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Papers by Panagiota (Naya) Giannakopoulou

Gold nanoparticles (NPs) are widely utilised for bio-imaging applications owing to their facile s... more Gold nanoparticles (NPs) are widely utilised for bio-imaging applications owing to their facile synthesis, ease of surface functionalisation and bio-conjugation, as well as bio-compatibility. However, when embedded in highly heterogeneous and fluo- rescing environments such as biological cells and tissues, these NPs have to be large (typically >50 nm diameter) to be distinguished optically against backgrounds via their linear absorption and scattering at the surface plasmon resonance (SPR). As a result, cell imaging protocols often adopt the use of fluorophore tags attached onto the NP, and assume that the fluorophore is a reliable reporter. These constructs are routinely used in correlative microscopy techniques such as Correlative Light Elec- tron microscopy (CLEM) and presume that the fluorophore colocalises well with the electron dense NP. In this thesis, I present the application of our recently developed four-wave-mixing (FWM) imaging modality to investigate the spatial cor...

Single nanoparticle tracking using optical microscopy is a powerful technique with many applicati... more Single nanoparticle tracking using optical microscopy is a powerful technique with many applications in biology, chemistry and material sciences. Despite significant advances, localising objects with nanometric position precision in a scattering environment remains challenging. Applied methods to achieve contrast are dominantly fluorescence based, with fundamental limits in the emitted photon fluxes arising from the excited-state lifetime as well as photobleaching. Here, we show a new four-wave mixing interferometry technique, whereby the position of a single non-fluorescing gold nanoparticle of 25\,nm radius is determined with 16\,nm precision in plane and 3\,nm axially from rapid single-point measurements at 1\,ms acquisition time by exploiting optical vortices. The precision in plane is consistent with the photon shot-noise, while axially it is limited by the nano-positioning sample stage, with an estimated photon shot-noise limit of 0.5\,nm. The detection is background-free even inside biological cells. The technique is also uniquely sensitive to particle asymmetries of only 0.5\% ellipticity, corresponding to a single atomic layer of gold, as well as particle orientation. This method opens new ways of unraveling single-particle trafficking within complex 3D architectures.

The nonlinear optical properties of a series of novel boron dipyrromethene derivatives (BODIPY) a... more The nonlinear optical properties of a series of novel boron dipyrromethene derivatives (BODIPY) are investigated by means of the Z-scan technique under 4 ns and 35 ps, visible and infrared laser pulses. All studied BODIPYs were found to exhibit strong saturable absorption and insignificant nonlinear refraction under visible excitation, while no response was observed under infrared laser excitation. The nonlinear optical parameters of the BODIPYs have been determined, revealing large nonlinear absorption coefficient values.

We demonstrate time-correlated single photon counting (TCSPC) in microfluidic droplets under high... more We demonstrate time-correlated single photon counting (TCSPC) in microfluidic droplets under high-throughput conditions. We discuss the fundamental limitations in the photon acquisition rate imposed by the single photon detection technique and show that it does not preclude accurate fluorescence lifetime (FLT) measurements at a droplet throughput exceeding 1 kHz with remarkable sensitivity. This work paves the way for the implementation of innovative biomolecular interaction assays relying on the FLT detection of nanosecond-lived fluorophores for high-throughput biotechnological applications, including high-throughput screening or cell sorting potentially allowed by droplet microfluidics or other fast sample handling facilities.

Conference Presentations by Panagiota (Naya) Giannakopoulou

In recent years, gold nanoparticles (NPs) have attracted enormous attention for their life-scienc... more In recent years, gold nanoparticles (NPs) have attracted enormous attention for their life-science applications as drug delivery vehicles, novel optical labels or local probes and sensors 1-3. Gold NPs are excellent optical labels as they exhibit high optical cross sections near their morphology-dependent surface plasmon resonance (SPR) frequencies 4. Owing to their high photostability and biocompatibility, together with their photothermal properties and electron microscopy contrast, gold NPs are among the most promising systems utilised in the biomedical research community 5, 6. In this work, we present a study which combines two optical imaging modalities: confocal fluorescence and Four-wave mixing (FWM) microscopy in order to shed light on the endosomal trafficking of ligand-conjugated gold nanoparticles 7. The samples consisted of fluorescently labelled Transferrin (Tf)-conjugated gold nanoparticles (AuNPs) of two different diameter sizes (15nm and 40nm) taken up by HeLa cells via clathrin mediated endocytosis. Resonant FWM microscopy, a coherent third-order non-linear imaging technique recently developed in our laboratory, enabled us to detect single gold nanoparticles completely background free 8. We studied the correlation between fluorescent confocal imaging of the Tf labelled ligand and the label-free FWM imaging of the AuNPs to elucidate the stability of the Tf-AuNP construct during cellular uptake, and whether cellular trafficking separates the ligand from the AuNP cargo. This study opens new perspectives to correlative microscopy and aims to devise a protocol for specifically transporting gold NPs into cells.

