic microbeads and on a few-layers transition metal dichalcogenide sample.
Multispectral fluorescence lifetime microscopy (FLIM) is a valuable tool for biomedical and environmental applications. A multidimensional acquisition scheme (space, time, spectrum) provides high information content and the drawback of long acquisition/processing times. Compressive Sensing (CS) combined with Single-Pixel Camera (SPC) acquisition scheme has been proposed as a strategy to reduce the number of measurements. We present a multispectral FLIM system based on SPC, CS and data fusion (DF) with a high-resolution camera to strongly reduce the acquisition time. We adopted a novel method for TCSPC to increase the count-rate. The system is characterized and validated on a cellular sample.
Time-resolved multispectral fluorescence microscopy provides a 4D hypercube dataset with high specificity for cellular examination. However, this is generally obtained by significantly increasing the measurement time, which is quite limiting for in vivo measurement or with photosensitive sample. It is possible to reduce the measurement effort with a novel microscopy framework exploiting compressive sensing based on single-pixel camera. In this work, we present a compressive sensing system and validate it with a cellular sample. Data fusion with a high-resolution camera image allows us to tackle the well-known problem of low-resolution in single-pixel imaging.
In this presentation we show a method capable of measuring and correcting field dependent aberration in a microscope setup without a dedicated wavefront sensor using a pupil conjugated deformable lenses in combination with anisoplanatic deconvolution.
Structured Illumination microscopy is a super-resolution imaging technique based on sample fluorescence excitation with a spatially modulated light pattern. The pattern properties as well as the capability to shift it over sample determine the quality of the final images. At the current state of the art, pattern generation and translation require bulky and non-trivial optical setups. Here we propose an integrated optical device for the versatile generation and translation of the light pattern. This device can be used as light source for a standard microscope, upgrading it to a super-resolution system.
Heterogeneity plays an important role in medicine and biology, which can be investigated by exploiting single cell analysis (SCA). Among SCA methods, imaging cytometry allows the analysis of individual 2D and 3D spatial features. Here we present a femtosecond laser fabricated optofluidic automated platform encompassing a thermo-optic phase shifter, cylindrical lenses and a microfluidic network to generate and shift a dual-color patterned light sheet within a microchannel where the samples of interest flow. The device can be used as add-on and can provide an acquisition rate of about 1 cell/second, or subnuclear resolution at the single cell level.
Fourier-plane optical microscopy is a powerful technique for studying the angularly-resolved optical properties of a plethora of materials and devices. The information about the direction of the emission of light by a sample is extracted by imaging the objective back focal plane on a two-dimensional detector, via a suitable optical system. This imaging technique is able to resolve the angular spectrum of the light over a wide angular field of view, but typically it doesn’t provide any spectral information, since it integrates the light intensity over a broad wavelength range. On the other hand, advanced hyperspectral imaging techniques are able to record the spectrum of the transmitted/reflected/emitted light at each pixel of the detector. In this work, we combine an innovative hyperspectral imaging system with Fourier-space microscopy, and we apply the novel device to the characterization of planar organic microcavities. In our system, hyperspectral imaging is performed by Fourier-transform spectroscopy thanks to an innovative common-path birefringent interferometer: it generates two delayed replicas of the light field, whose interference pattern is recorded as a function of their delay. The Fourier Transform of the resulting interferogram yields the intensity spectrum for each element of the microscope angular field-of-view. This system provides an angle-resolved hyperspectral view of the microcavities. The hyperspectral Fourier-space image clearly evidences the cavity modes both in photoluminescence and reflection, whose energy has a parabolic dependence on the emission angle. From the hyperspectral image, we reconstruct a 3D view of the parabolic cavity dispersion across the whole Fourier space.
Integrated optical switches and modulators allow performing reconfigurability in integrated circuits, resulting as fundamental components in different fields ranging from optical communications to sensing and metrology. Among different methods, the thermo-optic effect has been successfully used to fabricate optical modulators by femtosecond laser micromachining (FLM) in glass substrates, proving high stability, no losses dependance but long switching time. In this work, we present an integrated optical switch realized by FLM with a switching time of less than 1 ms: which is about 1 order of magnitude faster than the other devices present in literature. This result has been achieved by carefully optimizing the geometry and the position of resistors and trenches near the waveguides through simulation and experimental validation. In addition, by means of an optimization of the applied voltage signal, we have demonstrated a further significant temporal improvement, measuring a switching time of less than 100 μs.
Multidimensional fluorescence microscopy techniques produce dataset rich of information (space, emission spectrum and lifetime) to investigate photophysical processes in biological samples. To acquire a 4D dataset, one promising microscope design is based on the single-pixel camera scheme and on compressive sensing acquisitions, thanks to which the measurement time can be reduced. Within this framework, a computational step is required to move from the acquisition space to the pixel space and, subsequently, the analysis can be carried out exploiting the high dimensionality. In this work we present an experimental system and a fast-fit method that can produce a map of fluorophore concentrations in parallel to the measurement routine.
Multispectral fluorescence lifetime imaging microscopy (λFLIM) is a high sensitivity technique for multifold applications. We present a λFLIM system and a compressive sensing data acquisition strategy, which allows one to reduce the measurement time.
