The new generation of the bh DCS-120 FLIM system features unprecedented temporal resolution, unprecedented timing reproducibility, high spatial resolution, high sensitivity, and near-ideal photon efficiency. Fluorescence lifetimes can be detected down to 10 ps. The decay data can be resolved into 4096 time channels, with a minimum time bin width of 405 fs. Pixel numbers as high as 4096 x 4096 are available. The system is available in a confocal version with excitation by ps diode lasers, or as a two-photon version with a Ti:Sa laser or a femtosecond fibre laser. The DCS-120 FLIM system uses fast scanning by galvanometer mirrors, confocal or non-descanned detection, detection, and FLIM by bh's multidimensional TCSPC technique. Data acquisition functions include precision dual-channel FLIM, laser wavelength multiplexing, multi-wavelength FLIM, time-series FLIM, ultra-fast time-series recording by temporal-mosaic FLIM, spatial mosaic FLIM, Z stack FLIM, phosphorescence lifetime imaging (PLIM), and fluorescence lifetime-transient scanning (FLITS). Data analysis is performed by next generation SPCImage NG. Lifetime analysis is running on a GPU, reducing calculation time from previously several minutes to a few seconds. Other features are the availability of image segmentation via a phasor plot or via 2D time-domain histograms, and automatic modelling of the system IRF. Together with the extraordinary timing stability of the recording system, repeated recalibration of the IRF is no longer necessary. Target applications of the new DCS-120 system are molecular imaging in live cells and tissues, protein-interaction experiments by FRET, metabolic imaging by NADH / FAD decay parameters, identification of cancer cells, and the recording of dynamic physiological processes of cell metabolism and neurology. https://www.becker-hickl.com
We describe a metabolic-imaging system based on simultaneous recording of lifetime images of NAD(P)H and FAD. The system uses two-photon excitation by a dual-wavelength femtosecond fibre laser. The two wavelengths of the laser, 780 nm and 880 nm, are multiplexed synchronously with the frames or the lines of the scan. The recording system uses two parallel TCSPC FLIM channels, detecting from 420 to 475 nm and 480 to 600 nm. By using the multiplexing functions of the TCSPC modules, separate images for NAD(P)H and FAD are recorded. A third image is obtained for the SHG of the 880 nm laser wavelength. Data analysis delivers images of the amplitude-weighted lifetime, tm, the component lifetimes, t1 and t2, the amplitudes of the components, a1 and a2, the amplitude ratio, a1/a2, and the fluorescence-lifetime redox ratio (FLIRR), a2nadh/a1fad. We demonstrate the performance of the system for metabolic imaging of mammalian skin.
In this work, we have tested the optimal estimation (OE) algorithm for the reconstruction of the optical properties of a two-layered liquid tissue phantom from time-resolved single-distance measurements. The OE allows a priori information, in particular on the range of variation of fit parameters, to be included. The purpose of the present investigations was to compare the performance of OE with the Levenberg–Marquardt method for a geometry and real experimental conditions typically used to reconstruct the optical properties of biological tissues such as muscle and brain. The absorption coefficient of the layers was varied in a range of values typical for biological tissues. The reconstructions performed demonstrate the substantial improvements achievable with the OE provided a priori information is available. We note the extreme reliability, robustness, and accuracy of the retrieved absorption coefficient of the second layer obtained with the OE that was found for up to six fit parameters, with an error in the retrieved values of less than 10%. A priori information on fit parameters and fixed forward model parameters clearly improves robustness and accuracy of the inversion procedure.
The nEUROPt protocol is one of two new protocols developed within the European project nEUROPt to characterize the performances of time-domain systems for optical imaging of the brain. It was applied in joint measurement campaigns to compare the various instruments and to assess the impact of technical improvements. This protocol addresses the characteristic of optical brain imaging to detect, localize, and quantify absorption changes in the brain. It was implemented with two types of inhomogeneous liquid phantoms based on Intralipid and India ink with well-defined optical properties. First, small black inclusions were used to mimic localized changes of the absorption coefficient. The position of the inclusions was varied in depth and lateral direction to investigate contrast and spatial resolution. Second, two-layered liquid phantoms with variable absorption coefficients were employed to study the quantification of layer-wide changes and, in particular, to determine depth selectivity, i.e., the ratio of sensitivities for deep and superficial absorption changes. We introduce the tests of the nEUROPt protocol and present examples of results obtained with different instruments and methods of data analysis. This protocol could be a useful step toward performance tests for future standards in diffuse optical imaging.
