Lifetime MAP reconstruction in frequency-domain fluorescence lifetime imaging microscopy

International audienceWe propose a robust statistical framework for reconstructing lifetime map corrupted by vesicle motion in frequency domain FLIM imaging. Instrumental noise is taken into account to improve lifetime estimation. Robust M-estimators and MLestimators allow to jointly estimate motion and lifetime. Performances are demonstrated on simulated and real samples

GPU acceleration of time-domain fluorescence lifetime imaging

Fluorescence lifetime imaging microscopy (FLIM) plays a significant role in biological sciences, chemistry, and medical research. We propose a Graphic Processing Units (GPUs) based FLIM analysis tool suitable for high-speed and flexible time-domain FLIM applications. With a large number of parallel processors, GPUs can significantly speed up lifetime calculations compared to CPU-OpenMP (parallel computing with multiple CPU cores) based analysis. We demonstrate how to implement and optimize FLIM algorithms on GPUs for both iterative and non-iterative FLIM analysis algorithms. The implemented algorithms have been tested on both synthesized and experimental FLIM data. The results show that at the same precision the GPU analysis can be up to 24-fold faster than its CPU-OpenMP counterpart. This means that even for high precision but time-consuming iterative FLIM algorithms, GPUs enable fast or even real-time analysis

Nanosecond compressive fluorescence lifetime microscopy imaging via the RATS method with a direct reconstruction of lifetime maps

RAndom Temporal Signals (RATS) method has proven to be a useful and versatile method for measuring photoluminescence (PL) dynamics and fluorescence lifetime imaging (FLIM). Here, we present two fundamental development steps in the method. First, we demonstrate that by using random digital laser modulation in RATS, it is possible to implement the measurement of PL dynamics with temporal resolution in units of nanoseconds. Secondly, we propose an alternative approach to evaluating the FLIM measurements based on a single-pixel camera experiment. In contrast to the standard evaluation, which requires a lengthy iterative reconstruction of PL maps for each timepoint, here we use a limited set of predetermined PL lifetimes and calculate the amplitude maps corresponding to each lifetime. The alternative approach significantly saves post-processing time and, in addition, in a system with noise present, it shows better stability in terms of the accuracy of the FLIM spectrogram. Besides simulations that confirmed the functionality of the extension, we implemented the new advancements into a microscope optical setup for mapping PL dynamics on the micrometer scale. The presented principles were also verified experimentally by mapping a LuAG:Ce crystal surface

In vivo fluorescence lifetime tomography of a FRET probe expressed in mouse

Förster resonance energy transfer (FRET) is a powerful biological tool for reading out cell signaling processes. In vivo use of FRET is challenging because of the scattering properties of bulk tissue. By combining diffuse fluorescence tomography with fluorescence lifetime imaging (FLIM), implemented using wide-field time-gated detection of fluorescence excited by ultrashort laser pulses in a tomographic imaging system and applying inverse scattering algorithms, we can reconstruct the three dimensional spatial localization of fluorescence quantum efficiency and lifetime. We demonstrate in vivo spatial mapping of FRET between genetically expressed fluorescent proteins in live mice read out using FLIM. Following transfection by electroporation, mouse hind leg muscles were imaged in vivo and the emission of free donor (eGFP) in the presence of free acceptor (mCherry) could be clearly distinguished from the fluorescence of the donor when directly linked to the acceptor in a tandem (eGFP-mCherry) FRET construct

Measurements of absolute concentrations of NADH in cells using the phasor FLIM method.

We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. The method requires the measurement of a solution of NADH at a known concentration. The phasor representation of the fluorescence decay accounts for the differences in quantum yield of the free and bound form of NADH, pixel by pixel of an image. The concentration of NADH in every pixel in a cell is obtained after adding to each pixel in the phasor plot a given amount of unmodulated light which causes a shift of the phasor towards the origin by an amount that depends on the intensity at the pixel and the fluorescence lifetime at the pixel. The absolute concentration of NADH is obtained by comparison of the shift obtained at each pixel of an image with the shift of the calibrated solution

NANOSCALE INVESTIGATION OF NUCLEAR STRUCTURES BY TIME-RESOLVED MICROSCOPY

The eukaryotic cell nucleus is composed by heterogeneous biological structures, such as the nuclear envelope (NE) and chromatin. At a morphological level, chromatin organization and its interactions with nuclear structures, such as nuclear lamina (NL) and nuclear pore complex (NPC), are suggested to play an essential role in the regulation of gene activity, which involves the packaging of the genome into transcriptionally active and inactive sites, bound to healthy cell proliferation and maintenance. However, the processes governing the relation between nuclear structures and gene regulation are still unclear. For this reason, the advanced microscopy methods represent a powerful tool for imaging nuclear structures at the nanometer level, which is essential to understand the effect of nuclear interactions on genome function. The nanometer information may be achieved either through the advanced imaging techniques in combination with fluorescence spectroscopy or with the help of super-resolution methods, increasing the spatial resolution of the conventional optical microscopy. In this thesis, I implemented a double strategy based on a novel FLIM-FRET assay and super resolution SPLIT-STED method for the investigation of the chromatin organization and nuclear envelope components (lamins and NPC) at the nanoscale, in combination with the phasor analysis. The phasor approach can be applied to several fluorescence microscopy techniques abled to provide an image with an additional information in a third channel. Phasor plot is a graphical representation, which decodes the fluorescence dynamics encoded in the image, revealing a powerful tool for the data analysis in time-resolved imaging. The Chapter 1 of the thesis is characterized by an Introduction, which provides an overview on the chromatin organization at the nanoscale and the description of the several advanced fluorescence microscopy techniques used for its investigation. They are broadly divided into two main categories: the advanced imaging techniques, such as Fluorescence Correlation Spectroscopy (FCS), single particle tracking (SPT) and Fluorescence Recovery After Photobleaching (FRAP), Forster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) and the super-resolution techniques, which include Stimulated Emission Depletion (STED), Structured Illumination Microscopy (SIM) and single molecule localization microscopy (SMLM). Following, Chapter 2 focus on the capabilities of the phasor approach in time-resolved microscopy, as a powerful tool for the analysis of the experimental data. After a description of the principles of time-domain and frequency-domain measurements, in this section are explained the rules of the phasor analysis and its applications in different fluorescence microscopy techniques. In Chapter 3, I present a FRET assay, based on the staining of the nuclei with two DNA-binding dyes (e.g. Hoechst 33342 and Syto Green 13) by using frequency-domain detection of FLIM and the phasor analysis in live interphase nuclei. I show that the FRET level strongly depends on the relative concentration of the two fluorophores. I describe a method to correct the values of FRET efficiency and demonstrate that, with this correction, the FLIM-FRET assay can be used to quantify variations of nanoscale chromatin compaction in live cells. In Chapter 4, the phasor analysis is employed to the improvement of the resolving power of the super-resolution STED microscopy. I describe a novel method to investigate nuclear structures at the nanometer level, known as SPLIT (Separation of Photons by Lifetime Tuning), developed by my group in last years. By using the phasor approach, the SPLIT technique decodes the variations of spectroscopic parameters of fluorophores, such as lifetime and fluorescence intensity, due to the effect of the modulated depletion power of the STED technique, increasing the resolving power. In this chapter, I develop the concept of the SPLIT method modulating the excitation pattern during the image acquisition to overcome its limitation linked to the photobleaching effect and the signal-to-noise ratio

Wide-Field Multi-Parameter FLIM: Long-Term Minimal Invasive Observation of Proteins in Living Cells.

Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes