Fast volumetric fluorescence lifetime imaging of multicellular systems using single-objective light-sheet microscopy

The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8-7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence lifetime measurements of low quantum efficiency sub-nanosecond fluorophores.

PurposeTo describe different patterns of macular pigment (MP) seen in fluorescence lifetime imaging ophthalmoscopy (FLIO) and to analyze ex vivo fluorescence characteristics of carotenoids.MethodsA total of 31 eyes of young healthy subjects, 4 eyes from patients with albinism, 36 eyes with macular telangiectasia type 2 (MacTel), 24 eyes with retinitis pigmentosa, and 1 eye with a macular hole were included in this clinic-based, cross-sectional study. All subjects underwent Heidelberg Engineering FLIO and MP measurements (dual-wavelength autofluorescence). Fundus autofluorescence (FAF) lifetimes of a 30° retinal field were detected in two spectral channels (SSC: 498–560 nm; LSC: 560–720 nm), and amplitude-weighted mean fluorescence lifetimes (τm) were calculated. Additionally, autofluorescence lifetimes of known dilutions of lutein and zeaxanthin were measured in a cuvette in free- and protein-associated states.ResultsMP shows a significant inverse correlation to foveal FAF lifetimes measured with FLIO (SSC: r = −0.608; P < 0.001). Different distribution patterns can be assigned to specific disease-related changes. Two patients with albinism, who did not have MP, were found to be missing short FAF lifetimes. In solvent, lutein and zeaxanthin show very short autofluorescence lifetimes (∼50–60 ps; SSC), as do their respective binding proteins (∼40–50 ps; SSC). When combining carotenoids with their specific binding proteins, the decay times shift to longer means (∼70–90 ps; SSC).ConclusionsThis study expands upon previous findings of an impact of MP on short FAF lifetimes by describing ex vivo autofluorescence lifetimes of carotenoids and different in vivo autofluorescence patterns that can be associated with certain diseases.

Fluorescence lifetime imaging microscopy (FLIM) and spectral imaging are two broadly applied methods for increasing dimensionality in microscopy. However, their combination is typically inefficient and slow in terms of acquisition and processing. By integrating technological and computational advances, we developed a robust and unbiased spectral FLIM (S-FLIM) system. Our method, Phasor S-FLIM, combines true parallel multichannel digital frequency domain electronics with a multidimensional phasor approach to extract detailed and precise information about the photophysics of fluorescent specimens at optical resolution. To show the flexibility of the Phasor S-FLIM technology and its applications to the biological and biomedical field, we address four common, yet challenging, problems: the blind unmixing of spectral and lifetime signatures from multiple unknown species, the unbiased bleedthrough- and background-free Förster resonance energy transfer analysis of biosensors, the photophysical characterization of environment-sensitive probes in living cells and parallel four-color FLIM imaging in tumor spheroids.

Three-dimensional imaging at high-spatiotemporal resolutions and over large penetration depths is key for unmasking the dynamics and structural organization of complex biological systems. However, the need to axially shift the focus, with consequent limitations in imaging speed, and signal degradation at large depths due to scattering effects, makes this task challenging. Here, we present a novel approach in 2-photon excitation microscopy that allows fast volumetric imaging and enhanced signal-to-background (S/B) in thick tissue. Our technique is based on ultrafast beam shaping at each pixel by means of an acoustic optofluidic lens. Shaping the excitation beam with different phase profiles enables both high-speed axial focus shifting, for continuous volumetric imaging, and controlled aberrated imaging, advantageous for out-of-focus background removal. We provide a theoretical description of our approach, and demonstrate volumetric imaging of neuronal cells from a mouse brain slice with enhancements in S/B up to a factor of 10 over a depth of 600 μm.

