Photothermal Microscopy Research Articles

Fast 3D visualization of endogenous brain signals with high-sensitivity laser scanning photothermal microscopy.

A fast, high-sensitivity photothermal microscope was developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope. We confirmed a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrated simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 μs. The fluorescence image visualized neurons expressing yellow fluorescence proteins, while the photothermal signal detected endogenous chromophores in the mouse brain, allowing 3D visualization of the distribution of various features such as blood cells and fine structures probably due to lipids. This imaging modality was constructed using compact and cost-effective laser diodes, and will thus be widely useful in the life and medical sciences.

Characterization of Sintered Mixed Oxides by Photothermal Microscopy

A new photothermal microscopy device is presented and used to map the thermal diffusivity of mixed oxides. The particular cases of $$\hbox {UO}_{2}+10~\%\hbox {Gd}_{2}\hbox {O}_{3}$$ and $$\hbox {UO}_{2}+7.5~\%\hbox {Dy}_{2}\hbox {O}_{3}$$ are discussed, showing how to determine the homogeneity of the prepared samples, distinguish interdiffused oxides from mixed oxides, recognize and characterize clusters, and determine the relative abundance of different species and its spatial distribution. Histograms can be generated that provide a quantitative determination of the homogeneity. Adding an independent determination of the thermal diffusivity as a function of the composition made in bulk material allows the conversion of the diffusivity map into a concentration map, providing direct quantitative determination of the homogeneity and spatial distribution in concentration units. The new device can be added as an accessory to an optical microscope entering through a standard C mount camera port. Two semiconductor lasers within the viewing spectroscopic window of any microscope (650 nm wavelength for the pump and 785 nm for the probe) that were coupled to a single-mode fiber were used, and the reflected signal was collected using the same fiber. With this construction, the resulting equipment was robust and reliable.

Optical detection of individual ultra-short carbon nanotubes enables their length characterization down to 10 nm.

Ultrashort single-walled carbon nanotubes, i.e. with length below ~30 nm, display length-dependent physical, chemical and biological properties that are attractive for the development of novel nanodevices and nanomaterials. Whether fundamental or applicative, such developments require that ultrashort nanotube lengths can be routinely and reliably characterized with high statistical data for high-quality sample production. However, no methods currently fulfill these requirements. Here, we demonstrate that photothermal microscopy achieves fast and reliable optical single nanotube analysis down to ~10 nm lengths. Compared to atomic force microscopy, this method provides ultrashort nanotubes length distribution with high statistics, and neither requires specific sample preparation nor tip-dependent image analysis.

Photothermal microscopy: imaging of energy dissipation from photosynthetic complexes.

An idea of a photothermal imaging microscopy (PTIM) is proposed, along with its realization based on a dependence of fluorescence anisotropy of dye molecules on heat emission in their nearest vicinity. Erythrosine B was selected as a fluorophore convenient to report thermal deactivation of the excited pigment-protein complex isolated from the photosynthetic apparatus of plants (LHCII), owing to the relatively large spectral gap between the fluorescence emission bands of chlorophyll a and a probe. Comparison of the simultaneously recorded images based on fluorescence lifetime of LHCII and fluorescence anisotropy of erythrosine shows a high rate of thermal energy dissipation from the aggregated forms of the complex and, possibly, thermal energy transmission along the protein supramolecular structures. Relatively high resolution of this novel microscopic technique, comparable to the fluorescence lifetime microscopy, enables its application in a nanoscale imaging and in nanothermography.

Measurement of Nanoparticle Adlayer Properties by Photothermal Microscopy.

Many nanoparticle applications require molecular adlayers that impart desirable interfacial characteristics. Such characteristics are crucial in controlling the interaction of the nanoparticle with the environment or other nanoparticles; however, departures from bulk values are expected for adlayer properties and in situ methods to evaluate the magnitude of these departures, preferably on the scale of a single nanoparticle, are needed. Here we investigate the potential of single-particle photothermal microscopy for measuring the thermal properties of nanoparticle-supported, layer-by-layer grown polyelectrolytes. We show that nanometer changes in adlayer thickness can be detected this way, and the water content of the nanoparticle-supported adlayers can be estimated.