We present a microscopy method capable of measuring aberrations in all the poits of the field of view and to correct for the field-dependent aberrations in a closed loop multi conjugated AO system using two deformable lenses and no wavefront sensor.
The investigation of artworks of cultural heritage is generally aimed at the characterization of the constituent materials and the evaluation of their state of conservation. Research may shed light on the pigments and their potential deterioration mechanisms, and on the conservation treatments. Laboratory analysis on micro-samples taken from the artwork is still an invaluable practice for a deep understanding of the paint layer composition. In this context, a powerful technique is spectral microscopy, which acquires the spectrum for each point in the image of a sample. To acquire a continuous spectrum, one very efficient method is based on Fourier-transform (FT) spectroscopy as it allows massive parallelization on all the image pixels. Here we introduce a hyperspectral microscope based on an innovative FT spectrometer; the device is compact, robust, with high throughput and broad spectral coverage. In our microscope, light is collected by an infinity-corrected objective, propagates in the innovative spectrometer and is imaged on a silicon monochrome CMOS camera by a tube lens. The typical spectral resolution of the microscope, which can be flexibly adjusted for each measurement, is 3 THz (4 nm at 600 nm). We show very compact implementations of the hyperspectral microscope and their use for wide-field imaging of reflection, fluorescence and, interestingly, fluorescence-free Raman spectra. Thanks to the high throughput, the acquisition time of our microscope is significantly shorter than traditional raster-scanning approaches.
In this work we present a microscope on chip based on Light Sheet Fluorescence Microscopy, capable to automatically perform 3D and dual-color imaging of specimens diluted in a liquid suspension. A microfluidic channel is used for automatic sample delivery, while integrated optical components such as optical waveguides and lenses are used to illuminate the sample flowing in the channel. The device is fabricated by femtosecond laser micromachining in a glass substrate. Benefiting from the versatility of the fabrication technique we present two prototypes that have been optimized for different samples such as single cells and Drosophila embryos.
Multispectral Fluorescence Lifetime Imaging Microscopy (FLIM) is a fundamental tool to study multifold processes in biology and material science. The growing demand for acquisition time reduction requires the parallel acquisition of a multi-dimensional dataset and the exploitation of compressive sensing techniques. In this work we present a multispectral FLIM set-up based on wide-field structured illumination coupled with a spectrometer and a novel time-resolved parallel 18x1 SPAD array detector, working in a single pixel camera scheme. We show the system characterization and its imaging properties varying the compression ratio.
We introduce a Fourier-transform hyperspectral microscope based on an ultrastable birefringent interferometer. The microscope enables wide field acquisition with broad spectral coverage, tunable spectral resolution, high sensitivity and short acquisition time. We present the prototype of an add-on to a commercial microscope. We provide examples of applications in biology and solid state physics. The microscope is suited for fluorescence and Raman imaging.
The reconstruction of an object hidden behind a scattering curtain is a modern topic in the field of imaging, which has stimulated an active scientific production over the past few years. However, most of the work done in the field was in addressing the reconstruction of a bi-dimensional object. Here, instead, we tackle the reconstruction of a three-dimensional fluorescent sample hidden behind an opaque layer. To do so, we show that the auto-correlation operation well behave in projection tomography, letting us to reconstruct a three-dimensional auto-correlation of the object. By having access to such information, it is possible to implement a phase retrieval algorithm to roll back to the actual reconstruction of the specimen.
Tomographic inspection of fluorescent labels distributed within a specimen is an important aspect in biology. Light sheet fluorescent microscopy (LSFM) offers a powerful and simple tool to selectively slice the sample and let us directly obtain a tomographic view of the specimen. However, due to non-isotropic resolution of the technique along the axial scanning, one may want to combine different views of the object and add deconvolution to the process in order to achieve higher resolution. Typically, multi-view Bayesian methods based on Richardson-Lucy deconvolution are used for this task once the datasets are exactly registered against each other. In this work, instead, we begin to investigate how to avoid the alignment procedure and use a direct algorithm to form a multi-view tomographic reconstruction. To do this, we developed a new framework based on auto-correlation analysis that let us achieve deconvolved reconstructions starting from blurred auto-correlations. Since the latter are insensitive to shifts, we can combine the auto-correlations coming from multi-view acquisitions without taking care of the registration procedure.
Time-resolved multispectral imaging has recently found many applications ranging from biomedical to environmental field. Multidimensional approach measuring spectral and ultrafast temporal dynamics of fluorescence signal combined with spatial information (imaging) allows one to characterize biological processes at both microscopic and macroscopic level, representing a fundamental step towards development of diagnostic strategies. Long acquisition time is the main drawback of multidimensional approach because it is not compatible with biological system dynamics. In order to reduce the measurement times, it is necessary to parallelize the acquisition (hardware level) and to optimize the acquisition strategy to reduce the measurements number while preserving the information content. In this work we have developed a time-resolved multispectral fluorescence imaging system based on a spad array combined with compression techniques which allows to reduce the number of time-resolved acquisitions by a factor < 70%. The system is based on a double DMD configuration (excitation and detection) coupled to a 32x1 SPAD array, each one with its own TCSPC circuit, placed after an imaging spectrometer. This allows one to use the spatial modulation of the excitation/detection light to acquire images at different wavelengths following the single pixel camera (SPC) scheme. In order to compress the number of acquisitions, a CW fluorescence image is acquired through a CCD and Hadamard transform is applied to select most significative coefficients. The patterns related to these coefficients are subsequently used for SPC acquisition for time and spectral resolution. A Total-Variation based algorithm is used for the reconstruction of the 4D images.
We introduce a Fourier-transform hyperspectral microscope based on an ultrastable birefringent interferometer. The microscope enables wide field acquisition with broad spectral coverage, tunable spectral resolution, high sensitivity and short acquisition time. We present two prototype implementations and an add-on for a commercial microscope. We provide examples of applications in biology and solid state physics.
Lab on a Chip devices are compact and portable chips mainly constituted by a network of microfluidic channels. They aim at substituting bulk laboratory instrumentations, with the advantages of increasing the automation and the sensitivity of the analysis, reducing the costs and opening the possibility of performing measurements at the Point of Care. Among different Lab on Chips, optofluidic ones have the advantages of optical investigation, but the integration of optical and microfluidic components in a single substrate is very challenging from a technological point of view. A recent fabrication technique, known as femtosecond laser micromachining (FLM), has proven to be ideal for the realization of these devices, allowing the fabrication of the whole device in a single irradiation step. Here, we will present a platelet counter and a microscope on chip, that fully take advantage from the versatility of FLM. To succeed in these works a fundamental aspect to address is the capability to control the sample positioning in the microfluidic channel. A single particle per time should pass in the detection region to avoid the overlooking of specimen. Moreover, a precise control of the sample orientation and position in the channel cross section is needed for imaging. The 3D capabilities of FLM have been fundamental in the realization of advanced fluidic layouts capable of sample manipulation with no need of any additional external field. We have successfully proven red blood cells and platelets counting, as well as single cells, cellular spheroids and drosophila embryos 3D imaging.
Drosophila Melanogaster is a sample of high biological interest that is being widely used as biological model, due to the relatively short life cycle, short genome and ease in culturing. In this work we present a microscope on chip capable of processing Drosophila embryos to obtain three dimensional fluorescent images at high throughput. This device, based on light sheet microscopy, uses a plane of light intercepting the sample channel to optically and noninvasively section the embryos while flowing. This permits to automatically acquire for each sample the stack of images necessary for the subsequent 3D reconstruction with no need of any manual sample positioning and alignment. The whole chip is fabricated in a glass substrate by femtosecond laser micromachining. The device has been optimized for the specific morphology of the sample. Indeed, the highly elliptical shape of the embryos (about 100 x 500 μm2) might affect the image quality degrading both the vertical and the axial resolution of the system. To overcome this issue, we have first optimized the layout of the fluidic channel to precisely control the sample orientation by means of hydrodynamic forces. Thereafter, we have optimized the properties of the optical circuit, to realize two opposite light sheets impinging on the sample, perfectly overlapped, with a high signal to noise ratio. With these actions, we have been able to obtain high quality Drosophila reconstruction.
Diffuse Optical Tomography (DOT) is a tool for 3D reconstruction of absorption and scattering inside a tissue. Typically, this method requires a dense distribution of sources and detectors, thus hampering the possibility of fully exploring a time-resolved detection. Recently, techniques based on structured-light illumination and compressing detection have been developed, opening the possibility of fully exploiting a source/detector spatial modulation for compression at the measurement stage. Here we propose a combined Continuous-Wave (CW) and time-domain (TD) adaptive scheme based on the singular-value decomposition (SVD) for optimal-patterns calculation. Patterns are firstly computed based on a fast acquisition via a CCD, and consequently projected for time-resolved measurements.
Time-resolved imaging is a valuable tool for biomedical applications such as Diffused Optical Tomography (DOT) and Fluorescence Lifetime Imaging (FLIM). The first one characterizes and localizes absorption/scattering heterogeneities, which can be representative of tumors, and is routinely used for brain functional imaging. FLIM provides relevant information (e.g. pH, ion concentration and FRET) in cell biology and find application in molecular imaging for preclinical studies in small animals. Beyond biomedical applications, time-resolved imaging is exploited for environmental monitoring, LIDAR and characterization of combustion processes.
Structured light illumination and compressive-sensing detection have been recently proposed as new strategies to preserve information content while significantly reducing the number of measurements. One possible implementation of this approach is the Single Pixel Camera (SPC), where the inner product between the image of the subject and appropriate patterns is measured by using a spatial modulator (e.g. DMD, SLM) and focusing the light on a single pixel detector.
In this work, we present a time-resolved imaging system for DOT applications based on structured light illumination and SPC detection, implementing an adaptive scheme based on Singular-Value Decomposition for optimal generation of input/output patterns. Moreover, a novel scheme of time-resolved camera, with ps temporal resolution, is proposed and experimentally validated. The device consists of a high-density matrix of single photon detection elements which can be selectively enabled/disabled. Spatial modulator and detector are combined into a single chip improving cost and compactness. In conclusion, the proposed time-resolved imaging approach can have significant impact on biomedical, environmental and LIDAR applications as an alternative to gated cameras or scanning systems.
