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.
Fluorescence Lifetime Imaging (FLIM) was introduced in 1993 by the company Becker & Hickl and is no longer a myth. However, some myths have arisen around the topic of FLIM over time. Since the Mythbusters have not yet dealt with this topic, I would like to take up and classify some of these myths in this presentation. This includes questions like, what significantly influences the measurement time in my FLIM experiment or also in which kind of FLIM experiment does a high temporal precision help me. As an example for a FLIM hot topic, I will also show metabolic imaging. Finally, I will briefly introduce new Becker & Hickl hardware and what it can do for you.
Bacterial infections often pose a time-sensitive concern; however, the current diagnostic methods are comprised of lengthy processes. This study explores label-free imaging techniques based on two-photon excitation of intrinsic molecules found in various bacterial species. Specifically, two-photon excitation microscopy (TPEF) and fluorescence lifetime imaging microscopy (FLIM) are employed. These methods have been extensively utilized in the area of animal cells, yielding favorable outcomes, yet their application to bacteria remains largely unexplored. Analogous to the work on animal cells, initial attention is directed towards the metabolic coenzymes, namely nicotinamide adenine dinucleotide (phosphate) (NADPH) and flavin adenine dinucleotide (FAD). The extra time component of the FLIM setup was used to further investigate differences between the decay curves of the emitted autofluorescence in their different growth phases, corresponding to different internal microenvironments. Bacteria were excited at 740nm and 900nm and TPEF and FLIM signals were recorded with fitting bandpass filters, respectively. Different species (Escherichia coli K12, Pseudomonas fluorescens and Staphylococcus aureus) and different growth phases were examined to identify characteristic signal combinations. The two image channels and both wavelengths are compared and plotted against each other. The results show that the bacteria can already be classified by their autofluorescence signals. The growth phase seems to have less influence than the species. Based on this data, a classification is made to uniquely identify them. For this purpose, the database will next be expanded to include additional species and the classification will be automated.
Using Becker & Hickl TCSPC FLIM systems with fast TCSPC modules and fast detectors, we found ultra-fast fluorescence decay components in biological material, such as mushroom spores, pollen grains, plant tissue, and malignant melanoma. The component decay times are on the order of 10 to 50 ps, the amplitudes range from about 60% to more than 99%. In general, the shortest lifetimes and the largest amplitudes of the fast component were found in strongly coloured material, such as black mushroom spores and melanoma tissue. However, the lifetime not always correlates with the colour. In particular, a fast decay component was not found in tissue from basal cell papilloma, although it is dark brown. This may open a way to identify melanoma tissue and melanoma cells, and thus provide a new tool to investigate melanoma progression.
Clinical practitioners consider an abnormal cell metabolism as hallmark of carcinogenesis. Cellular energy metabolism is accessible by imaging of the fast autofluorescence decay of the endogenous fluorophore NADH. This technique is called metabolic fluorescence lifetime imaging (metabolic FLIM), best performed with multiphoton excitation and the rapid, precise and quantitative TCSPC technology from Becker&Hickl GmbH. However, conventional multiphoton FLIM microscopes rely on surface layer tissue access or excised tissue samples. Imaging inside the body for medical diagnostics is routinely accomplished with endoscopes. Here we present multiphoton metabolic FLIM of NADH performed through an endoscope, with significant applications in oncology and beyond.
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