There is a demand for technologies able to assess the perfusion of surgical flaps quantitatively and reliably to avoid ischemic complications. The aim of this study is to test a new high-speed high-definition laser Doppler imaging (LDI) system (FluxEXPLORER, Microvascular Imaging, Lausanne, Switzerland) in terms of preoperative mapping of the vascular supply (perforator vessels) and postoperative flow monitoring. The FluxEXPLORER performs perfusion mapping of an area 9×9 cm with a resolution of 256×256 pixels within 6 s in high-definition imaging mode. The sensitivity and predictability to localize perforators is expressed by the coincidence of preoperatively assessed LDI high flow spots with intraoperatively verified perforators in nine patients. 18 free flaps are monitored before, during, and after total ischemia. 63% of all verified perforators correspond to a high flow spot, and 38% of all high flow spots correspond to a verified perforator (positive predictive value). All perfused flaps reveal a value of above 221 perfusion units (PUs), and all values obtained in the ischemic flaps are beneath 187 PU. In summary, we conclude that the present LDI system can serve as a reliable, fast, and easy-to-handle tool to detect ischemia in free flaps, whereas perforator vessels cannot be detected appropriately.
Laser Doppler Imaging (LDI) is a non-invasive optical technique used for assessing the blood flow in biological tissue. This
technique allows visualizing 2D distribution of blood flow over an extended area of tissue, e.g. human skin. We have
developed a new high-speed instrument for full-field blood flow laser Doppler imaging. The new imager delivers high-resolution
flow images every 0.7 to 11 seconds, depending on the number of points in the acquired time-domain signal (32-512 points) and the image resolution (256×256 or 512×512 pixels). This new imaging modality utilizes a digital integrating
CMOS image sensor to detect Doppler signals in a plurality of points over the area illuminated by a divergent laser beam of
a uniform intensity profile. The integrating property of the detector improves the signal-to-noise ratio of the measurements,
which results in high-quality flow images.
We made a series of measurements in vitro to test the performance of the system in terms of bandwidth, SNR, etc.
Subsequently we give some examples of flow-related images measured on human skin, thus demonstrating the performance
of the imager in vivo. The perspectives for future implementations of the imager for clinical and physiological applications
will be discussed.
Laser Doppler (LDI) and laser speckle imaging (LSI) are two optical non-invasive techniques that are used to obtain 2D maps of blood flow in biological tissues. Each of these techniques has some benefits and drawbacks for measuring the blood flow. LSI is a true real-time imaging technique, but less sensitive to changes of flow parameters such as speed and concentration. In contrast, LDI has superior measurement accuracy but it is not a real-time technique. Recently we have developed a blood-flow imaging system that combines both imaging modalities with a gain in speed and accuracy. Using a single integrating CMOS image sensor for measuring both the Doppler signal spectrum and the image speckle contrast flow-map images are produced. In LDI mode, the flow map refresh rate is 1.2 seconds per 256x256 pixels image. In LSI mode the frame rate is 10 flow-map images per second. We present the basic design and in-vivo performance of this new hybrid imaging system. Subsequently, we discuss the potential of the new instrument for future implementations into clinical research.
We describe the design and performance of a new full-field high-speed laser Doppler imaging system developed for mapping and monitoring of blood flow in biological tissue. The total imaging time for 256x256 pixels region of interest is 1.2 seconds. An integrating CMOS image sensor is utilized to detect Doppler signal in a plurality of points simultaneously on the sample illuminated by a divergent laser beam of a uniform intensity profile. The integrating property of the detector improves the signal-to-noise ratio of the measurement, which results in high-quality flow-images provided by the system. The new technique is real-time, non-invasive and the instrument is easy to use.
The wide range of applications is one of the major challenges for a future application of the imager. High-resolution high-speed laser Doppler perfusion imaging is a promising optical technique for diagnostic and assessing the treatment effect of the diseases such as e.g. atherosclerosis, psoriasis, diabetes, skin cancer, allergies, peripheral vascular diseases, skin irritancy and wound healing. We present some biological applications of the new imager and discuss the perspectives for the future implementations of the imager for clinical and physiological applications.
KEYWORDS: Doppler effect, Blood circulation, Skin, Imaging systems, 3D metrology, 3D image processing, In vivo imaging, Tissue optics, Wound healing, Digital micromirror devices
We present a combination of topography measurements based on digital fringe projection and blood flow imaging based on Laser Doppler Imaging (LDI). Both techniques are optical, non-contact and high-speed whole-field methods well suited for in-vivo measurements on the skin. Laser Doppler perfusion imaging is an interferometric technique used for visualization of two-dimensional (2D) maps of blood flow. Typically the measured sample has a surface with a specific 3D relief. In many cases the sample relief can be of importance for correct interpretation of the obtained perfusion data. We combined the topography and the blood flow data obtained from the same object. The structural information provided by the topography is completed by the functional images provided by LDI.
