This conference presentation was prepared for the Multiphoton Microscopy in the Biomedical Sciences XXIII conference at SPIE BIOS, 2023.

The majority of cancer cells have high frequencies of DNA damage response defects which results in a deficient repair mechanism which can give rise to oncogenes that regulate cellular metabolism. In this project, the effects of tumor suppressor protein p53 and translesional synthesis protein REV3L are studied to relate DNA damage signaling and repair to cellular metabolism by using the fluorescence lifetime of the metabolic coenzyme NADH. Our results show that restoring function to p53 and silencing REV3L simultaneously suppressed the cancerous metabolic phenotype and resulted in the greatest amount of cancer cell death

We demonstrate optical redox ratio and fluorescence lifetime imaging microscopy of intrinsic metabolic co-factors NAD(P)H and FAD to quantify metabolic changes in human immune cells from peripheral blood. This approach is attractive because it does not require cell surface labels or transfection, enabling rapid assessment of single cell metabolism. Multiphoton microscopy provides near infrared excitation of these autofluorescent molecules, thereby maximizing cell viability. Newly trained neural networks automatically segment single cells for analysis of heterogeneity within and between patients. Overall, this approach is attractive for both basic research and patient management in cancer and immunology.

Wound re-epithelialization is complex and imperfect. Understanding which cells are involved in this process, how they are spatially arranged, and when they contribute to wound healing is a longstanding scientific and clinical challenge. We used a recently developed fast large area multiphoton exoscope for in vivo imaging of human skin to study the process of wound healing in vivo in human skin. We monitored the re-epithelization of wounds generated by removal of the epidermis following a suction blistering procedure and identified the morphological and metabolic signatures of epidermal and dermal cells involved in the healing process.

FLIM of metabolic coenzymes, as NAD(P)H and FAD, is now widely accepted to be one of the most important imaging methods for cell metabolism. However, new algorithms are needed to circumvent various problems and to image cell metabolism and redox state from fluorescence lifetimes. The significance of a metabolic index based on NAD(P)H FLIM will be explained and compared with the fluorescence lifetime induced redox ratio (FLIRR). The importance of FMN will be discussed and the FLIRR approach will be extended. Using a three channel TCSPC system simultaneous metabolic NADH/FAD/FMN FLIM and oxygen PLIM/dFLIM (delayed fluorescence) could be realized.

Cellular metabolism is dysregulated in many diseases. Single-cell measurements of metabolism are important since cellular heterogeneity influences patient outcomes. Single-cell segmentation and analysis of fluorescence lifetime images of the metabolic coenzymes, reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD), provides a label-free method to interrogate metabolism at a cellular level. To facilitate cell-level analysis, we are developing automated segmentation algorithms. Additionally, we are creating and testing models for predicting metabolic phenotypes from fluorescence lifetime metrics. Our applications of single-cell metabolic phenotyping include evaluating responses of cancer cells to chemotherapy and characterizing macrophage phenotypes.

Second-harmonic generation (SHG) is uniquely capable of imaging collagen non-invasively with high-resolution, making it ideal to evaluate tissue organization in health and disease. For this, quantitative data analysis is essential. Different approaches have been proposed to quantify tissue organization from SHG images. Nevertheless, these methods have never been objectively evaluated or compared. In this study, we performed a comprehensive analysis on the performance of different metrics in computer-generated SHG images with increasing levels of disorganization to evaluate the advantages and limitations of each approach.

We developed a multimodal optical metabolic imaging system that integrates SRS, MPF, and SHG, for studying aging and diseases. We quantitatively measured the metabolic dynamics in cells and animals under various conditions, and further combined deuterium oxide probing with stimulated Raman scattering (DO-SRS) for visualizing newly synthesized protein and lipid molecules, in addition to macromolecules (protein and lipid, NADH and Flavin, collagen) imaged with the label free multimodal microscopy.

Macroscopic fluorescence lifetime FRET imaging (MFLI-FRET) presents a much-needed analytical tool to non-invasively quantify drug-receptor engagement in tumors and other organs in preclinical studies. We demonstrate the specificity and sensitivity of MFLI-FRET for direct and robust measurement of trastuzumab-HER2 target engagement in various types of breast and ovarian cancer tumor xenograft models.

I will present a new fluorescence imaging method called LEAD (line excitation array detection) microscopy, capable of providing 0.8 million frames per second. This method performs line-scanning of excitation laser beam using a chirped signal-driven longitudinal acousto-optic deflector to create a virtual light-sheet, and images the field-of-view with a linear photomultiplier tube array to generate a 66×14 pixel frame each scan cycle. I will present an implementation of the LEAD microscopy as a blur-free 3D imaging flow cytometer with 3.5-micron resolution and signal-to-background ratios >200. I will also present its conceptual implementation as an ultrafast two-photon LEAD microscopy (2p-LEAD).