Performance assessment of instruments devised for clinical applications is of key importance for validation and quality assurance. Two new protocols were developed and applied to facilitate the design and optimization of instruments for time-domain optical brain imaging within the European project nEUROPt. Here, we present the “Basic Instrumental Performance” protocol for direct measurement of relevant characteristics. Two tests are discussed in detail. First, the responsivity of the detection system is a measure of the overall efficiency to detect light emerging from tissue. For the related test, dedicated solid slab phantoms were developed and quantitatively spectrally characterized to provide sources of known radiance with nearly Lambertian angular characteristics. The responsivity of four time-domain optical brain imagers was found to be of the order of 0.1 m2 sr. The relevance of the responsivity measure is demonstrated by simulations of diffuse reflectance as a function of source-detector separation and optical properties. Second, the temporal instrument response function (IRF) is a critically important factor in determining the performance of time-domain systems. Measurements of the IRF for various instruments were combined with simulations to illustrate the impact of the width and shape of the IRF on contrast for a deep absorption change mimicking brain activation.
The optical properties of the human head in the range from 600 nm to 1100 nm have been non-invasively in-vivo investigated by various research groups using different diffuse optics techniques and data analysis methods.
Novel protocols were developed and applied in the European project “nEUROPt” to assess and compare the performance
of instruments for time-domain optical brain imaging and of related methods of data analysis. The objective of the first
protocol, “Basic Instrumental Performance”, was to record relevant basic instrumental characteristics in a direct way.
The present paper focuses on the second novel protocol (“nEUROPt” protocol) that was devoted to the assessment of
sensitivity, spatial resolution and quantification of absorption changes within inhomogeneous media. It was implemented
with liquid phantoms based on Intralipid and ink, with black inclusions and, alternatively, in two-layered geometry.
Small black cylinders of various sizes were used to mimic small localized changes of the absorption coefficient. Their
position was varied in depth and lateral direction to address contrast and spatial resolution. Two-layered liquid phantoms
were used, in particular, to determine depth selectivity, i.e. the ratio of contrasts due to a deep and a superficial
absorption change of the same magnitude. We introduce the tests of the “nEUROPt” protocol and present exemplary
results obtained with various instruments. The results are related to measurements with both types of phantoms and to
the analysis of measured time-resolved reflectance based on time windows and moments. Results are compared for the
different instruments or instrumental configurations as well as for the methods of data analysis. The nEUROPt protocol
is also applicable to cw or frequency-domain instruments and could be useful for designing performance tests in future
standards in diffuse optical imaging.
KEYWORDS: Brain, Luminescence, Indocyanine green, In vivo imaging, Tissues, Neuroimaging, Biomedical optics, Fluorescence spectroscopy, Picosecond phenomena, Signal to noise ratio
Non-invasive detection of fluorescence from the optical tracer indocyanine green is feasible in the adult human brain when employing a time-domain technique with picosecond resolution. A fluorescence-based assessment may offer higher signal-to-noise ratio when compared to bolus tracking relying on changes in time-resolved diffuse reflectance. The essential challenge is to discriminate the fluorescence originating from the brain from contamination by extracerebral fluorescence and hence to reconstruct the bolus kinetics; however, a method to reliably perform the necessary separation is missing. We present a novel approach for the decomposition of the fluorescence contributions from the two tissue compartments. The corresponding sensitivity functions pertaining to the brain and to the extracerebral compartment are directly derived from the in-vivo measurement. This is achieved by assuming that during the initial and the late phase of bolus transit the fluorescence signal originates largely from one of the compartments. Solving the system of linear equations allows one to approximate time courses of a bolus for each compartment. We applied this method to repetitive measurements on two healthy subjects with an overall 34 boluses. A reconstruction of the bolus kinetics was possible in 62% of all cases.