KEYWORDS: Doppler effect, Imaging systems, Signal processing, Digital signal processing, CMOS sensors, Sensors, Skin, Signal detection, CMOS cameras, Blood circulation
An apparatus for parallel, full-field, laser Doppler perfusion imaging has been developed and applied for measurements of blood flow in human skin. This new imaging system allows obtaining 2D flow maps or to monitor flux signals from a plurality of separate predetermined points simultaneously with a 2D matrix of photodetectors. The uniform area illumination is achieved with a highly multimode optical fiber. The detection part of the system is based on an intelligent CMOS camera (iMVS-155) with a built-in digital signal processor. The imaging time of the system is 4 times quicker than for conventional scanning laser Doppler imager. The performance of the system was tested on artificial samples demonstrating linear response to the velocity up to 3 mm/sec. Also, the results of the measurements obtained with the new system on human skin are reported.
We present parallel single molecule detection (SMD) and fluorescence correlation spectroscopy (FCS) experiments with a fully integrated complementary metal oxide semiconductor (CMOS) single-photon 2×2 detector array. Multifocal excitation is achieved with a diffractive optical element (DOE). Special emphasis is placed on parallelization of the total system. The performance of the novel single-photon CMOS detector is investigated and compared to a state-of-the-art single-photon detecting module [having an actively quenched avalanche photodiode (APD)] by measurements on free diffusing molecules at different concentrations. Despite the order of magnitude lower detection efficiency of the CMOS detector compared to the state-of-the-art single-photon detecting module, we achieve single molecule sensitivity and reliably determine molecule concentrations. In addition, the CMOS detector performance for the determination of the fraction of slowly diffusing molecules in a primer solution (two-component analysis) is demonstrated. The potential of this new technique for high-throughput confocal-detection-based systems is discussed.
We present multipoint Fluorescence Correlation Spectroscopy (FCS) experiments with a fully integrated Complementary Metal Oxide Semiconductor (CMOS) single photon 2x2 detector array. Multifocal excitation was achieved with a diffractive optical element (DOE). Special emphasis was put on parallelization of the total system. In particular the performance of the single-photon CMOS detector was investigated and compared to a state-of-the art single-photon detecting module (actively quenched avalanche photo diode) by measurements on free diffusing molecules at different concentrations. The potential of our new technique for high throughput FCS based systems is discussed.
In this paper we present recent spectroscopic studies using a Solid Immersion Lens for Fluorescent Correlation Spectroscopy measurements. We compare the performance of the Solid Immersion Lens confocal microscope built-up in our group to the performance of a conventional confocal microscope used for FCS. The novelty of the new SIL-FCS microscope is a system containing a conventional objective (NA = 0.6) combined with a Solid Immersion Lens used for single molecule experiment. Important parameters for single molecule experiments such as collection efficiency and excitation field confinement are investigated for different modes of the SIL objective system.
KEYWORDS: CMOS sensors, Microscopy, Digital signal processing, Signal processing, Spatial frequencies, Imaging systems, Microscopes, Signal detection, Objectives, Structured light
Real-time optically sectioned microscopy is demonstrated using an AC-sensitive detection concept realized with smart CMOS image sensor and structured light illumination by a continuously moving periodic pattern. We describe two different detection systems based on CMOS image sensors for the detection and on-chip processing of the sectioned images in real time. A region-of-interest is sampled at high frame rate. The demodulated signal delivered by the detector corresponds to the depth discriminated image of the sample. The measured FWHM of the axial response depends on the spatial frequency of the projected grid illumination and is in the μm-range. The effect of using broadband incoherent illumination is discussed. The performance of these systems is demonstrated by imaging technical as well as biological samples.
Monitoring biological relevant reactions on the single molecule level by the use of fluorescent probes has become one of the most promising approaches for understanding a variety of phenomena in living organisms. By applying techniques of fluorescence spectroscopy to labelled molecules a manifold of different parameters becomes accessible i.e. molecular dynamics, energy transfer, DNA fingerprinting, etc... can be monitored at the molecular level.
However, many of these optical methods rely on oversimplified assumptions, for example a three-dimensional Gaussian observation volume, perfect overlap volume for different wavelength, etc. which are not valid approximations under many common measurement conditions. As a result, these measurements will contain significant, systematic artifacts, which limit their performance and information content.
Based on Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Spectroscopy we will present representative examples including a thorough signal analysis with a strong emphasis on the underlying optical principles and limitations. An outlook to biochip applications, parallel FCS and parallel Lifetime measurements will be given with cross links to optical concepts and technologies used in industrial inspection.
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