This conference presentation was prepared for SPIE BiOS, 2024.

Our two-photon light-sheet microscopy approach combines better engineering of the detection objective’s point-spread-function, with optimized computational tools for hyperspectral and fluorescence lifetime imaging, to study living specimens. Our goal is to animate the wealth of high-throughput molecular data to better understand complex events, ranging from embryonic development to disease processes. This two-photon SPIM approach generates images with more than a10-fold improved imaging speed & sensitivity than conventional light sheet microscopes, resulting in 4D (3D over time) cell and molecular images that offer sufficient speed and resolution to unambiguous trace cell lineages, movements and signals in intact systems. Our new multispectral image analysis tools exceed the performance of our previous work by orders of magnitude in speed, error propagation and accuracy. Novel denoising strategies for hyperspectral and FLIM datasets, some using machine learning, permit imaging at far lower light levels, yielding rapid and unambiguous analyses without perturbing even fragile multiplex-labeled specimens.

Multi-photon imaging offer a large potential to characterize both the morphology and molecular composition (= morphochemistry) of cells or tissue. Particularly interesting is the establishment of multimodal nonlinear microscopy for medical purposes. Investigating tissues of different morphochemistry underlines the versatility of non-linear multimodal imaging, e.g., to distinguish between healthy tissue and tissue altered by tumors, or drugs. Here, we show that the interplay between multimodal nonlinear imaging (CARS, TPEF, SHG) either in a microscopic or endoscopic setting in combination with innovative artificial intelligence approaches and fs-laser ablation opens exciting new ways for an intraoperative histopathological tissue analysis and selective removal.

Users and suppliers of multiphoton microscopy recognize the rapid growth in use-cases of non-linear imaging techniques – from advanced neuroscience to embryology, from H&E histology augmentation/replacement to clinical endoscopy. This variety of applications, together with the expanding number of probes, beam manipulation, delivery and excitation schemes, is a strong driving force for the continuous advancements in parameters and functionality of the commercial femtosecond laser sources used for these applications. In this presentation we will review novel developments in femtosecond lasers and their interplay with emerging techniques. Among these, we will look at 3-photon excitation microscopy, 2-photon holographic imaging and optogenetic stimulation, and their enablement of all-optical investigation of neural activities. We will also describe how an increasing level of optical integration helps to streamline the laser usage in the laboratory and (pre-)clinical environment.

Multiphoton microscopy has seen widespread adoption from the research community to address the challenges associated with image collection many hundreds of micrometers into living tissue. Over time, technology advancements have contributed to the evolution of 2-photon, 3-photon and other nonlinear microscopy techniques. Deep Tissue imaging necessitates wavelengths spanning from the visible to the near-IR to achieve desirable resolution. Excitation requires a femtosecond laser pulse to minimize out-of-focus fluorescence. Image collection requires novel broadband microscope objectives which must transmit both the near-IR excitation wavelengths and the subsequent visible fluorescence. During this presentation, a concise overview of the historical development of these technologies will be presented through real-life examples that demonstrate how modern research facilities are leveraging these advancements.

2-Photon imaging has dramatically expanded its reach in neuroscience-related applications thanks to the development of genetically encoded functional probes that report cellular activity and activates/silence individual neurons (optogenetics). The combination of 3D spatial resolution afforded by 2-photon imaging and the proliferation of advanced scanning and scanless methodologies enable powerful parallel functional imaging of thousands of individually selected neurons. Faster and parallel imaging of large cell populations requires femtosecond lasers with higher average power and energy, but also improved pulse-to-pulse stability all along the required tuning ranges. Light Conversion has pioneered the development of femtosecond wavelength-tunable devices as well as of high-energy and -power Ytterbium lasers ad amplifiers. In this presentation we highlight how these developments are powering advanced imaging techniques using reporting and optogenetic probes.

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.

PicoQuant`s new confocal microscope Luminosa combines state-of-the-art hardware with cutting edge software to deliver high quality data while simplifying daily operation. Luminosa’s rapidFLIM hardware can record several frames per second, which the software handles with a novel dynamic binning format. In combination with GPU-accelerated algorithms, this enables high-speed automated analysis of FLIM images. We demonstrate the combination of PicoQuant’s latest multi-channel TCSPC device and a cooled high-performance 23-pixel SPAD-array developed jointly with Pi Imaging Technologies for time-resolved confocal image scanning microscopy (FLIM-ISM). We also discuss how advanced data processing can be applied to FLIM-ISM for additional performance gains. For certain applications widefield imaging offers unique advantages. Here we show how the combination of a powerful PicoQuant laser and the novel SPAD512S camera from Pi Imaging Technology facilitates time-resolved widefield imaging. We benchmark the camera’s performance, demonstrating video rate acquisition speeds with minimal photobleaching.