Fluorescence lifetime imaging in PDT. An overview

Imaging of Hydroxychloroquine Toxicity with Fluorescence Lifetime Imaging Ophthalmoscopy

Exposure of nanosecond pulsed electric fields (nsPEFs) to live cells is an increasing research interest in biology and medicine. Despite extensive studies, a question still remains as to how effects of application of nsPEF on intracellular functions are different between cancerous cells and normal cells and how the difference can be detected. Herein, we have presented an approach of autofluorescence lifetime (AFL) microscopy of flavin adenine dinucleotide (FAD) to detect effects of application of nsPEF having 50 ns of a pulse width, nsPEF(50), on intracellular function in lung cancerous cells, A549 and H661, which show nsPEF(50)-induced apoptosis, and normal cells, MRC-5, in which the field effect is less or not induced. Then, the application of nsPEF(50) is shown to increase the lifetime of FAD autofluorescence in lung cancerous cells, whereas the electric field effects on the autofluorescence of FAD was not significant in normal healthy cells, which indicates that the lifetime measurements of FAD autofluorescence are applicable to detect the field-induced change in intracellular functions. Lifetime and intensity microscopic images of FAD autofluorescence in these lung cells were also acquired after exposure to the apoptosis-inducer staurosporine (STS). Then, it was found that the AFL of FAD became longer after exposure not only in the cancerous cells but also in the normal cells. These results indicate that nsPEF(50) applied to lung cells induced apoptotic cell death only in lung cancerous cells (H661 and A549) but not in lung normal cells (MRC-5), whereas STS induced apoptotic cell death both in lung cancerous cells and in lung normal cells. The lifetime microscopy of FAD autofluorescence is suggested to be very useful as a sensitive detection method of nsPEF-induced apoptotic cell death.

Diagonally Scanned Light-Sheet Microscopy for Fast Volumetric Imaging of Adherent Cells

Colorectal cancer (CRC) ranks among the most prevalent malignancies worldwide, driving a quest for comprehensive characterization methods. We report a characterization of the ex vivo autofluorescence lifetime fingerprint of colorectal tissues obtained from 73 patients that underwent surgical resection. We specifically target the autofluorescence characteristics of collagens, reduced nicotine adenine (phosphate) dinucleotide (NAD(P)H), and flavins employing a fiber-based dual excitation (375 nm and 445 nm) optical imaging system. Autofluorescence-derived parameters obtained from normal tissues, adenomatous lesions, and adenocarcinomas were analyzed considering the underlying clinicopathological features. Our results indicate that differences between tissues are primarily driven by collagen and flavins autofluorescence parameters. We also report changes in the autofluorescence parameters associated with NAD(P)H that we tentatively attribute to intratumoral heterogeneity, potentially associated to the presence of distinct metabolic subpopulations. Changes in autofluorescence signatures of malignant tumors were also observed with lymphatic and venous invasion, differentiation grade, and microsatellite instability. Finally, we characterized the impact of radiative treatment in the autofluorescence fingerprints of rectal tissues and observed a generalized increase in the mean lifetime of radiated adenocarcinomas, which is suggestive of altered metabolism and structural remodeling. Overall, our preliminary findings indicate that multiparametric autofluorescence lifetime measurements have the potential to significantly enhance clinical decision-making in CRC, spanning from initial diagnosis to ongoing management. We believe that our results will provide a foundational framework for future investigations to further understand and combat CRC exploiting autofluorescence measurements.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-74224-8.

PurposeTo investigate the autofluorescence lifetimes as well as spectral characteristics of soft drusen and retinal hyperpigmentation in age-related macular degeneration (AMD).MethodsForty-three eyes with nonexudative AMD were included in this study. Fluorescence lifetime imaging ophthalmoscopy (FLIO), which detects autofluorescence decay over time in the short (SSC) and long (LSC) wavelength channel, was performed. The mean autofluorescence lifetime (τm) and the spectral ratio (sr) of autofluorescence emission in the SSC and LSC were recorded and analyzed. In total, 2760 soft drusen and 265 hyperpigmented areas were identified from color fundus photographs and spectral domain optical coherence tomography (SD-OCT) images and superimposed onto their respective AF images. τm and sr of these lesions were compared with fundus areas without drusen. For clearly hyperfluorescent drusen, the local differences compared to fundus areas without drusen were determined for lifetimes and sr.ResultsHyperpigmentation showed significantly longer τm (SSC: 341 ± 81 vs. 289 ± 70 ps, P < 0.001; LSC: 406 ± 42 vs. 343 ± 42 ps, P < 0.001) and higher sr (0.621 ± 0.077 vs. 0.539 ± 0.083, P < 0.001) compared to fundus areas without hyperpigmentation or drusen. No significant difference in τm was found between soft drusen and fundus areas without drusen. However, the sr was significantly higher in soft drusen (0.555 ± 0.077 vs. 0.539 ± 0.081, P < 0.0005). Hyperfluorescent drusen showed longer τm than surrounding fundus areas without drusen (SSC: 18 ± 42 ps, P = 0.074; LSC: 16 ± 29 ps, P = 0.020).ConclusionsFLIO can quantitatively characterize the autofluorescence of the fundus, drusen, and hyperpigmentation in AMD.Translational Relevance The experimental FLIO technique was applied in a clinical investigation. As FLIO yields information on molecular changes in AMD, it might support future diagnostics.

