The hallmark of metabolic alteration of increase glycolysis, i.e. Warburg effect, in cancer cells together with atypical extracellular matrix structure may be responsible for tumor cell aggressiveness and drug resistance. Here we apply the phasor approach technique in fluorescence lifetime imaging microscopy (FLIM) as a novel method to measure metabolic alteration as a function of ECM mechanics. We imaged and compared triple-negative breast cancer (TNBC) cells to non-cancerous cells on various ECM stiffness. Dysregulation of mitochondrial motion may contribute to the fueling of bioenergy demands in metastatic cancer. To measure mitochondria motion and analyze their fusion and fission events, we developed a new algorithm called “mitometer” that is unbiased, and allows for automated segmentation and tracking of mitochondria in live cell 2D and 3D time-lapse images.

Fluorescence fluctuation spectroscopy (FFS) offers a window to observe and investigate dynamics of bio-molecular organisation. Furthermore, FFS acquisitions uniquely allow obtaining insights into other properties of the sample such as concentration and brightness (counts per molecule) of the fluorescently labelled species simultaneously. Here, we exploit the large number of measurements and statistics obtained from scanning fluorescence fluctuation spectroscopy (sFFS) acquisitions to elucidate bio-molecular organisation. We demonstrate the versatility and sensitivity of the approach using computer simulations, synthetic biological membranes and living cells.

Graphene-induced energy transfer (GIET) was recently introduced for sub-nanometric axial localization of fluorescent molecules. GIET exploits the near-field energy transfer from an excited fluorophore to a single sheet of graphene. This alters the fluorescence decay-time of the emitter and can be easily determined by fluorescence lifetime imaging microscopy (FLIM). The axial resolution of GIET implies to study of biological membranes. We present the measurement of the thickness of synthetic model membranes and demonstrate changes upon the addition of cholesterol. Furthermore, we are able to show the flipping of lipids from one leaflet to the other and determine the rates of this dynamics. In addition, we used GIET for mapping quasi-stationary states of the mitochondrial membranes before and during ATP synthesis. Upon activation, the inner membrane clearly approaches the outer membrane and the inter-membrane space is reduced by ∼2 nm.

In this work, we developed a planar dielectric antenna for analytes diffusing in aqueous solution. The so-called optofluidic antenna can collect more than 86% of all photons from a randomly oriented dipole-like emitter. The antenna involves a sub-micrometer water channel capped with air where the analytes are interrogated. The small dimension of the water channel in combination with the water/air interface confines the motion of the analytes, resulting in a slowing down of the translational diffusion. We characterize the photonic properties of the optofluidic antenna by investigating different dye molecules using fluorescence correlation spectroscopy. Moreover, we demonstrate the performance of our antenna by studying the dynamical behavior of the Holliday junction (HJ) at the single-molecule level using multiparameter fluorescence detection, which allows us to identify the HJ’s different FRET states in real-time.

Detection and imaging of single molecules have always been important tasks in fundamental science as well as practical applications. In this work we developed a novel approach to ultra-sensitive, ultra-fast detection and imaging of single molecules. Our approach allowed for detection of single troponin-T (cTnT) molecules (a clinically important marker for cardiac muscle damage) in human blood serum 1000 times faster than the existing techniques. We also performed imaging studies of single cTnT molecules and their motion in serum in real time and demonstrated the capability of this technique to measure ultra-low, clinically significant cTnT concentrations of 1 pg/mL.

Identifying biomolecules of interest in cryogenic electron tomography (CET) reconstructions is made challenging by the lack of non-perturbative and specific labeling methods compatible with CET. Combining fluorescence and CET is a promising approach to overcome this labeling limitation. However, diffraction-limited fluorescence data has insufficient resolution to provide clear labeling in the crowded cellular environment. Super-resolution fluorescence techniques achieve resolution on the tens of nanometers scale, making them compatible with the length scales of interest in labeling biomolecules in CET. I will discuss the development of a cryogenic single-molecule based super-resolution imaging approach that achieves an average localization precision of less than ten nanometers and is compatible with the latest CET methods.

We present fluorophore labels that transiently and repetitively bind to their targets as probes for various types of super-resolution fluorescence microscopy. Such labels show a weak (~10 µM – 100 nM) affinity to a target and are kept in an imaging buffer that constitutes a reservoir with a high concentration of intact probes, enabling repetitive binding to the same target (we refer to these labels as “exchangeable labels”). This dynamic labeling approach minimizes photobleaching and yields a constant fluorescence signal over time, which has been beneficially exploited in SMLM [1-4], STED [5, 6], and super-resolution optical fluctuation imaging (SOFI) [7]. Multi-color, 3D, and live cell imaging, as well as imaging of large fields of view, is facilitated [4]. We further present the implementation of neural networks for multi-emitter localization to achieve multi-color SMLM with short acquisition times of one minute [8].

Structured Illumination Microscopy (SIM) is a widely used super-resolution microscopy method, capable of imaging at twice the diffraction limit of conventional widefield microscopes. We developed a new method to assess in silico the spatio-temporal resolution limits of SIM, and demonstrated that its capacity to reconstruct super-resolved information is substantially worse than the time required to acquire a full stack of raw frames. We also applied our method to gauge the efficacy of a reconstruction method termed “rolling SIM” which claimed to improve the temporal resolution of SIM, and we showed that this is not the case.

Simultaneous investigations of the spatial arrangement and relative composition of proteins are of crucial importance in live-cell imaging. It was recently shown that Image Scanning Microscopy (ISM) can double the confocal resolution. Additionally, fluorescence lifetime based imaging (FLIM) opens an additional dimension in multispecies identification and separation. We combine these two techniques using an array of single-photon detectors together with a multichannel TCSPC and time tagging system for time-resolved single photon detection via tens of channels. First results illustrate the spatial resolution improvement and the selection of suitable fluorescent dyes to allow for lifetime based multiplexing.