Reduction of distortion in photothermal microscopy and its application to the high-resolution three-dimensional imaging of nonfluorescent tissues.

A scheme for reducing image distortion in photothermal microscopy is presented. In photothermal microscopy, the signal shape exhibits twin peaks corresponding to the focusing or defocusing of the probe beam when a sample is scanned in the axial direction. This causes a distortion when imaging a structured sample in the axial plane. Here, we demonstrate that image distortion caused by the twin peaks is effectively suppressed by providing a small offset between two the focal planes of the pump and the probe beams. Experimental results demonstrate improvement in resolution, especially in the axial direction, over conventional optical microscopy-even with the focal offset. When a dry objective lens with a numerical aperture of 0.95 is used, the full width at half the maximum of the axial point spread function is 0.6 μm, which is 50% (62%) smaller than the focal spot sizes of the pump (probe) beam. Herein, we present high-resolution three-dimensional imaging of thick biological tissues based on the present scheme.

Photothermal raster image correlation spectroscopy of gold nanoparticles in solution and on live cells.

Raster image correlation spectroscopy (RICS) measures the diffusion of fluorescently labelled molecules from stacks of confocal microscopy images by analysing correlations within the image. RICS enables the observation of a greater and, thus, more representative area of a biological system as compared to other single molecule approaches. Photothermal microscopy of gold nanoparticles allows long-term imaging of the same labelled molecules without photobleaching. Here, we implement RICS analysis on a photothermal microscope. The imaging of single gold nanoparticles at pixel dwell times short enough for RICS (60 μs) with a piezo-driven photothermal heterodyne microscope is demonstrated (photothermal raster image correlation spectroscopy, PhRICS). As a proof of principle, PhRICS is used to measure the diffusion coefficient of gold nanoparticles in glycerol : water solutions. The diffusion coefficients of the nanoparticles measured by PhRICS are consistent with their size, determined by transmission electron microscopy. PhRICS was then used to probe the diffusion speed of gold nanoparticle-labelled fibroblast growth factor 2 (FGF2) bound to heparan sulfate in the pericellular matrix of live fibroblast cells. The data are consistent with previous single nanoparticle tracking studies of the diffusion of FGF2 on these cells. Importantly, the data reveal faster FGF2 movement, previously inaccessible by photothermal tracking, and suggest that inhomogeneity in the distribution of bound FGF2 is dynamic.

Quantitative photothermal phase imaging of red blood cells using digital holographic photothermal microscope.

Photothermal microscopy (PTM), a noninvasive pump-probe high-resolution microscopy, has been applied as a bioimaging tool in many biomedical studies. PTM utilizes a conventional phase contrast microscope to obtain highly resolved photothermal images. However, phase information cannot be extracted from these photothermal images, as they are not quantitative. Moreover, the problem of halos inherent in conventional phase contrast microscopy needs to be tackled. Hence, a digital holographic photothermal microscopy technique is proposed as a solution to obtain quantitative phase images. The proposed technique is demonstrated by extracting phase values of red blood cells from their photothermal images. These phase values can potentially be used to determine the temperature distribution of the photothermal images, which is an important study in live cell monitoring applications.

Single-particle absorption spectroscopy by photothermal contrast.

Removing effects of sample heterogeneity through single-molecule and single-particle techniques has advanced many fields. While background free luminescence and scattering spectroscopy is widely used, recording the absorption spectrum only is rather difficult. Here we present an approach capable of recording pure absorption spectra of individual nanostructures. We demonstrate the implementation of single-particle absorption spectroscopy on strongly scattering plasmonic nanoparticles by combining photothermal microscopy with a supercontinuum laser and an innovative calibration procedure that accounts for chromatic aberrations and wavelength-dependent excitation powers. Comparison of the absorption spectra to the scattering spectra of the same individual gold nanoparticles reveals the blueshift of the absorption spectra, as predicted by Mie theory but previously not detectable in extinction measurements that measure the sum of absorption and scattering. By covering a wavelength range of 300 nm, we are furthermore able to record absorption spectra of single gold nanorods with different aspect ratios. We find that the spectral shift between absorption and scattering for the longitudinal plasmon resonance decreases as a function of nanorod aspect ratio, which is in agreement with simulations.