Diffuse Optical Tomography (DOT)is a powerful tool for the reconstruction of optical properties inside a diffusive medium, such as biological tissues. In particular, in the last years, techniques based on structured light illumination and compressive sensing detection have been developed. In this work a time-resolved system based on structured light illumination and compressive detection has been developed and used for DOT. Moreover, a data-driven algorithm for optimal pattern generation based on the Singular-Value Decomposition has been implemented and validated.
In this report, we discuss the interest of quality metrics for imaging and image processing of multi-views in light sheet fluorescent 3D microscopy. Various metrics of focus are tested on real and simulated data so as to automatically assess the informational quality of the images. Application of such metrics are given for several information tasks including online control of acquisition, fast registration or image fusion. Illustrations are given for typical samples of interest for in vivo imaging with light sheet microscopy such as spheroids or organoids. We point to the reader softwares freely available under FIJI which enable to test the computation of a basic quality metric, for registration and fusion.
A time-resolved Diffuse Optical Tomography system based on multiple view
acquisition, pulsed structured light illumination and detection with spatial compression is
proposed. Reconstructions on heterogeneous tissue mimicking phantoms are presented.
Selective plane illumination microscopy (SPIM) is an optical sectioning technique that allows imaging of biological samples at high spatio-temporal resolution. Standard SPIM devices require dedicated set-ups, complex sample preparation and accurate system alignment, thus limiting the automation of the technique, its accessibility and throughput. We present a millimeter-scaled optofluidic device that incorporates selective plane illumination and fully automatic sample delivery and scanning. To this end an integrated cylindrical lens and a three-dimensional fluidic network were fabricated by femtosecond laser micromachining into a single glass chip. This device can upgrade any standard fluorescence microscope to a SPIM system.
We used SPIM on a CHIP to automatically scan biological samples under a conventional microscope, without the need of any motorized stage: tissue spheroids expressing fluorescent proteins were flowed in the microchannel at constant speed and their sections were acquired while passing through the light sheet. We demonstrate high-throughput imaging of the entire sample volume (with a rate of 30 samples/min), segmentation and quantification in thick (100-300 μm diameter) cellular spheroids.
This optofluidic device gives access to SPIM analyses to non-expert end-users, opening the way to automatic and fast screening of a high number of samples at subcellular resolution.
Diffuse Optical Tomography (DOT) can be described as a highly multidimensional problem generating a huge data set with long acquisition/computational times. Biological tissue behaves as a low pass filter in the spatial frequency domain, hence compressive sensing approaches, based on both patterned illumination and detection, are useful to reduce the data set while preserving the information content. In this work, a multiple-view time-domain compressed sensing DOT system is presented and experimentally validated on non-planar tissue-mimicking phantoms containing absorbing inclusions.
Light sheet fluorescence microscopy has proven to be a powerful tool to image fixed and chemically cleared samples, providing in depth and high resolution reconstructions of intact mouse organs. We applied light sheet microscopy to image the mouse intestine. We found that large portions of the sample can be readily visualized, assessing the organ status and highlighting the presence of regions with impaired morphology. Yet, three-dimensional (3-D) sectioning of the intestine leads to a large dataset that produces unnecessary storage and processing overload. We developed a routine that extracts the relevant information from a large image stack and provides quantitative analysis of the intestine morphology. This result was achieved by a three step procedure consisting of: (1) virtually unfold the 3-D reconstruction of the intestine; (2) observe it layer-by-layer; and (3) identify distinct villi and statistically analyze multiple samples belonging to different intestinal regions. Even if the procedure has been developed for the murine intestine, most of the underlying concepts have a general applicability.
We report on a new source able to provide probe pulses in the UV visible range and on the demonstration of its
application to hyperspectral (fluorescence lifetime) imaging measurements. The source is able to generate UV (down to
300 nm) and blue light exploiting high-order mode propagation in a microstructured fiber pumped by a Ti:Sapphire laser.
We believe that further optimization of pump wavelength, fiber length and fiber zero-dispersion wavelength could
generate light well below 300 nm using a simple and stable set-up and become a useful tool for biomedical imaging. We
demonstrated its versatility using the source for FLIM-FRET measurement a 460 nm and hyperspectral FRET-FLIM
measurements.
We present a compact time-resolved spectrometer suitable for optical spectroscopy from 400 nm to 1 μm wavelengths.
The detector consists of a monolithic array of 16 high-precision Time-to-Digital Converters (TDC) and Single-Photon
Avalanche Diodes (SPAD). The instrument has 10 ps resolution and reaches 70 ps (FWHM) timing precision over a 160
ns full-scale range with a Differential Non-Linearity (DNL) better than 1.5 % LSB. The core of the spectrometer is the
application-specific integrated chip composed of 16 pixels with 250 μm pitch, containing a 20 μm diameter SPAD and
an independent TDC each, fabricated in a 0.35 μm CMOS technology. In front of this array a monochromator is used to
focus different wavelengths into different pixels. The spectrometer has been used for fluorescence lifetime spectroscopy:
5 nm spectral resolution over an 80 nm bandwidth is achieved. Lifetime spectroscopy of Nile blue is demonstrated.
