Photothermal spectroscopy

Photothermal spectroscopy is a group of high sensitivity spectroscopy techniques used to measure optical absorption and thermal characteristics of a sample. The basis of photothermal spectroscopy is the change in thermal state of the sample resulting from the absorption of radiation. Light absorbed and not lost by emission results in heating. The heat raises temperature thereby influencing the thermodynamic properties of the sample or of a suitable material adjacent to it. Measurement of the temperature, pressure, or density changes that occur due to optical absorption are ultimately the basis for the photothermal spectroscopic measurements. As with photoacoustic spectroscopy, photothermal spectroscopy is an indirect method for measuring optical absorption, because it is not based on the direct measure of the light which is involved in the absorption. In another sense, however, photothermal (and photoacoustic) methods measure directly the absorption, rather than e.g. calculate it from

Coherent Imaging of Cellular Dynamics

Miguel Sison

Mitochondrial dynamics refers to the processes of fusion, fission, and transport that aid mitochondria in accomplishing their many roles; including ATP production, oxygen sensing, and homeostasis. Due to their involvement in numerous essential cellular activity, dysfunctional mitochondria have been implicated in a wide range of human diseases. Confocal microscopy using fluorophores for molecular specificity remains the gold standard of intracellular imaging. However, fluorescent labels can be toxic to the cell upon prolonged exposure and still suffer from photobleaching. These compromise the application of confocal fluorescence microscopy for true long lasting time-lapse imaging of living samples. Optical coherence microscopy (OCM) exploits the intrinsic variation in the scattering properties of the sample to achieve fast, label-free, and highly sensitive three-dimensional imaging. Unfortunately, being label-free means OCM lacks specificity and coherence based imaging techniques have no counterpart to fluorescent markers. The invention of the photothermal optical lock-in OCM (poli-OCM) brought about the possibility of specific OCM imaging using gold nanoparticles (AuNP) as photothermal bio-markers. The use of AuNPs as specific contrast agents has substantial advantages stemming from their well-established biocompatibility and photostability. Microscopic techniques that offer fast three-dimensional imaging over extended time durations would do well in revealing previously inaccessible knowledge on mitochondria. In this work, we quantify mitochondrial dynamics based on specific poli-OCM and surface functionalization of AuNPs. In realizing mitochondria specific poli-OCM imaging, it is necessary to functionalize AuNP with mitochondria targeting capabilities. We present copolymer surface coatings that provide the AuNPs with improved stability, solubility, and cellular uptake on top of mitochondria labeling. We further optimize the utilization of these AuNP labels for poli-OCM imaging. We also demonstrated poli-OCM imaging with differently structured gold nanolabels, which could lead to the realization of multimodal imaging using a single bio-marker. The two quantification techniques we developed were based on (1) temporal autocorrelation analysis combined with a classical diffusion model and (2) single particle tracking. Autocorrelation analysis is the foundation of fluorescence correlation spectroscopy (FCS); a technique extensively used for analyzing dynamic phenomena in chemistry and biophysics. We extend this analysis to three-dimensional poli-OCM imaging allowing us to map quantified mitochondrial diffusion parameters in three dimensions within the cell. We also investigate how the size of the mitochondria with respect to the point spread function (PSF) of the poli-OCM impacts the result of our autocorrelation analysis. Single particle tracking complements our temporal autocorrelation analysis since recent advances in localization and tracking algorithms have demonstrated precision better than the size of the PSF. Finally, we demonstrate the possibility of using mitochondria specific poli-OCM imaging with the quantification techniques we developed in studying the Cockayne syndrome (CS). CS is a very rare and fatal genetic disease that has been associated with mitochondrial dysfunction. To our knowledge, no study has been conducted focusing on quantifying the effect of CS on mitochondrial dynamics.

EPFL2017

Fluorescent Nanodiamond–Gold Hybrid Particles for Multimodal Optical and Electron Microscopy Cellular Imaging

Theo Lasser, Weina Liu, Miguel Sison

There is a continuous demand for imaging probes offering excellent performance in various microscopy techniques for comprehensive investigations. of cellular processes by more than, one technique. FluoreScent nanodianiond gold nanoparticles (END Au) constitute a new class of "all in -one" hybrid particles providing.unique features fOr multimodal imaging including optical imaging, eleCtron microscopy, and, and potentially even quantum, sensing. COnfocal and optical coherence microscopy of the END Au allow fast investigations inside living cells via emission, scattering, and photothermal imaging techniques because, the END 'emission is not quenched by AuNPs. In electron microscopy, transmission electron microscopy (TEM) and. scanning transmission electron microscopy (STEM) analysis of END Au reveals greatly enhanced contrast due to the gold particles as well as an extraordinary flickering behavior in three-diniensional Cellular environments originating from the nanodiamonds. The unique multimodal imaging characteristics of END Au enable detailed studies inside cells ranging from statistical distributions at the entire cellular level (micrometers) down to the tracking of individual particles in sub cellular organelles (nanometers). Herein, the processes of endosomal membrane uptake and release of FNDs were elucidated for the first time by the imaging of individual END Au hybrid nanoparticles with single-particle resolution. Their convenient preparation, the availability of various surface groups, their flexible detection modalities, and their single-particle contrast in combination with the capability for endosomal penetration and low cytotoxicity make FND Au unique candidates for multimodal optical electronic imaging applications with great potential for emerging techniques, such as quantum sensing inside living cells.

Amer Chemical Soc2016

Coherent Imaging of Cellular Dynamics

Miguel Sison

EPFL2017

Fluorescent Nanodiamond–Gold Hybrid Particles for Multimodal Optical and Electron Microscopy Cellular Imaging

Theo Lasser, Weina Liu, Miguel Sison

Amer Chemical Soc2016

Coherent Imaging of Cellular Dynamics

Three dimensional time lapse imaging of live cell mitochondria with photothermal optical lock-in optical coherence microscopy (Conference Presentation)

Fluorescent Nanodiamond–Gold Hybrid Particles for Multimodal Optical and Electron Microscopy Cellular Imaging

Three dimensional time lapse imaging of live cell mitochondria with photothermal optical lock-in optical coherence microscopy (Conference Presentation)

Coherent Imaging of Cellular Dynamics

Fluorescent Nanodiamond–Gold Hybrid Particles for Multimodal Optical and Electron Microscopy Cellular Imaging