Biological imaging with nonlinear photothermal microscopy using a compact supercontinuum fiber laser source.

Nonlinear photothermal microscopy is applied in the imaging of biological tissues stained with chlorophyll and hematoxylin. Experimental results show that this type of organic molecules, which absorb light but transform dominant part of the absorbed energy into heat, may be ideal probes for photothermal imaging without photochemical toxicity. Picosecond pump and probe pulses, with central wavelengths of 488 and 632 nm, respectively, are spectrally filtered from a compact supercontinuum fiber laser source. Based on the light source, a compact and sensitive super-resolution imaging system is constructed. Further more, the imaging system is much less affected by thermal blurring than photothermal microscopes with continuous-wave light sources. The spatial resolution of nonlinear photothermal microscopy is ~ 188 nm. It is ~ 23% higher than commonly utilized linear photothermal microscopy experimentally and ~43% than conventional optical microscopy theoretically. The nonlinear photothermal imaging technology can be used in the evaluation of biological tissues with high-resolution and contrast.

TAT and HA2 facilitate cellular uptake of gold nanoparticles but do not lead to cytosolic localisation.

The methods currently available to deliver functional labels and drugs to the cell cytosol are inefficient and this constitutes a major obstacle to cell biology (delivery of sensors and imaging probes) and therapy (drug access to the cell internal machinery). As cell membranes are impermeable to most molecular cargos, viral peptides have been used to bolster their internalisation through endocytosis and help their release to the cytosol by bursting the endosomal vesicles. However, conflicting results have been reported on the extent of the cytosolic delivery achieved. To evaluate their potential, we used gold nanoparticles as model cargos and systematically assessed how the functionalisation of their surface by either or both of the viral peptides TAT and HA2 influenced their intracellular delivery. We evaluated the number of gold nanoparticles present in cells after internalisation using photothermal microscopy and their subcellular localisation by electron microscopy. While their uptake increased when the TAT and/or HA2 viral peptides were present on their surface, we did not observe a significant cytosolic delivery of the gold nanoparticles.

Mie and potential scattering by a refractive 1/r inhomogeneity: Electromagnetic scattering by an infinite Coulomb-like scatterer

The weak electromagnetic scattering of a special type of gradient index scatterer resembles the situation of charged particle scattering off an electrostatic Coulomb potential. Accordingly, plane-wave scattering for a radially symmetric decaying perturbation of the refractive index n(r)=n0−ν/k0r is studied. The experimental realization of this specific scattering is found in photothermal single particle microscopy. Vectorial scattering in Lorenz–Mie theory recovers the properties in the Born approximation of the corresponding potential scattering scenario. Shaped beams within the generalized Lorenz–Mie theory are found to resolve known pathologic properties of the problematic 1/r potential.

Label-free imaging of melanoma with nonlinear photothermal microscopy.

Nonlinear photothermal microscopy, in which the intensity of the pump heating beam is modulated at f and the photothermal signal is extracted from the probe beam with a lock-in amplifier referred to 2f, is applied to the imaging of mouse melanoma without any staining. The pump and probe pulses, with central wavelengths of 488 and 632 nm, and a pulse duration of ∼100 ps, are filtered from a compact commercial supercontinuum fiber laser source. An auto-balanced detector is applied to accumulate the signal and remove the laser noise of the probe. The spatial resolution of the nonlinear photothermal imaging is enhanced by ∼18% in both theoretical calculations and experiments, compared with a linear photothermal mechanism, and the resolution enhancement is theoretically ∼42% compared with conventional optical microscopy. This imaging technique shows possibilities for the clinical evaluation of melanoma with a high contrast and spatial resolution.