Presently time-resolved optical spectroscopy is applied with increasing success for non-invasive medical diagnostics mainly up to 1100 nm. We extended the investigation range beyond this limit, employing a supercontinuum fiber laser source and a Single-Photon Avalanche Diode in InGaAs/InP operated in gated mode. First in-vivo measurements were performed on the forearm and the breast of two healthy volunteers, reaching up to 1360 nm.
Optical Projection Tomography (OPT) is a three dimensional imaging technique that is particularly suitable for studying
millimeter sized biological samples and organisms. Similarly to x-ray computed tomography, OPT is based on the
acquisition of a sequence of images taken through the sample at many angles (projections). Assuming the linearity of the
optical absorption process, the projections are combined to reconstruct the 3-D volume of the sample, typically using a
filtered back-projection algorithm. OPT has been applied to in-vivo imaging of zebrafish (Danio rerio). The instrument
and the protocol for in vivo imaging of zebrafish embryos and juvenile specimens are described.
Light scattering remains a challenge for in vivo OPT, especially when samples at the upper size limit, like zebrafish at
the adult stage, are under study. We describe Time-Gated Optical Projection Tomography (TGOPT), a technique able to
reconstruct adult zebrafish internal structures by counteracting the scattering effects through a fast time-gate. The time
gating mechanism is based on non-linear optical upconversion of an infrared ultrashort laser pulse and allows the
detection of quasi-ballistic photons within a 100 fs temporal gate. This results in a strong improvement in contrast and
resolution with respect to conventional OPT. Artifacts in the reconstructed images are reduced as well. We show that
TGOPT is suited for imaging the skeletal system and nervous structures of adult zebrafish.
Fluorescence molecular tomography (FMT) is quite demanding in terms of acquisition/computational times due to the huge amount of data. Different research groups have proposed compression approaches regarding both illumination (wide field structured light instead of raster point scanning) and detection (compression of the acquired images). The authors have previously proposed a fast FMT reconstruction method based on the combination of a multiple-view approach with a full compression scheme. This method had been successfully tested on a cylindrical phantom and is being generalized in this paper to samples of arbitrary shape. The devised procedure and algorithms have been tested on an ex-vivo mouse.
KEYWORDS: Picosecond phenomena, Luminescence, Clocks, Field programmable gate arrays, Electronics, Power supplies, Fluorescence lifetime imaging, Time metrology, Interfaces, Data acquisition
We present a low-power Time-to-Digital Converter (TDC) chip, fabricated in a standard cost-effective 0.35 μm CMOS
technology, which provides 160 ns dynamic range, 10 ps timing resolution and Differential Non-Linearity better than
0.01 LSB rms. This chip is the core of a compact TDC module equipped with an USB 2.0 interface for user-friendly
control and data-acquisition. The TDC module is suitable for a wide variety of applications such as Fluorescence
Lifetime Imaging (FLIM), time-resolved spectroscopy, Diffuse Optical Spectroscopy (DOS), Optical Time-Domain
Reflectometry (OTDR), quantum optics, etc. In particular, we show the application of our TDC module to fluorescence
lifetime measurements.
In recent years, an increasing effort has been devoted to the optimization of acquisition and reconstruction schemes for fluorescence molecular tomography (FMT). In particular, wide-field structured illumination and compression of the measured images have enabled significant reduction of the data set and, consequently, a decrease in both acquisition and processing times. FMT based on this concept has been recently demonstrated on a cylindrical phantom with a rotating-view scheme that significantly increases the reconstruction quality. In this work, we generalize the rotating-view scheme to arbitrary geometries and experimentally demonstrate its applicability to murine models. To the best of our knowledge this is the first time that FMT based on a rotating-view scheme with structured illumination and image compression has been applied to animals.
In this paper we report imaged neuronal rat cells in a confocal laser scanning microscope by simultaneous generation of
the three requested wavelengths obtained by a UV-extended supercontinuum source. This is to the best of our
knowledge that such a measure was performed using a microstructured fiber pumped by a standard Ti:Sapphire laser.
We observed efficient UV light generation when a novel pumping scheme was used. The pump wavelength is close to
the zero-dispersion wavelength of the fiber first high-order mode and offset axial pumping is used. By tuning the pump
wavelength and power level we were able to generate mW-power levels in the visible wavelength interval down and of
about hundreds of microwatt in the UV wavelength interval down to 300 nm. The pump alignment was very simple and
very stable. We believe that further optimization of pump wavelength, fiber length and fiber zero-dispersion wavelength
could generate light well below 300 nm using a simple and stable set-up. To demonstrate the potentiality of this
technique we imaged neuronal rat cells in a confocal laser scanning microscope by simultaneous generation of the three
requested wavelengths.
We introduce flow optical projection tomography, an imaging technique capable of visualizing the vasculature of living specimens in 3-D. The method detects the movement of cells in the bloodstream and creates flow maps using a motion-analysis procedure. Then, flow maps obtained from projection taken at several angles are used to reconstruct sections of the circulatory system of the specimen. We therefore demonstrate an in vivo, 3-D optical imaging technique that, without the use of any labeling, is able to reconstruct and visualize the vascular network of transparent and weakly scattering living specimens.
