Folding and Unfolding of Exogenous G-Rich Oligonucleotides in Live Cells by Fluorescence Lifetime Imaging Microscopy of o-BMVC Fluorescent Probe

Guanine-rich oligonucleotides (GROs) can self-associate to form G-quadruplex (G4) structures that have been extensively studied in vitro. To translate the G4 study from in vitro to in live cells, here fluorescence lifetime imaging microscopy (FLIM) of an o-BMVC fluorescent probe is applied to detect G4 structures and to study G4 dynamics in CL1-0 live cells. FLIM images of exogenous GROs show that the exogenous parallel G4 structures that are characterized by the o-BMVC decay times (≥2.4 ns) are detected in the lysosomes of live cells in large quantities, but the exogenous nonparallel G4 structures are hardly detected in the cytoplasm of live cells. In addition, similar results are also observed for the incubation of their single-stranded GROs. In the study of G4 formation by ssHT23 and hairpin WT22, the analyzed binary image can be used to detect very small increases in the number of o-BMVC foci (decay time ≥ 2.4 ns) in the cytoplasm of live cells. However, exogenous ssCMA can form parallel G4 structures that are able to be detected in the lysosomes of live CL1-0 cells in large quantities. Moreover, the photon counts of the o-BMVC signals (decay time ≥ 2.4 ns) that are measured in the FLIM images are used to reveal the transition of the G4 formation of ssCMA and to estimate the unfolding rate of CMA G4s with the addition of anti-CMA into live cells for the first time. Hence, FLIM images of o-BMVC fluorescence hold great promise for the study of G4 dynamics in live cells.

Simple phasor-based deep neural network for fluorescence lifetime imaging microscopy

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to probe the molecular environment of fluorophores. The analysis of FLIM images is usually performed with time consuming fitting methods. For accelerating this analysis, sophisticated deep learning architectures based on convolutional neural networks have been developed for restrained lifetime ranges but they require long training time. In this work, we present a simple neural network formed only with fully connected layers able to analyze fluorescence lifetime images. It is based on the reduction of high dimensional fluorescence intensity temporal decays into four parameters which are the phasor coordinates, the mean and amplitude-weighted lifetimes. This network called Phasor-Net has been applied for a time domain FLIM system excited with an 80 MHz laser repetition frequency, with negligible jitter and afterpulsing. Due to the restricted time interval of 12.5 ns, the training range of the lifetimes was limited between 0.2 and 3.0 ns; and the total photon number was lower than 106, as encountered in live cell imaging. From simulated biexponential decays, we demonstrate that Phasor-Net is more precise and less biased than standard fitting methods. We demonstrate also that this simple architecture gives almost comparable performance than those obtained from more sophisticated networks but with a faster training process (15 min instead of 30 min). We finally apply successfully our method to determine biexponential decays parameters for FLIM experiments in living cells expressing EGFP linked to mCherry and fused to a plasma membrane protein.

Raman Imaging and Fluorescence Lifetime Imaging Microscopy for Diagnosis of Cancer State and Metabolic Monitoring

Hurdles for effective tumor therapy are delayed detection and limited effectiveness of systemic drug therapies by patient-specific multidrug resistance. Non-invasive bioimaging tools such as fluorescence lifetime imaging microscopy (FLIM) and Raman-microspectroscopy have evolved over the last decade, providing the potential to be translated into clinics for early-stage disease detection, in vitro drug screening, and drug efficacy studies in personalized medicine. Accessing tissue- and cell-specific spectral signatures, Raman microspectroscopy has emerged as a diagnostic tool to identify precancerous lesions, cancer stages, or cell malignancy. In vivo Raman measurements have been enabled by recent technological advances in Raman endoscopy and signal-enhancing setups such as coherent anti-stokes Raman spectroscopy or surface-enhanced Raman spectroscopy. FLIM enables in situ investigations of metabolic processes such as glycolysis, oxidative stress, or mitochondrial activity by using the autofluorescence of co-enzymes NADH and FAD, which are associated with intrinsic proteins as a direct measure of tumor metabolism, cell death stages and drug efficacy. The combination of non-invasive and molecular-sensitive in situ techniques and advanced 3D tumor models such as patient-derived organoids or microtumors allows the recapitulation of tumor physiology and metabolism in vitro and facilitates the screening for patient-individualized drug treatment options.

Fluorescence Lifetime Imaging Microscopy of Porphyrins in Helicobacter pylori Biofilms

Bacterial biofilm constitutes a strong barrier against the penetration of drugs and against the action of the host immune system causing persistent infections hardly treatable by antibiotic therapy. Helicobacter pylori (Hp), the main causative agent for gastritis, peptic ulcer and gastric adenocarcinoma, can form a biofilm composed by an exopolysaccharide matrix layer covering the gastric surface where the bacterial cells become resistant and tolerant to the commonly used antibiotics clarithromycin, amoxicillin and metronidazole. Antimicrobial PhotoDynamic Therapy (aPDT) was proposed as an alternative treatment strategy for eradicating bacterial infections, particularly effective for Hp since this microorganism produces and stores up photosensitizing porphyrins. The knowledge of the photophysical characteristics of Hp porphyrins in their physiological biofilm microenvironment is crucial to implement and optimize the photodynamic treatment. Fluorescence lifetime imaging microscopy (FLIM) of intrinsic bacterial porphyrins was performed and data were analyzed by the ‘fit-free’ phasor approach in order to map the distribution of the different fluorescent species within Hp biofilm. Porphyrins inside bacteria were easily distinguished from those dispersed in the matrix suggesting FLIM-phasor technique as a sensitive and rapid tool to monitor the photosensitizer distribution inside bacterial biofilms and to better orientate the phototherapeutic strategy.

Temperature profile characterization with fluorescence lifetime imaging microscopy in a thermophoretic chip

This study introduces a thermophoretic lab-on-a-chip device to measure the Soret coefficient. We use resistive heating of a microwire on the chip to induce a temperature gradient, which is measured by fluorescence lifetime imaging microscopy (FLIM). To verify the functionality of the device, we used dyed polystyrene particles with a diameter of 25 nm. A confocal microscope is utilized to monitor the concentration profile of colloidal particles in the temperature field. Based on the measured temperature and concentration differences, we calculate the corresponding Soret coefficient. The same particles have been recently investigated with thermal diffusion forced Rayleigh scattering (TDFRS) and we find that the obtained Soret coefficients agree with literature results. This chip offers a simple way to study the thermophoretic behavior of biological systems in multicomponent buffer solutions quantitatively, which are difficult to study with optical methods solely relying on the refractive index contrast. Graphic abstract