We report on the development of a scanning non-contact brain imager, based on a novel technique in time-resolved nearinfrared
spectroscopy, i.e. the null source-detector distance approach. Our concept is designed to image an area of about
10 cm2 with small adjustable scanning steps, i.e. a high density of mapping points can be realized. The feasibility of the
proposed method was tested with a single-point confocal optical setup without beam scanning so far. A set of test
measurements was performed on a liquid phantom with a small black polyvinyl chloride (PVC) cylinder as a target,
which was translated in X direction to emulate the optical scanning and estimate lateral spatial resolution, and in Z
direction to estimate the depth sensitivity of the instrument. The problem of dominance of early photons at null sourcedetector
separation was solved by applying a fast time-gated detector to detect late only photons. Two fast-gated
detectors, a newly developed state-of-art time-gated single-photon avalanche photodiode (tgSPAD) and commercially
available fast-gated intensified CCD (iCCD) camera, were compared against each other. It was shown that, due to its
better dynamic range, the tgSPAD is capable to detect later photons than the iCCD camera, and hence, a scanning system
equipped with the time-gated SPAD has better depth sensitivity. Thus the time-gated SPAD is the detector of choice for
further development of the non-contact confocal brain scanner.
Functional near-infrared spectroscopy (fNIRS) is a commonly used technique to measure the cerebral vascular response
related to brain activation. It is known that systemic physiological processes, either independent or correlated with the
stimulation task, can influence the optical signal making its interpretation challenging. The aim of the present work is to
investigate the impact of task-evoked changes in the systemic physiology on fNIRS measurements for a cognitive
paradigm. For this purpose we carried out simultaneous measurements of time-domain fNIRS on the forehead and
systemic physiological signals, i.e. mean blood pressure, heart rate, respiration, galvanic skin response, scalp blood flow
(flux) and red blood cell (RBC) concentration changes. We performed measurements on 15 healthy volunteers during a
semantic continuous performance task (CPT). The optical data was analyzed in terms of depth-selective moments of
distributions of times of flight of photons through the tissue. In addition, cerebral activation was localized by a
subsequent fMRI experiment on the same subject population using the same task. We observed strong non-cerebral task-evoked
changes in concentration changes of oxygenated hemoglobin in the forehead. We investigated the temporal
behavior and mutual correlations between hemoglobin changes and the systemic processes. Mean blood pressure (BP),
galvanic skin response (GSR) and heart rate exhibited significant changes during the activation period, whereby BP and
GSR showed the highest correlation with optical measurements.
We present a multi-laboratory comparison of several independent forward solvers used for photon migration
through layered media. Two main categories of forward solvers are presented: Monte Carlo procedures and
solutions of the diffusion equation for the time domain. For Monte Carlo we have included four independent
codes. For the solutions of the diffusion equation, we have presented: two semi-analytical approaches based
on the Green's function method and one solution obtained with the finite element method. The comparisons
between the different time-dependent solutions were performed for a two-layer medium.
To facilitate the design and optimization of instruments for time-domain optical brain imaging within the European
project "nEUROPt", the performance of various instruments is assessed and compared. This type of instruments relies on
picosecond lasers with high repetition rates, fast detectors and time-correlated single photon counting. The first step of
the assessment included a number of basic tests that are related to parameters of the source, to the differential
nonlinearity of the timing electronics and to the temporal instrument response function (IRF). An additional test has been
devised to measure the responsivity of the detection system, i.e. the overall efficiency to collect and detect light
emerging from tissue. Dedicated solid slab phantoms have been developed and quantitatively spectrally characterized to
provide sources of known radiance with nearly Lambertian angular characteristics. The wavelength-dependent
transmittance factor of these phantoms was of the order of 1020/(W s m2sr). Measurements of the responsivity of the
detection systems of three time-domain optical brain imagers tested yielded similar values of the order of 0.1 mm2sr.
We present results of a clinical study on bedside perfusion monitoring of the human brain by optical bolus tracking. We measure the kinetics of the contrast agent indocyanine green using time-domain near-IR spectroscopy (tdNIRS) in 10 patients suffering from acute unilateral ischemic stroke. In all patients, a delay of the bolus over the affected when compared to the unaffected hemisphere is found (mean: 1.5 s, range: 0.2 s to 5.2 s). A portable time-domain near-IR reflectometer is optimized and approved for clinical studies. Data analysis based on statistical moments of time-of-flight distributions of diffusely reflected photons enables high sensitivity to intracerebral changes in bolus kinetics. Since the second centralized moment, variance, is preferentially sensitive to deep absorption changes, it provides a suitable representation of the cerebral signals relevant for perfusion monitoring in stroke. We show that variance-based bolus tracking is also less susceptible to motion artifacts, which often occur in severely affected patients. We present data that clearly manifest the applicability of the tdNIRS approach to assess cerebral perfusion in acute stroke patients at the bedside. This may be of high relevance to its introduction as a monitoring tool on stroke units.
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