Gold nanoparticles (NPs) are widely utilised for bio-imaging applications owing to their facile s... more Gold nanoparticles (NPs) are widely utilised for bio-imaging applications owing to their facile synthesis, ease of surface functionalisation and bio-conjugation, as well as bio-compatibility. However, when embedded in highly heterogeneous and fluo- rescing environments such as biological cells and tissues, these NPs have to be large (typically >50 nm diameter) to be distinguished optically against backgrounds via their linear absorption and scattering at the surface plasmon resonance (SPR). As a result, cell imaging protocols often adopt the use of fluorophore tags attached onto the NP, and assume that the fluorophore is a reliable reporter. These constructs are routinely used in correlative microscopy techniques such as Correlative Light Elec- tron microscopy (CLEM) and presume that the fluorophore colocalises well with the electron dense NP. In this thesis, I present the application of our recently developed four-wave-mixing (FWM) imaging modality to investigate the spatial cor...

Single nanoparticle tracking using optical microscopy is a powerful technique with many applicati... more Single nanoparticle tracking using optical microscopy is a powerful technique with many applications in biology, chemistry and material sciences. Despite significant advances, localising objects with nanometric position precision in a scattering environment remains challenging. Applied methods to achieve contrast are dominantly fluorescence based, with fundamental limits in the emitted photon fluxes arising from the excited-state lifetime as well as photobleaching. Here, we show a new four-wave mixing interferometry technique, whereby the position of a single non-fluorescing gold nanoparticle of 25\,nm radius is determined with 16\,nm precision in plane and 3\,nm axially from rapid single-point measurements at 1\,ms acquisition time by exploiting optical vortices. The precision in plane is consistent with the photon shot-noise, while axially it is limited by the nano-positioning sample stage, with an estimated photon shot-noise limit of 0.5\,nm. The detection is background-free even inside biological cells. The technique is also uniquely sensitive to particle asymmetries of only 0.5\% ellipticity, corresponding to a single atomic layer of gold, as well as particle orientation. This method opens new ways of unraveling single-particle trafficking within complex 3D architectures.

The nonlinear optical properties of a series of novel boron dipyrromethene derivatives (BODIPY) a... more The nonlinear optical properties of a series of novel boron dipyrromethene derivatives (BODIPY) are investigated by means of the Z-scan technique under 4 ns and 35 ps, visible and infrared laser pulses. All studied BODIPYs were found to exhibit strong saturable absorption and insignificant nonlinear refraction under visible excitation, while no response was observed under infrared laser excitation. The nonlinear optical parameters of the BODIPYs have been determined, revealing large nonlinear absorption coefficient values.

We demonstrate time-correlated single photon counting (TCSPC) in microfluidic droplets under high... more We demonstrate time-correlated single photon counting (TCSPC) in microfluidic droplets under high-throughput conditions. We discuss the fundamental limitations in the photon acquisition rate imposed by the single photon detection technique and show that it does not preclude accurate fluorescence lifetime (FLT) measurements at a droplet throughput exceeding 1 kHz with remarkable sensitivity. This work paves the way for the implementation of innovative biomolecular interaction assays relying on the FLT detection of nanosecond-lived fluorophores for high-throughput biotechnological applications, including high-throughput screening or cell sorting potentially allowed by droplet microfluidics or other fast sample handling facilities.

In recent years, gold nanoparticles (NPs) have attracted enormous attention for their life-scienc... more In recent years, gold nanoparticles (NPs) have attracted enormous attention for their life-science applications as drug delivery vehicles, novel optical labels or local probes and sensors 1-3. Gold NPs are excellent optical labels as they exhibit high optical cross sections near their morphology-dependent surface plasmon resonance (SPR) frequencies 4. Owing to their high photostability and biocompatibility, together with their photothermal properties and electron microscopy contrast, gold NPs are among the most promising systems utilised in the biomedical research community 5, 6. In this work, we present a study which combines two optical imaging modalities: confocal fluorescence and Four-wave mixing (FWM) microscopy in order to shed light on the endosomal trafficking of ligand-conjugated gold nanoparticles 7. The samples consisted of fluorescently labelled Transferrin (Tf)-conjugated gold nanoparticles (AuNPs) of two different diameter sizes (15nm and 40nm) taken up by HeLa cells via clathrin mediated endocytosis. Resonant FWM microscopy, a coherent third-order non-linear imaging technique recently developed in our laboratory, enabled us to detect single gold nanoparticles completely background free 8. We studied the correlation between fluorescent confocal imaging of the Tf labelled ligand and the label-free FWM imaging of the AuNPs to elucidate the stability of the Tf-AuNP construct during cellular uptake, and whether cellular trafficking separates the ligand from the AuNP cargo. This study opens new perspectives to correlative microscopy and aims to devise a protocol for specifically transporting gold NPs into cells.