Multimodal imaging with optical microscopy that can image both fluorescence-labeled and label-free structures in the same sample enables correlating the two. We previously developed one-photon bi-functional microscopy that can reconstruct 3D fluorescence and 3D refractive index from the same fluorescence images captured in epi-mode, but this technique only works for samples consisting of sparsely labeled fluorescence structures and weak scattering media. Our new work jointly develops multiphoton microscopy and a physics-based computational model for simultaneously imaging fluorescence and refractive index in strong scattering samples.

In this talk, I will present optical imaging platforms and methodologies that aim to empower label-free in vivo microscopy. Label-free in vivo microscopy promises to be a versatile tool for studying and diagnosing diseases in living animals and humans. Part of the challenge of label-free in vivo microscopy lies in the lack of simultaneous contrast, limited signal generation efficiency, and nonintuitive interpretation. This talk will cover how we attempt to address these challenges by resorting to light engineering and algorithms.

Fluorescence microscopy based on non-linear optical phenomena has become a new perspective optical tool in biological imaging. This contribution is aimed at synthesis and micro/spectroscopic characterization of novel S,N-heteroarene-functionalized BODIPY-class dyes with potential application as fluorescence and NLO markers in high-resolution multiphoton microscopy. The molecules have been classified with respect to their NLO response and most perspective ones has been tested for potential applications on cultured cancer cells. Supported by: EU-H2020 871124 (Laserlab-Europe) and 810701 (LAMatCU), Slovak research grant agencies APVV-17-0324, APVV-21-0503, VEGA No. 1/0669/22 and VEGA No. 2/0070/21.

Fluorescence Lifetime Imaging (FLIM) has become more attractive in recent years as it offers increased specificity in many assays as well as the possibility of multiplexing the read out of many markers with a small number of detectors. Here we present how FLIM modalities are implemented in Luminosa, the new single-photon counting confocal microscope by PicoQuant. Thanks to a dynamic binding format and GPU-based algorithms FLIM images of 1024x1024 can be analyzed in a few seconds. The FLIM analysis workflow suggests the best fitting model based on statistical arguments and requires minimal user interaction making these modalities become accessible to new users who can then confidently start working with FLIM and incorporate it into their research toolbox combining the strengths of phasor plots with decay fitting.

A tension-sensitive biosensor was used to detect changes in applied force across the mechanosensitive focal adhesion protein vinculin. When the biosensor is under tension, two fluorescent proteins separate, decreasing the amount of Förster Resonance Energy Transfer (FRET) observed. By measuring FRET using time-correlated single-photon counting fluorescence lifetime imaging microscopy (TCSPC-FLIM) we observe the loss of FRET, as a direct consequence of an applied intracellular force across the biosensor. Mouse embryonic fibroblasts (MEFs) transfected with a vinculin construct encoding the Tension Sensing Module (TSM), demonstrate how force-transduction changes within maturing adhesions in both fixed and live cells.

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.

Ultrafast laser sources are a key enabling technology for nonlinear excitation in multiphoton microscopy. Rapid developments in the last five years have realized a wider scope of laser platforms that address emerging opportunities for (pre)clinical applications in nonlinear microscopy, along with additional optical functionality such as integrated power modulation options and higher energy regimes that enable deeper imaging via 3-photon excitation. In this presentation we will review the latest developments in ultrafast lasers and their impacts in the field of nonlinear microscopy.

Hands-free ultrafast lasers have become the primary light source for many biological imaging applications. Multiphoton imaging has benefitted from these developments by offering an easy-to-use platform that reaches a wider audience. Techniques such as Fluorescent Resonant Energy Transfer (FRET), Fluorescence Lifetime Imaging Microscopy (FLIM), Coherent Anti-Stokes Raman Scattering (CARS), and Stimulated Raman Scattering (SRS) can easily be performed using these powerful tools. In this presentation, the latest developments in ultrafast lasers for multiphoton imaging will be presented.

This conference presentation was prepared for the Multiphoton Microscopy in the Biomedical Sciences XXIII conference at SPIE BiOS, 2023.

We will present the initiative in imaging a hugely expanded drosophila brain via expansion microscopy, which has brought unprecedented opportunities and challenges. 100X expansion has been demonstrated in enlarging the typically 0.6x0.3x0.2 mm brain to 60x30x20 mm, which promises electron microscopy resolution (approximately 3 nm) with optical microscopy (approximately 300 nm) to resolve the synapses connection. A highly sped-up imaging method integrated with an ultrasound-activated microtome is crucial to support the initiative. Critically, photon statistics set the fundamental considerations in selecting the contrasts for optical imaging.

Laser scanning multiphoton microscopy is widely used in biomedical research. The commonly available system can support a frame rate of up to tens of Hz. Recent advances in function indicators demand a frame rate of hundreds of Hz, beyond the capabilities of common systems. Although a few novel solutions have been developed recently, they often require highly specialized sources and hardware. To broadly enable high-speed imaging, we developed an optical gearbox system that is compatible with commonly available hardware. Here, we present the results of the gearbox based high-speed in vivo functional imaging.