We present a novel approach to achieve high-speed depth-resolved two-photon imaging through the development of a deep-learning-based temporal-focusing two-photon microscope utilizing the De-scattering with Excitation Patterning (DEEP) method, referred to as DEEP-Line. DEEP-Line incorporates a line-scanning scheme, widefield detection utilizing a high-speed Silicon Photomultiplier array, and employs deep-learning-based image reconstruction. The performance of our system is validated using diverse biological samples. Our imaging method achieves orders of magnitude improvement in speed by reducing excitation patterns to several tens and employing MHz parallel detections. Furthermore, our approach can enable fluorescence lifetime imaging and enhances axial resolution.

Multiphoton imaging typically requires a complex multi-device laser system, a large optical table, and skilled staff. This reality encumbers neuroscience and other biomedical applications. Here, we will present our direction to provide a turn-key solution for three-photon (3P) imaging. The application-optimized laser provides the anticipated reliability and simplicity but also maintains tunability in wavelength and dispersion control, which seems quite important in the current state of the application. We will explicitly dig into details of long-term power and pulse-to-pulse energy stability, and how that directly impacts your imaging results.

Despite the long development of light microscopy, it remains challenging to visualize fragile living systems without labeling or specific sample preparation. Taking hint from the supercontinuum generation in ultrafast laser engineering, we generated ‘supercontinuum’ fluorescence from unlabeled live samples before nonlinear phototoxicity onset. Our supercontinuum intrinsic fluorescence imaging (SCIFI) identified drug cytotoxicity in real-time, demystified embryogenesis before/after implantation, performed omics-like cell phenotyping or tissue histopathology, tracked freely moving small organisms, scanned brain area for targeted patching, and provided new insights on photosynthesis. This demonstration paves the way for a mobile microscopy facility to freely exploit molecular biology across life sciences.

This conference presentation was prepared for SPIE BiOS, 2024.

Two-photon excitation fluorescence microscopy (2PEM) has transformed biological imaging, offering deeper tissue penetration, reduced photodamage, and improved 3D resolution. Here, we explore a cutting-edge 15 fs ultrafast laser for enhanced 2PEM. Our setup includes a single-photon avalanche diode (SPAD) array for image scanning microscopy (ISM) and a 775 nm pulsed laser for STED microscopy. The ultrashort excitation pulse enables higher peak powers and enhanced image clarity due to wider bandwidth. ISM with a 7x7 SPAD array (Genoa Instruments s.r.l., Genoa, Italy) provides crucial insights into imaging conditions, ensuring high-resolution and high-SNR imaging under sub-optimal and deep imaging scenarios. Additionally, our 775 nm pulsed laser implements STED for super-resolution 2PEF imaging beyond the diffraction limit. Integrating these techniques in a single microscope platform unlocks powerful nanoscale investigations in tissues, uncovering complex cellular interactions and paving the way for breakthroughs in neurobiology, developmental biology, and cancer research.

Optical microscopy has been indispensable for physiologically visualizing neuronal structure and function, while it remains a challenge since the limited diffraction resolution and restricted imaging depth. Nonlinear multifocal structured illumination microscopy (MSIM) provides resolution-doubled images and good penetration, but its performance is seriously degraded in deep tissues. Thus, we present a non-inertial scanning nonlinear MSIM system combined with adaptive optics (AO) to realize super-resolution imaging with aberration correction in vivo. It is indispensable for visualizing various synaptic spines and detailed morphological changes of neurons in vivo. Using the nonlinear MSIM for SHG imaging, collagen fiber alignments were quantified in various tissues, demonstrating its feasibility for identifying collagen characteristics.

Non-linear optical (NLO) microscopy techniques like coherent anti-Stokes Raman scattering (CARS) are highly effective tools for label-free vibrational imaging, allowing for chemical analysis of biological samples in their native state. We introduce video-rate wide-field CARS microscopy over a vast field of view (tens of micrometers) to enable real-time analysis of fast biological dynamics with down to few-millisecond time resolution. We generate stable broadband Stokes pulses in a YAG crystal using an amplified ytterbium laser source delivering 260-fs high-energy (μJ-level) pulses in the near-infrared. Our system combines fast and tunable single-wavelength real-time imaging with the acquisition of hypercubes, chemically more information-rich.