Gold Nanoparticles: Recent Advances in the Biomedical Applications

Among the multiple branches of nanotechnology applications in the area of medicine and biology, Nanoparticle technology is the fastest growing and shows significant future promise. Nanoscale structures, with size similar to many biological molecules, show different physical and chemical properties compared to either small molecules or bulk materials, find many applications in the fields of biomedical imaging and therapy. Gold nanoparticles (AuNPs) are relatively inert in biological environment, and have a number of physical properties that are suitable for several biomedical applications. For example, AuNPs have been successfully employed in inducing localized hyperthermia for the destruction of tumors or radiotherapy for cancer, photodynamic therapy, computed tomography imaging, as drug carriers to tumors, bio-labeling through single particle detection by electron microscopy and in photothermal microscopy. Recent advances in synthetic chemistry makes it possible to make gold nanoparticles with precise control over physicochemical and optical properties that are desired for specific clinical or biological applications. Because of the availability of several methods for easy modification of the surface of gold nanoparticles for attaching a ligand, drug or other targeting molecules, AuNPs are useful in a wide variety of applications. Even though gold is biologically inert and thus shows much less toxicity, the relatively low rate of clearance from circulation and tissues can lead to health problems and therefore, specific targeting of diseased cells and tissues must be achieved before AuNPs find their application for routine human use.

Sensitivity enhancement of photothermal microscopy with radially segmented balanced detection.

A novel detection method is proposed for highly sensitive photothermal microscopic imaging. This method is based on the characteristics of an angular-dependent photothermal signal; it improves signal intensity by up to two times and rejects the intensity noise of the probe beam. The subdiffraction resolution photothermal imaging of mouse skin melanoma is demonstrated using a laser diode-based photothermal microscopy system to evaluate this method. We confirm that the signal intensity is enhanced 1.7 times compared with the conventional detection method. Moreover, the intensity noise of the laser diode used for the probe beam is effectively reduced by approximately 31 dB, even for a sample with non-uniformity of the refractive index and stationary absorption. This method is implemented by means of a commonly used balanced detector and is thus potentially useful for high-speed imaging.

Simultaneous dual-wavelength imaging of nonfluorescent tissues with 3D subdiffraction photothermal microscopy.

Multi-wavelength microscopic imaging is essential to visualize a variety of nanoscale cellular components with high specificity and high spatial resolution. However, previous techniques are based on fluorescence, and thus cannot visualize nonfluorescent species, which are much less suffered from photodamage or photobleaching and hence are intrinsically useful in wider range of optical microscopy. Here, we show that simultaneous multi-wavelength imaging of nonfluorescent species can be achieved with the use of a photothermal microscope. Dual-wavelength subdiffraction imaging of biological tissues stained with hematoxylin and eosin is demonstrated. Three-dimensional label-free imaging of mouse melanoma tissue section is also presented to demonstrate the effectiveness of the enhanced spatial resolution. Our technique can be implemented using cost-effective and compact laser diodes and is applicable for various types of both fluorescent and nonfluorescent tissues.

Targeting Cell Membrane Lipid Rafts by Stoichiometric Functionalization of Gold Nanoparticles With a Sphingolipid-Binding Domain Peptide.