We use SHG microscopy to study collagen alterations in idiopathic pulmonary fibrosis (IPF) in human tissues and in vitro models. Texture analysis successfully classified the fiber morphology in normal and IPF tissues at near 100% accuracy. SHG polarization measurement indicated reduced chirality in the collagen triple helix in IPF in both tissues and spheroid models. We have created a “collagen atlas” in normal and IPF lung using the combination of SHG and THG to image large areas in annotated histology slides. The combined collagen and cellular organization data formed an accurate classification scheme and identified the most sensitive features.

Collagen is ubiquitously found inside the human body and is the main structural protein in the Extracellular Matrix (ECM) of various tissues. In this work, we focus on the improvement instrumentation and methods used for obtaining collagen structural information in the SHG microscopy. We proposed dual liquid crystals based polarization-resolved SHG approach to address the deadlock, which breaks limitation of only working for modulating excitation angle of linear polarization but not directionality for all polarization state control. We assessed the SHG polarization excited responses of the isolated collagen type I and type II gels with quantitative analysis methods at different scale levels using our system and the results showed the potential of Dual-LC PSHG microscopy to differentiate the type of collagen based on the macroscopic SHG responses.

Ultrasonic methods hold immense promise for functional measurements and interventions during in vivo studies due to the beneficial scattering properties of acoustic waves. Here we present the design and application of a system that integrates into standard multiphoton microscopes, which co-linearly combines two-photon fluorescence imaging and precise acoustic sonication. By applying this system across a range of samples, we observed a novel gigantic Acousto-Optic (AO) modulation of two-photon excited fluorescence, and also study the mechanisms of functional ultrasound neuromodulation, an emerging non-invasive neurostimulation technique. This work demonstrates simple photonic/acoustic methods to study novel phenomenon in the awake mouse.

Spatiotemporal focusing has enabled widefield, axially confined multiphoton excitation. Its main advantage of an improved performance through scattering media, useful for deep imaging and optogenetics, however, has largely been evaluated empirically because of a lack of a suitable computational model. To overcome this we have developed a full-wave simulation describing two-photon microscopy image formation through scattering media with spatiotemporal focusing. This model provides full freedom to vary imaging parameters and observe quantities not generally accessible in experiment. We use this model to reveal new insights into the properties of spatiotemporal focusing in the presence of scattering media.

We demonstrate a highly multimodal nonlinear micro-endoscope for real-time, label-free imaging of biological tissues. The endoscope can perform two and three photon excited fluorescence, second, third harmonic and CARS imaging for different excitation wavelengths. Ultrashort pulses are delivered to the sample by a double-clad antiresonant hollow core fiber over the 800-1800 nm spectral band. The fiber tip is placed into a doubly resonant piezoelectric tube which allows a spiral scanning on the sample. The endoscope distal head containing the scanning device and the GRIN micro-objective is 1.5 mm in diameter and 35 mm long. Real-time nonlinear imaging at 10 frame/s is demonstrated.

We developed a pulse-picking multimodal nonlinear optical microscope that increases the sensitivity of label-free chemical imaging at low average laser power. Using a function-generator-controlled acousto-optic modulator, we collinearly combine two excitation wavelengths and can flexibly control the number of pulses at each pixel. The pulse-picking method gives over 1000x signal improvement for CARS and 20x for two-photon excitation fluorescent and second-harmonic generation at the same low average power. By varying the peak and average power of laser pulses, we evaluated the laser phototoxicity and found the optimal power window with the best sensitivity and minimum phototoxicity for tissue samples and live cells.

Over the last decade, there has been rapid development of mode-locked femtosecond fiber lasers with the clear goal of providing an easy-to-use alternative to older technologies for two-photon microscopy applications. Single-wavelength fiber lasers have the potential to revolutionize two-photon fluorescence microscopy by making simple and cost-efficient light sources available to everyone. With short, clean pulses, these robust, turn-key lasers enable unmatched brightness in two-photon microscopy without unwanted heating of the sample. This technology enables the laser to be used in next-generation mobile applications, such as with the MINI2P microscope for measurement in freely-moving, unrestrained mice. This talk will explore some of these applications.

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.

For patients suffering from cancer or neurodegenerative diseases research into the underlying causes is of the highest importance. Here, we report on our efforts to optimize higher harmonic generation microscopy complemented with multi-photon (auto)fluorescence, to image for long period of times on acute human tissue slices. Results for time-lapse imaging of the induction of myelin-swelling in Multiple Sclerosis brains during gradual high sodium perfusion and fibrosis induced in samples obtained from lobectomy, in the context of pulmonary fibrosis will be presented.

This conference presentation was prepared for the Multiphoton Microscopy in the Biomedical Sciences XXIII conference at SPIE BiOS, 2023.

This conference presentation was prepared for the Multiphoton Microscopy in the Biomedical Sciences XXIII conference at SPIE BiOS, 2023.

This conference presentation was prepared for the Multiphoton Microscopy in the Biomedical Sciences XXIII conference at SPIE BiOS, 2023.

This conference presentation was prepared for the Multiphoton Microscopy in the Biomedical Sciences XXIII conference at SPIE BiOS, 2023.