A multiplane two-photon microscope with non-descanned detection provides imaging speeds up to 200 MP/s with four spectral channels (800 MSpectra/s) with area imaging speeds of up to 30 mm2/s. This enables high volumetric throughput deep-tissue imaging at speeds approaching light-sheet imaging without sacrificing resilience to scattering and high axial resolution. Multiplane excitation is achieved by multifocal scanner-synchronous strip-scanning with tilted translation, while spherical lens arrays with custom SiPM array boards provide non-descanned detection that is both inexpensive and easy to spectrally multiplex.

We have shown that diseases including cancers and fibroses have significant changes in the collagen fibril and fiber structure. We have used machine learning methods to classify normal and diseased tissues based on the fiber morphology in SHG images, however the important features remain unknown. Using the StyleGAN framework, we trained the latent space and used PCA for this determination. We found that curvature and density were the most important attributes in ovarian cancer. We previously demonstrated qualitative differences in the fibril size and spacing in several tissues. We now developed a more complete computational model of 3D phasematching. This in conjunction with wavelength- dependent measurements of the spatial emission pattern, will afford the extraction of average fibril size and packing. Collectively these determinations will be broadly applicable to identifying characteristic diagnostic targets in a wide range of diseases.

Anaplasma phagocytophilum is the intracellular bacteria that causes Human Granulocytic Anaplasmosis (HGA). While the skin is known as the entry site for the bacteria, HGA is characterized by systemic infection of A. phagocytophilum, but it is unknown how a localized infection of A. phagocytophilum leads to HGA. Here, we use a multimodal microscopy approach to image collagen and immune cell alterations due to A. phagocytophilum infection. Second-harmonic generation imaging revealed collagen fiber density decreased at the tick bite of infected skin. Additionally, significant differences in autofluorescence lifetime were observed due to A. phagocytophilum infection of human myeloid HL-60 cells.

Imaging with third harmonic generation (THG) provides a powerful label-free approach to probing biological systems, particularly looking at structures formed by lipids. We present the first demonstration of THG widefield holographic microscopy. Holographic microscopy preserves the amplitude and phase of the measured field, which enables us to construct a synthetic spatial aperture with expanded field of view for complex valued THG images. Additionally, we implement a matrix approach postprocessing algorithm to reconstruct aberration-free THG holographic images by estimating and correcting for phase aberrations and scattering distortions.

We investigate the chiral response of type I collagen using polarimetric second harmonic generation (SHG) microscopy. By flipping the sample, we observe changes in the sign of the amplitude of the chiral susceptibility ratio (C), while its retardancy phase with respect to the achiral ratio (R) remains unchanged. Our findings suggest that the chiral response of collagen originates from the interaction between electric dipole moments and laser radiation. This study demonstrates the potential of polarimetric SHG microscopy for studying collagen properties and visualizing disease-related alterations.

Fluorescence lifetime imaging (FLIM) of metabolic coenzymes, as NAD(P)H and FAD and oxygen induced phosphorescence lifetime imaging (PLIM) of phosphorescent drugs or delayed FLIM (dFLIM) is now widely accepted to be one of the most important imaging methods for cell metabolism and oxygen. Different algorithms are developed and optimized for simultaneous FLIM/PLIM/dFLIM to get reproducible results. The interaction of light, drug and oxygen during PDT (photodynamic therapy) induces a cellular response, which could be followed by various techniques. The aim of this work was to simultaneously image the cellular response and oxygen consumption in living cells with subcellular resolution.

This study employs the optical redox ratio and fluorescence lifetime imaging microscopy of intrinsic metabolic coenzymes NAD(P)H and FAD to assess the metabolic state of human neutrophils in vitro, under a variety of activation conditions and targeted metabolic pathway inhibition at the single-cell level. By measuring effector functions such as oxidative burst, neutrophil activation could be confirmed. In addition, identical metabolic changes were detected in zebrafish larvae neutrophils following activation, confirming the validity of these findings in vivo.

This study addresses challenges in Optical Metabolic Imaging due to dim signals, overlapping spectra, and similar lifetimes of NADH and FAD autofluorescent molecules. A Phasor-based S-FLIM-SHG microscope is introduced, enabling simultaneous acquisition of Hyperspectral Imaging (HSI), Fluorescence Lifetime Imaging Microscopy (FLIM), and Second Harmonic Generation imaging (SHG). The microscope's design efficiently detects scattered photons from complex samples and it is particularly competent at detection SHG signal. A novel 5D-snapshot (x, y, z, τ, λ). metabolic imaging method is proposed, significantly reducing acquisition times and enhancing measurement accuracy. The method's versatility is demonstrated across diverse sample types, with potential implications for advancing optical metabolic imaging capabilities.