A non-membrane protein-based nanoparticle agent for the tracking of lipid rafts on live cells is produced by stoichiometric functionalization of gold nanoparticles with a previously characterized sphingolipid- and cell membrane microdomain-binding domain peptide (SBD). The SBD peptide is inserted in a self-assembled monolayer of peptidol and alkane thiol ethylene glycol, on gold nanoparticles surface. The stoichiometric functionalization of nanoparticles with the SBD peptide, essential for single molecule tracking, is achieved by means of non-affinity nanoparticle purification. The SBD-nanoparticles have remarkable long-term resistance to electrolyte-induced aggregation and ligand-exchange and have no detectable non-specific binding to live cells. Binding and diffusion of SBD-nanoparticles bound to the membrane of live cells is measured by real-time photothermal microscopy and shows the dynamics of sphingolipid-enriched microdomains on cells membrane, with evidence for clustering, splitting, and diffusion over time of the SBD-nanoparticle labeled membrane domains. The monofunctionalized SBD-nanoparticle is a promising targeting agent for the tracking of lipid rafts independently of their protein composition and the labelling requires no prior modification of the cells. This approach has potential for further functionalization of the particles to manipulate the organization of, or targeting to microdomains that control signaling events and thereby lead to novel diagnostics and therapeutics.

Probing local bias-induced transitions using photothermal excitation contact resonance atomic force microscopy and voltage spectroscopy.

Nanomechanical properties are closely related to the states of matter, including chemical composition, crystal structure, mesoscopic domain configuration, etc. Investigation of these properties at the nanoscale requires not only static imaging methods, e.g., contact resonance atomic force microscopy (CR-AFM), but also spectroscopic methods capable of revealing their dependence on various external stimuli. Here we demonstrate the voltage spectroscopy of CR-AFM, which was realized by combining photothermal excitation (as opposed to the conventional piezoacoustic excitation method) with the band excitation technique. We applied this spectroscopy to explore local bias-induced phenomena ranging from purely physical to surface electromechanical and electrochemical processes. Our measurements show that the changes in the surface properties associated with these bias-induced transitions can be accurately assessed in a fast and dynamic manner, using resonance frequency as a signature. With many of the advantages offered by photothermal excitation, contact resonance voltage spectroscopy not only is expected to find applications in a broader field of nanoscience but also will provide a basis for future development of other nanoscale elastic spectroscopies.

The spherical nucleic acids mRNA detection paradox

Abstract From the 1950s onwards, our understanding of the formation and intracellular trafficking of membrane vesicles was informed by experiments in which cells were exposed to gold nanoparticles and their uptake and localisation, studied by electron microscopy. In the last decade, building on progress in the synthesis of gold nanoparticles and their controlled functionalisation with a large variety of biomolecules (DNA, peptides, polysaccharides), new applications have been proposed, including the imaging and sensing of intracellular events. Yet, as already demonstrated in the 1950s, uptake of nanoparticles results in confinement within an intracellular vesicle which in principle should preclude sensing of cytosolic events. To study this apparent paradox, we focus on a commercially available nanoparticle probe that detects mRNA through the release of a fluorescently-labelled oligonucleotide (unquenching the fluorescence) in the presence of the target mRNA. Using electron, fluorescence and photothermal microscopy, we show that the probes remain in endocytic compartments and that they do not report on mRNA level. We suggest that the validation of any nanoparticle-based probes for intracellular sensing should include a quantitative and thorough demonstration that the probes can reach the cytosolic compartment.

The spherical nucleic acids mRNA detection paradox

Abstract From the 1950s onwards, our understanding of the formation and intracellular trafficking of membrane vesicles was informed by experiments in which cells were exposed to gold nanoparticles and their uptake and localisation, studied by electron microscopy. In the last decade, building on progress in the synthesis of gold nanoparticles and their controlled functionalisation with a large variety of biomolecules (DNA, peptides, polysaccharides), new applications have been proposed, including the imaging and sensing of intracellular events. Yet, as already demonstrated in the 1950s, uptake of nanoparticles results in confinement within an intracellular vesicle which in principle should preclude sensing of cytosolic events. To study this apparent paradox, we focus on a commercially available nanoparticle probe that detects mRNA through the release of a fluorescently labelled oligonucleotide (unquenching the fluorescence) in the presence of the target mRNA. Using electron, fluorescence and photothermal microscopy, we show that the probes remain in endocytic compartments and that they do not report on mRNA level. We suggest that the validation of any nanoparticle-based probes for intracellular sensing should include a quantitative and thorough demonstration that the probes can reach the cytosolic compartment.