Time-domain diffuse optical spectroscopy has become a powerful tool to study highly scattering media, mainly in the
fields of non-invasive medical diagnostics and quality assessment of food and pharmaceutical products. Up to now this
technique has been exploited mostly up to 1100 nm: we extend the spectral range by means of a continuously tunable
pulsed laser source at a high repetition rate and a custom InGaAs/InP Single-Photon Avalanche Diode operated in time-gated
mode, working up to 1700 nm. The characterization of the system is presented. As a first example of application,
we measured the absorption spectrum of collagen powder in the range 1100 - 1700 nm, which could prove useful for
breast density assessment.
Since DNA is not internalized efficiently by cells, the success of gene therapy depends
on the availability of carriers to efficiently deliver genetic material into target cells. Gene delivery
vectors can be broadly categorized into viral and non-viral ones. Non-viral gene delivery systems
are represented by cationic lipids and polymers rely on the basics of supramolecular chemistry
termed "self-assembling": at physiological pH, they are cations and spontaneously form lipoplexes
(for lipids) and polyplexes (for polymers) complexing nucleic acids. In this scenario, cationic
polymers are commonly used as non-viral vehicles. Their effectiveness is strongly related to key
parameters including DNA binding ability and stability in different environments. Time-resolved
fluorescence spectroscopy of SYBR Green I (DNA dye) was carried out to characterize cationic
polymer/DNA complex (polyplex) formation dispersed in aqueous solution. Both fluorescence
amplitude and lifetime proved to be very sensitive to the polymer/DNA ratio (N/P ratio, +/-).
Diffuse optical tomography (DOT) and Fluorescence mediated tomography
(FMT) are powerful in-vivo optical imaging techniques but they are affected
by long acquisition and computational times. Recently, the use of structured light has
been proposed in order to reduce the acquisition time and also the computational time
of the inverse problem. Additionally, it has been proposed to compress the measured
data set to reduce the reconstruction time. Here we present our experimental approach,
describing the instrument for structured illumination and wide field detection and we
discuss the advantages to use a finite elements based approach. Then, we introduce
the use of spatial wavelets. Our method is based on the projection of a small number
of wavelet patterns (Haar and Battle-Lemarie wavelets). The detected images are
wavelet transformed and the information content is compressed to achieve fast 3D
reconstruction. Experimental results are presented, showing fast reconstruction of
complex absorbing/fluorescent objects in thick diffusive samples. Implications for fast
small animal imaging are discussed.
Time domain diffuse optical spectroscopy provides the absorption and scattering properties of biological tissues
and diffusive materials. Few measurements are available at discrete wavelengths beyond 1100 nm, and just one
time-domain system continuously tuneable up to 1400 nm. We developed a time-domain system, based on a
continuously tuneable supercontinuum pulsed source, and a custom InGaAs/InP Single-Photon Avalanche Diode.
Operation was demonstrated in the 1100-1700 nm range with a spectral resolution of 15 nm, a temporal resolution
of 150 ps and a background of 6000 counts/s. A first example of application on the optical characterization of
collagen powder is given.
We report on generation of blue light exploiting high-order mode propagation in a microstructured fiber pumped by a
Ti:Sapphire close to the zero-dispersion wavelength of the first high-order mode. An new interesting regime was
observed with axial offset pump. With 230 mW of incident pump power we generated over 3 mW in the 450-510 nm
window achieving 50 μW/nm power density. In a final round of measurements we were able to show generation of a
peak at 350 nm. This complex regime has still to be fully investigated but we believe an optimized fiber design will
allow to efficiently extend the operation of Ti:Sapphire laser to UV/blue wavelength region.
The feasibility of in vivo measurements in the range of 1000 to 1100 nm and the potential benefits of operation in that wavelength range for diagnostic applications are investigated. To this purpose, an existing system for time-resolved diffuse spectroscopy is modified to enable in vivo studies to be carried out continuously from 600 to 1100 nm. The optical characterization of collagen powder is extended to 1100 nm and an accurate measurement of the absorption properties of lipid is carried out over the entire spectral range. Finally, the first in vivo absorption and scattering spectra of breast tissue are measured from 10 healthy volunteers between 600 and 1100 nm and tissue composition is evaluated in terms of blood parameters and water, lipid, and collagen content using a spectrally constrained global fitting procedure.
We discuss the spectral distortions occurring when time-resolved diffuse spectroscopy is performed illuminating
with a spectrally wide source. We show that the spectral region within the source bandwidth that exhibits
the lowest absorption will dominate the resulting time-resolved curve, leading to significant distortions on the
retrieved absorption spectrum (including shifts in peak positions). Due to the nonlinear behavior of the light
attenuation due to absorption, this effect becomes more pronounced when including longer and longer photon
path lengths. First, a theoretical treatment of the problem is given and then the distortion is described by timeresolved
reflectance simulations and experimental measurements of lipid and water samples. Finally, a spectrally
constrained data analysis is proposed to overcome the distortion and improve the accuracy of the estimation of
chromophore concentrations from absorption spectra. Measurements on a lipid sample show a reduction of the
error from 30% to 6%.
Temporal propagation of sinusoidally modulated light in in-homogeneous
diffusive samples is investigated experimentally and by finite element simulations, showing
that the amplitude and the phase of the sinusoid are affected by the presence of inclusions.