Fast Electro-Optic Fluorescence Lifetime Microscopy (EOFLIM) for super-resolution microscopy and brain surgery.

Semiconductors in the shape of nanostructures, including quantum dots (0-dimension) and nanoplatelets (2-dimension) demonstrate unique electronic and optoelectronic properties because of the quantum mechanism related to excitons confined in low dimensions. Such confinement can provide intriguing properties like high capability of photo-activated carriers, band gap tunability, splendid photoluminescence (PL) yield, narrow emission bandwidth, high absorption efficiency of light, fast fluorescence lifetime, etc. These properties attract great attention due to their high potential in applications like solar cells, light-emitting diodes (LEDs), lasers, photodetectors, catalysts, etc. Novel semiconductors including MoS2, MXene and perovskite have received increasing research interest in recent years. Unlike traditional semiconductor materials like Si, Ge, II-VI and III-V materials, the novel ones usually have low-dimensional form natively, which have emerged as very promising candidates for advanced electronic and optoelectronic devices. Among the various novel semiconductor materials, this thesis focuses on two types of them, which are MXene Sc2CO2 and cesium lead halide perovskite CsPbBr3. The two materials have some features in common. Both of them are direct band gap materials, which makes them available for optical applications. And they can be synthesized directly into two-dimensional structure, which o ers the opportunity to learn the influence of one-dimensional confinement. To extend the study into another dimension, the quantum dots of CsPbBr3 are also studied. The first part of the thesis focuses on the study of Sc2COx which is a type of semi-conductive MXene. The material is synthesized for the first time by using magnetron sputtering. X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) are performed to determine the composition of the sample. The band gap of Sc2COx sample is determined by ultraviolet-visible (UV-VIS) range absorption and photoluminescence (PL) spectrum. The Tauc plot indicates that Sc2COx is a direct band gap material. The high sensitivity of scandium to oxygen makes the sample contain both scandium carbide and scandium oxide. Thermal annealing introduces more oxidation into the sample. The structure of MXene will be broken when the annealing temperature is higher than 600C. The second part of the thesis focuses on the study of perovskite CsPbBr3 in forms of nanoplatelets and quantum dots. For CsPbBr3 nanoplatelets, the effect of thickness of cesium lead halide perovskite CsPbBr3 nanoplatelets (NPLs) on their electronic structure and optical properties are investigated using an 8-band k · p model which is based on effective-mass envelope function theory with exciton binding energy consideration. We first reported the CsPbBr3 nanoplatelets' band structure and optical gain with exciton effect. As the thickness of NPLs decreases, their band gap increases and the band mixing is more obvious which influences the transition matrix element (TME). The optical gain of CsPbBr3 nanoplatelets is calculated by taking into account TME, Fermi factor, injected carrier density and thickness. A blue shift of peak position in optical gain can be observed as the thickness of NPLs decreases. For any given NPL with a certain thickness, there is a slight blue shift in the peak position of optical gain as the carrier density increases because of band filling effect. The maximum optical gain of thinner NPLs needs higher carrier density to reach saturation. To obtain high differential gain, the carrier density in thick NPLs has to maintain in a small range. Experimental work is carried out and the results agree with our theoretical results very well. For quantum dot, the size effect on the electronic structure and optical properties of cubic CsPbBr3 perovskite quantum dots are investigated by using an 8-band k · p model which is also based on effective-mass envelope function theory with exciton binding energy consideration. Quantum dots with smaller sizes have larger band gaps due to quantum confinement. The transition matrix element (TME) is also influenced by the size. The optical gain of such material is calculated by considering TME, Fermi factor, injected carrier density, quantum dot size, and dephasing rate. A higher density of the injected carrier is needed for smaller quantum dots to get a positive optical gain. The peak of optical gain has a blue shift as the size of a quantum dot decreases. When the carrier density increases for a quantum dot with a certain size, a blue shift in emission peak position can be observed due to the band filling effect. Smaller quantum dots have higher differential optical gain at carrier densities within a certain range. In a word, two direct band gap semiconductor materials, Sc2COx and CsPbBr3 are studied via theoretical and experimental approaches. Their band-structure and optical properties are presented, followed by the analysis of their potential in applications.