Herein, we will use Macroscopy Fluorescence Lifetime Forster Resonance Energy Transfer (MFLI FRET) to quantitatively image in vivo and non-invasively the efficacy of receptor-targeted drug delivery systems. We have established an antibody-to-target imaging pipeline to develop novel near-infrared (NIR)-labeled probes to interrogate and quantify the dynamics of drug-receptor binding in HER2 breast cancer tumor xenografts.

Fluorescence lifetime imaging (FLIM) is a sensitive tool to investigate protein-protein interactions in biology and may have some niche applications for early detection of cancer in liquid biospies. The gold-standard for such a technique is time-correlated single photon counting (TCSPC). For imaging, this has largely been exploited in laser scanning microscopy techniques. However, these imaging modalities are limited primarily by their particularly slow acquisition speeds compared to conventional imaging methodologies especially when the sample is sparse. We have developed a simple optical platform to exploit the sparseness of single molecule imaging and apply this to preliminary study of the heterodimer status of EGFR and HER3, in cell secreted vesicles (exosomes). In parallel we perform single EV trapping experiments with an ABEL trap to extend our spectroscopic analysis. We perform both fluorescence lifetime spectroscopy and SMLM on single exosomes in vitro to assess changes in the proclivity of heterodimer formation following Osimertinib treatment in lung cancer cells (H1975), which may elude to a possible functional role in cancer treatment resistance.

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.

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G-protein-coupled receptors (GPCRs) exhibit a variety of multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) can be applied to quantify the dynamics of individual receptor molecules in the presence or absence of different ligands. However, observation times of GPCRs, which are freely diffusing in solution, are limited to a few milliseconds. Here, we performed smFRET experiments on an active human A2A adenosine receptor reconstituted into a lipid nanodisc. We captured a single receptor with a confocal anti-Brownian electrokinetic trap (ABEL trap) and recorded smFRET time traces of up to seconds for individual receptors. Pulsed laser excitation enabled FRET donor lifetime recordings in parallel to confirm that conformational dynamics of the A2A adenosine receptor were causing smFRET fluctations. Influences of agonists as well as antagonists on transient conformational changes could be discriminated.

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.

Confocal microscopy is an essential tool in many academic disciplines due to its intrinsic sectioning capability. The combination with time-resolved single photon detectors and time-correlated single photon counting (TCSPC) devices has established it as the leading platform for time resolved investigation methods such as fluorescence lifetime imaging (FLIM). Recently, high-performance SPAD-arrays featuring few tens of pixels have become available. In this work we present the two central hardware building blocks: PicoQuant’s latest multi-channel TCSPC device and a cooled high-performance 23-pixel SPAD-array developed jointly with Pi Imaging Technologies. We discuss how these open up new possibilities in time-resolved confocal microscopy.

Chemical signaling is essential to information processing in the brain. The key events of this process in the mammalian brain are the exocytotic release of signaling molecules by one cell and its binding to a receptor molecule on the recipient cell, which then transduces the chemical message. Here we ask: if we take the receptors away, can there still be signaling? We find that the lipid bilayer responds to some signaling molecules, such as serotonin, and changes its order and mechanical properties. We then combine non-epifluorescence multiphoton UV with spectral confocal microscopy and show that these changes modulate membrane-mediated processes such as exo- and endocytosis. It also affects the functioning of non-cognate receptor proteins in the membrane. Nature appears to have optimized the membrane compositions of different organelles to tune them for neurotransmitter interactions. In summary, the lipid bilayer membrane can itself be a receptor for many signaling molecules. Potentially, our findings pave the way for a major new class of membrane-active but receptor-silent pharmacological agents that can affect biological function.

Quantitative time-resolved fluorescence techniques like FLIM are increasingly employed in fields like phase separation and cellular sensing. PicoQuant`s new microscope Luminosa combines state-of-the-art hardware with cutting edge software, delivering high quality data while simplifying daily operation. The software includes features like context-based workflows, sample-free auto-alignment and laser power calibration which improve reproducibility of experiments. We describe how FLIM is streamlined with Luminosa. Its rapidFLIM hardware records several frames per second with high count rates, which the software handles with a novel dynamic binning format. Combined with GPU-accelerated algorithms, this enables high-speed automated analysis of FLIM images with minimal user interaction. We will also show an outlook about how Luminosa can be used for combining FLIM with super-resolution modalities.

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.

This conference presentation was prepared for SPIE BiOS, 2024.

This conference presentation was prepared for SPIE BiOS, 2024.