Enhancement of imaging resolution with high spatial frequencies and early time-gating is
shown. Detection of the phase of the modulated light is proposed as a new method for the
accurate localization of tissue in-homogeneities. A fast 3-dimensional reconstruction based
on the detection of spatially modulated light at a limited number of spatial frequencies is
discussed.
KEYWORDS: Cameras, Tomography, 3D modeling, In vivo imaging, Brain activation, Optical fibers, Functional imaging, Optical tomography, Imaging systems, 3D metrology
We propose a system for 3D tomography using a single pulsed source and a time-gated camera for functional
imaging studies. Reconstructions were based on a linear model based on small perturbation assumption, applying
Tikhonov regularization. This approach was tested against simulations, demonstrating both detection and
localization capabilities. Preliminary measurements on realistic inhomogeneous phantoms showed good detection
sensibility, even for a low optical contrast, but poorer localization properties, possibly due to the still low SNR
of the system. Finally, an initial in vivo test on a motor cortex activation paradigm is presented.
A set-up for time-resolved transmittance and reflectance spectroscopy of diffusive media was upgraded to allow
measurements to be performed continuously from 600 to 1100 nm. Time-resolved diffuse optical spectroscopy of breast
was carried out on 10 healthy volunteers, demonstrating the feasibility of in vivo measurements up to 1100 nm. The
optical characterization of collagen was also extended revealing an absorption peak (around 1020-1030 nm), which could
prove of interest for the in vivo quantification of collagen.
A method was developed to estimate spectral changes of the absorption properties of turbid media from time-resolved
reflectance/transmittance measurements. It was derived directly from the microscopic Beer-Lambert law, and tested
against simulations and phantom measurements.
The mean penetration depth of diffusely reflected photons is dependent on the arrival time t of photons, but not
on the source-detector distance. Thus, all photons collected at the same t have the same depth sensitivity, and
can be used for the reconstruction.
Following this concept, we have implemented a system for 3D tomography using a single injection fiber
and a time-gated ICCD camera. The feasibility of the novel approach to reconstruct a local perturbation
was demonstrated both with simulations and phantom measurements. Finally, preliminary measurements were
performed in vivo following a standard protocol of motor cortex activation.
The use of spatially modulated light is finding application in biomedical optics having potential use in imaging
and tomography of tissues and small animals. We describe the
time-resolved propagation of spatial frequencies in
turbid media. We present a set-up based on a ps laser source, spatially modulated by a micro-mirror device and
a time-gated intensifier. We discuss the relevant information content that can be useful for imaging of tissues, in
terms of the spatial Fourier components of the propagating pulse. We demonstrate that high spatial frequencies
appear in the early time-gated signal whereas low frequencies persist for longer times and that the combined use
of high spatial frequencies and early time gates can be used to improve the resolution in imaging.
We describe the development of a compact time-resolved system for the measurement of the optical properties of highly scattering media over a bandwidth of 600-1100 nm. The instrument is based on a fiber laser generating supercontinuum radiation, that is spectrally dispersed and used to sequentially illuminate the sample. A single photon avalanche photo-diode in combination with time correlated single-photon counting is used to recover the time-dispersion curve at each wavelength. The calibration of the system and in-vivo applications are shown.
An experimental technique which allows one to perform pump-probe transient absorption spectroscopy in real-time is an
important tool to study irreversible processes. This is particularly interesting in the case of biological samples which
easily deteriorate upon exposure to light pulses, with the formation of permanent photoproducts and structural changes.
In particular pump-probe spectroscopy can provide fundamental information for the design of optical chromophores. In
this work a real-time pump-probe imaging spectroscopy system has been realized and we have explored the possibility to
further reduce the number of laser pulses by using a time-gated camera. We believe that the use of a time-gated camera
can provide an important step towards the final goal of pump-probe single shot spectroscopy.
The anisotropic light propagation in biological tissue is investigated in the steady-state and time domains. Monte
Carlo simulations performed for tissue that has anisotropic optical properties show that the steady-state and
time-resolved reflectance depends strongly on the measurement direction. We examined the determination of the
optical properties using an isotropic diffusion model and found that in the time domain, in contrast to steady-state
spatially-resolved reflectance measurements, the obtained absorption coefficient does not depend on the
measurement direction and is close to the correct value. We performed measurements of the steady-state and
time-resolved reflectance from porcine and bovine tendon which confirmed the theoretical findings. In addition,
we compared the results obtained from Monte Carlo simulations with the solutions of the anisotropic diffusion
theory for reflectance from semi-infinite media and for transmittance from slabs. In contrast to the literature,
we found that the anisotropic diffusion equation is, in general, not a valid approximation to the anisotropic light
propagation even in the diffusive regime.
We investigate anisotropic light propagation in biological tissue in steady-state and time domains. Monte Carlo simulations performed for tissue that consists of aligned cylindrical and spherical scatterers show that steady-state and time-resolved reflectance depends strongly on the measurement direction relative to the alignment of the cylinder axis. We examine the determination of optical properties using an isotropic diffusion model and find that in the time domain, in contrast to steady-state spatially resolved reflectance measurements, the obtained absorption coefficient does not depend on the measurement direction and is close to the true value. Contrarily, the derived reduced scattering coefficient depends strongly on the measurement direction in both domains. Measurements of the steady-state and time-resolved reflectance from bovine tendon confirm the theoretical findings.