The purpose of this study was to address the controversy whether the quality of volumetric high-resolution CT (HRCT) images is as good as that of axial nonvolumetric HRCT images by assessing the degree of motion artifact on images acquired with the two methods at MDCT of pediatric patients with known or suspected lung disease. A search of the hospital information system was conducted to identify the cases of pediatric patients with clinically suspected or known interstitial lung disease who underwent 16-MDCT of the chest with both volumetric and axial HRCT acquisitions (both 1.25-mm slice thickness) from March 2005 to July 2008. Two pediatric radiologists reviewed the images for the presence of motion artifacts at three anatomic levels (upper, middle, and lower lung zones). Motion artifacts were given numerical grades representing no artifact to severe artifact, and the paired Student's t test was used to compare the scores for the two acquisition methods. A total motion score for each acquisition was calculated by summing the scores for each of the three lung zones, and the scores for the two imaging methods were compared. Correlation between degree of motion artifact and age was evaluated. Effective radiation doses were estimated for volumetric and axial CT acquisitions. The study population consisted of 54 children (28 boys, 26 girls; mean age, 11.7 +/- 3.8 years; range, 5-18 years; eight inpatients, 46 outpatients) who underwent a total of 54 MDCT chest studies with volumetric and axial HRCT acquisitions. Motion artifact scores were higher for axial than for volumetric HRCT images of the upper (1.2 vs 1.0), middle (1.6 vs 1.2), and lower (2.2 vs 1.5) lung zones (p < 0.05 at each level). The total motion score of the axial HRCT images (mean, 5; range, 1-9) was higher than that of the volumetric HRCT images (mean, 3.6; range, 1-8) (p < 0.05). Younger age correlated with higher motion artifact score on axial HRCT images (r = -0.36, p < 0.01), whereas no correlation was found between age and motion artifact score on volumetric HRCT images (r = -0.12, p = 0.38). The effective radiation doses were 0.57 mSv for axial HRCT acquisition and 7.6 mSv for volumetric acquisition. The addition of axial acquisition increased the total radiation dose of the MDCT examination 7.1%. At CT of pediatric patients, reconstructed HRCT images from volumetric MDCT acquisition have significantly less motion artifact than images obtained with traditional axial acquisition.

Ca(2+) concentrations in biological cells are widely studied with fluorescent probes. The probes have a high selectivity for free calcium and exhibit marked changes in their photophysical properties upon binding. The differences in the fluorescent lifetime of the probes can now be used as a contrast mechanism for imaging purposes. This technique can be further exploited for the quantitative determination of ion concentrations within the cells. We describe the use of a fast fluorescence lifetime imaging method in combination with a standard confocal laser scanning microscope for the determination of Ca(2+) concentrations in single rat cardiac myocytes using the intensity probe Calcium Green.

Time-resolved photon counting methods have a finite bandwidth that restricts the acquisition speed of techniques like fluorescence lifetime imaging microscopy (FLIM). To enable faster imaging, computational methods can be employed to count photons when the output of a detector is directly digitized at a high sampling rate. Here, we present computational photon counting using a hybrid photodetector in conjunction with multithreshold peak detection to count instances where one or more photons arrive at the detector within the detector response time. This method can be used to distinguish up to five photon counts per digitized point, whereas previous demonstrations of computational photon counting on data acquired with photomultiplier tubes have only counted one photon at a time. We demonstrate in both freely moving C. elegans and a human breast cancer cell line undergoing apoptosis that this novel multithreshold peak detection method can accurately characterize the intensity and fluorescence lifetime of samples producing photon rates up to 223%, higher than previously demonstrated photon counting FLIM systems.