Fabry-Perot tomographs have captured compelling photoacoustic images as they combine small element sizes with high acoustic sensitivity and a broad frequency response. A photoacoustic tomograph based on a sCMOS camera and a Fabry-Perot sensor with uniform optical thickness was developed. The influence of camera parameters on the e.g., spatial distribution of the acoustic sensitivity was evaluated. The imaging capabilities were demonstrated by capturing images of PA phantoms.
A novel imaging system is presented, bridging 1P confocal- and MP-FLIM in one measurement setup, for a non-invasive, early and accurate detection of tumor tissues. Integrating a unit consisting of an acousto-optical modulator (AOM) and a second harmonic generator (SHG), together with a tunable Ti:Sa laser, provides stabilized femtosecond pulses from 340 nm to 1300 nm with high pointing precision for microscopy, for the first time. This facilitates high precision metabolic imaging of individual cells through 1P FLIM, and deep tissue imaging through MP-FLIM. While SHG reveales insights into non-centrosymmetric structures in tissues, simultaneously. The system offers 2 confocal FLIM detection channels, 2 non-descanned detection channels for MP FLIM and 1 detection channel for SHG light. This talk emphasizes the instrumentation of the system, followed by presenting obtained metabolic images and optimized analysis of data images.
KEYWORDS: Fluorescence lifetime imaging, 3D image processing, 3D metrology, Data analysis, Confocal microscopy, Cancer detection, Cancer, Autofluorescence
Fluorescence lifetime imaging (FLIM) is a versatile tool, even detecting cancers without staining agent from NADH and FAD autofluorescence. While confocal techniques provide wonderful images of 2D structures, they risk neglecting crucial details in the out-of-image-plane dimension. Thorough understanding of three-dimensional samples is obtained through “Z-stack measurements”, coupled with appropriate functional sectioning capabilities.
Here, we demonstrate the combination of Becker&Hickl’s advanced SPCimage NG data analysis suite with an easy-to-follow ImageJ/Fiji 3D rendering workflow to generate volume FLIM images of real samples. Combined with Becker&Hickl’s renowned lifetime measurement precision 3D FLIM elevates metabolic imaging and deep-tissue work.
Fluorescence lifetime imaging (FLIM) is a popular and versatile tool for in vivo research with minimal disruption of the biological system. Recently, the desire has grown to investigate dynamic processes with FLIM, creating a need for video rate FLIM contrast measurements. Because the potential applications are manifold, the different information depth of video FLIM and ‘detailed, slow’ FLIM measurements must be considered.
This talk highlights the complimentary nature of video rate and standard FLIM measurements with practical examples, measured with the same machine. Strengths and limitations of each measurement mode are highlighted, and best-practice analysis strategies are determined for each.
Fabry-Pérot (FP) sensors have enabled high resolution 3D photoacoustic (PA) imaging in backward mode. However, raster-scanning of the interrogation laser beam across the sensor can result in slow 3D image acquisition. To overcome this limitation, parallelized PA signal acquisition can be used for which FP sensors with uniform optical thickness are required. In this work, the optical thickness is tuned a) irreversibly through the use of a photopolymer host matrix and b) actively using embedded electro-optic (EO) chromophores. Polymer spacers (5 μm) were deposited using spin coating and sandwiched between two dielectric mirrors and transparent ITO electrodes. The employed polymer guest-host system consists of an EO chromophore (2-methyl-4-nitroaniline) and poly(vinyl cinnamate). EO tuneability was induced using contact poling and a tuneability of 68 pm was demonstrated. The optical thickness was homogenised by raster scanning a UV beam whilst varying the exposure time across a 4 mm2 detection aperture.
Fabry-Pérot (FP) polymer film sensors exhibit small element sizes, high acoustic sensitivity, transparency and flat frequency response to enable high resolution 3D photoacoustic (PA) imaging in backward mode. However, conventional raster scan interrogation can result in slow data acquisition (several min for 3D images) compared to parallelized piezoelectric detector arrays. To address this limitation, parallelization using a camera-based readout of FP sensors is investigated. This approach requires the optical thickness of the polymer spacer to be sufficiently uniform over the scan area to obtain high acoustic sensitivity for all active elements. Since the deposition of passive polymer layers with sufficient homogeneity of thickness is challenging, the use of electro-optically (EO) or piezoelectric (PE) tunable polymer film spacers is investigated. The spacers are sandwiched between two dielectric mirrors and transparent electrodes to form an FP sensor. In this work, spin coated guest-host systems consisting of EO chromophores (2-methyl-4-nitroaniline) embedded in a PMMA matrix, and thermally evaporated PE film spacers (PVDF) were examined. Both systems were electrically poled using a corona discharge. The optical transfer function, the transmission spectrum of the excitation passband from 600 nm to 1100 nm and the tuning range of the FP sensors were determined. Furthermore, the detection of PA waves was demonstrated. Tunable FP sensors in conjunction with camera-based interrogation techniques have the potential to provide 3D image acquisition times on the order of seconds.
Tomographic photoacoustic (PA) images acquired using a Fabry-Perot (FP) based scanner offer high resolution and image fidelity but can result in long acquisition times due to the need for raster scanning. To reduce the acquisition times, a parallelised camera-based PA signal detection scheme is developed. The scheme is based on using a sCMOScamera and FPI sensors with high homogeneity of optical thickness. PA signals were acquired using the camera-based setup and the signal to noise ratio (SNR) was measured. A comparison of the SNR of PA signal detected using 1) a photodiode in a conventional raster scanning detection scheme and 2) a sCMOS camera in parallelised detection scheme is made. The results show that the parallelised interrogation scheme has the potential to provide high speed PA imaging.
Cancer is one of the leading causes for morbidity and mortality worldwide. Therefore, efforts are concentrated on cancer detection in an early stage to enhance survival rates for cancer patients. A certain intraoperative navigation in the tumor border zone is also an essential task to lower the mortality rate after surgical treatment. Molecular spectroscopy methods proved to be powerful tools to differentiate cancerous and healthy tissue. Within our project comparison of different vibration spectroscopy methods were tested to select the better one or to reach synergy from their combination.
One key aspect was in special fiber probe development for each technique. Using fiber optic probes in Raman, MIR and NIR spectroscopy is a very powerful method for non-invasive in vivo applications. Miniaturization of Raman probes was achieved by deposition of dielectric filters directly onto the silica fiber end surfaces. Raman, NIR and MIR spectroscopy were used to analyze samples from kidney tumors. The differentiation between cancer and healthy samples was successfully obtained by multivariate data analysis.
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