We demonstrate the feasibility of white-light time-resolved optical mammography. The instrumentation is based on supercontinuum light generated in photonic crystal fiber and 32-channel parallel time-correlated single-photon-counting detection. Total measurement time is of the order of 10 min for typical clinical applications. Preliminary measurements performed on volunteers show the ability of the system to determine tissue constituent concentrations and structure over the entire breast area. Furthermore, measurements on a tissue-like sample demonstrate detection and characterization of inclusions.
We report on an instrument for time-resolved spectroscopy (TRS) based on white-light generation in a highly non-linear crystal fiber. TRS in the visible and near-infrared region at picosecond-to-nanosecond time scales has attracted increased interest in recent years owing to the possibility of spectroscopic analysis of turbid media, such as biological tissues. A self-mode-locked Ti:Sapphire oscillator pumped by an Ar:ion laser provides pulses 50 - 100 fs long, at 85 MHz repetition rate. The light is focused into a crystal fiber, which consists of a core surrounded by a mesh of air-filled holes. White light is generated by a combination of several non-linear effects in the fiber. We optimize the spectrum for measurements in the region 600 - 1000 nm. For detection, we use an imaging spectrometer coupled to a 16-channel photomultiplier tube, enabling simultaneous detection in 16 wavelength bands. We use time-correlated single-photon counting to record the signal, with a temporal resolution of ~160 ps. To demonstrate the system, we have performed measurements of the diffuse time-resolved reflectance of tissue phantoms made of epoxy resin with added scattering and absorbing materials. The data was evaluated using a light propagation model based on diffusion theory, to extract the scattering and absorption coefficients of the medium. The results corresponded very well with previous measurements on the phantoms performed using other TRS instruments.
The first, to our knowledge, in-vivo broadband spectroscopic characterization of breast tissue using different interfiber distances as well as transmittance measurements is presented. Absorption and scattering properties are measured on six healthy subjects, using time-resolved diffuse spectroscopy and an inverse model based on the diffusion equation. Wavelength-tunable picosecond-pulse lasers and time-correlated single-photon counting detection are employed, enabling fully spectroscopic measurements in the range 610 to 1040 nm. Characterization of the absorption and reduced scattering coefficients of breast tissue is made with the aim of investigating individual variations, as well as variations due to different measurement geometries. Diffuse reflectance measurements at different interfiber distances (2, 3, and 4 cm) are performed, as well as measurements in transmittance mode, meaning that different sampling volumes are involved. The results show a large variation in the absorption and scattering properties depending on the subject, correlating mainly with the water versus lipid content of the breast. Intrasubject variations, due to different interfiber distances or transmittance modes, correlate with the known structures of the breast, but these variations are small compared to the subject-to-subject variation. The intrasubject variations are larger for the scattering data than the absorption data; this is consistent with different spatial localization of the measurements of these parameters, which is explained by the photon migration theory.
In vivo absorption and reduced scattering spectra of the human calcaneous from 650 to 1000 nm were assessed using a laboratory system for time-resolved transmittance spectroscopy. Measurements were performed on the calcaneous of seven female volunteers ranging from 26 to 82 years of age. The analysis of the absorption spectra, using a linear combination of the key tissue absorbers (bone mineral, water, lipids, oxy- and deoxyhemoglobin), revealed a general decrease in bone mineral content and an increase in lipids with age, which is in agreement with the aging transformations that occur in bone tissues. The scattering spectra were less effective in detecting such changes in older subjects, showing only a minor decrease in the coefficient for these subjects. The capability to noninvasively quantify bone tissue composition suggests a possible use of optical biopsy for the diagnosis of bone pathologies such as osteoporosis, which are characterized by a progressive reduction and transformation of the mineral in the bone matrix.
Carbon coatings of thickness down to 2 nanometers are needed to increase the storage density in magnetic hard disks and reach the 100 Gbit/in2 target. Methods to measure the properties of these ultrathin hard films still have to be developed. We show that combining Surface Brillouin Scattering (SBS) and x-ray reflectivity measurements the elastic constants of such films are accessible. Tetrahedral amorphous carbon films of thickness down to about 2 nm were deposited on Si by an S bend filtered cathodic vacuum arc, achieving a continuous coverage on large areas free of macroparticles. Film thickness and mass density are measured by x-ray reflectivity: densities about 3 g/cm3 are found, indicating a significant sp3 content. The dispersion relations of surface acoustic waves are measured by SBS. We show that for thicknesses above approximately 4 nm these waves can be described by a continuum elastic model based on a single homogeneous equivalent film. The elastic constants can then be obtained by fitting the dispersion relations, computed for given film properties, to the measured dispersion relations. For thicknesses of 3 nm or less qualitative differences among films are well measurable, but quantitative results are less reliable. We have thus shown that we can grow and characterise nanometer size tetrahedral amorphous carbon films, which maintain their high density and peculiar mechanical properties down to around 4-nm thickness, satisfying the requirements set for the hard disk coating material.
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