Lipid droplets have become a major research topic recently, as they are found to be involved in obesity related diseases.
Most of this research has been focused on the localization of the proteins playing a role in lipid droplet formation or
breakdown. The role of different lipid species however remains unclear because it is difficult to distinguish different fatty
acids with the present microscopy techniques. Coherent Anti-Stokes Raman scattering (CARS) is the non-linear analogue
of spontaneous Raman scattering. Multiplex CARS microscopy can provide quantitative, chemical and physical
information, making it an excellent tool to study the composition and thermodynamic phase of lipid droplets. To
investigate the potential of CARS in this field, we have incubated HeLa cells with four different fatty acids, varying in
saturation. The fatty acids were internalized by the cells and stored as lipid droplets, which we imaged with multiplex
CARS microscopy. We were able to distinguish either of the fatty acids as such in lipid droplets inside the cells.
Furthermore, we found that solid phase fatty acids were fluidized when present in lipid droplets. This illustrates the
potential of CARS microscopy to elucidate the possible role of the chemistry of fatty acids in lipid droplet regulation.
Third Harmonic Generation (THG) from the vicinity of interfaces, using focused laser beams can be obtained virtually
from any inhomogeneous medium. Its sensitivity to the presence and extent of inhomogeneity in the focal volume has
already found a variety of applications ranging from material characterization to label free three-dimensional
microscopy of biological samples. In this presentation, we demonstrate a number of new applications of THG in the
microscopy of food samples and living cells. Also, we report on an anomalous behavior in the THG z-response. So far
the observations and theoretical predictions supported a single peak of THG signal, with the peak position
corresponding to the interface. We have observed an anomalous behavior where a single interface can give rise to two
peaks located across the interface. The simulations, which we carried out using a paraxial theory of THG and
measurements done on typical normally dispersive materials, suggest that this anomalous behavior is due to a particular
combination of χ(3) and the magnitude of dispersion.
A novel procedure is developed to describe and reproduce experimental coherent anti-Stokes Raman scattering (CARS)
data, with particular emphasis on highly congested spectral regions. The approach - exemplified here with high-quality
multiplex CARS data - makes use the maximum entropy method for phase retrieval. The retrieved imaginary part of the
nonlinear susceptibility is shown to be equal to the spontaneous Raman spectrum. The phase retrieval procedure does not
influence the noise contained in the spectra. The conversion of CARS to Raman data permits a quantitative interpretation
of CARS spectra. This novel approach is demonstrated for highly congested multiplex CARS spectra of sucrose, fructose
and glucose. This novel procedures enables extraction of vibrational information from multiplex CARS data without the
use of a priori information of the sample.
A method for noninvasively determining blood oxygenation in individual vessels inside bulk tissue would provide a powerful tool for biomedical research. We explore the potential of coherent anti-Stokes Raman scattering (CARS) spectroscopy to provide this capability. Using the multiplex CARS approach, we measure the vibrational spectrum in hemoglobin solutions as a function of the oxygenation state and observe a clear dependence of the spectral shape on oxygenation. The direct extraction of the Raman line shape from the CARS data using a maximum entropy method phase retrieval algorithm enables quantitative analysis. The CARS spectra associated with intermediate oxygenation saturation levels can be accurately described by a weighted sum of the fully oxygenated and fully deoxygenated spectra. We find that the degree of oxygenation determined from the CARS data agrees well with that determined by optical absorption. As a nonlinear optical technique, CARS inherently provides the 3-D imaging capability and tolerance to scattering necessary for biomedical applications. We discuss the challenges in extending the proof of principle demonstrated to in vivo applications.
This book provides a comprehensive account of the theory of image formation in a confocal fluorescence microscope as well as a practical guideline to the operation of the instrument, its limitations, and the interpretation of confocal microscopy data. The appendices provide a quick reference to optical theory, microscopy-related formulas and definitions, and Fourier theory.
The general utility of two compact femtosecond laser sources for Third Harmonic Generation (THG) and Multiphoton absorption microscopy is demonstrated. The effect of aberrations due to a dielectric interface on THG microscopy is investigated both from a theoretical and experimental point of view. The significance of these aberration issues in third order nonlinear optical susceptibility (χ(3)) measurements using THG microscopy is discussed.
KEYWORDS: Signal to noise ratio, Raman spectroscopy, Signal detection, Microscopy, Raman scattering, Polarization, Spectral resolution, Signal generators, Spectroscopy, Interference (communication)
In multiplex CARS microscopy the generated anti-Stokes signal is generated and detected simultaneously
over a significant part of the vibrational spectrum. The signal-to-noise ratio of the thus detected spectra is
limited only by shot-noise. This principle is demonstrated using a dilution series of 2-propanol in water.
It is derived theoretically and shown experimentally that for low solute concentrations - in contrast to
methods that suppress the non-resonant background - the CARS signal strength from a particular
vibrational mode depends linearly on its concentration. Furthermore, excellent agreement is shown
between the experimental data and fits to the theory. It is shown that this approach permits rapid (20 ms
acquisition) detection of a single lipid mono-layer, with sufficient signal-to-noise to determine the order
parameter for the acyl chain packing. Also it is demonstrated that this detection scheme provides an
absolute measure of the solute concentration.
Using both vibrational sum-frequency generation and fluorescence microscopy, the phase behavior of a DPPC lipid monolayer on water is investigated as a function of surface pressure. The vibrational specificity of the sum-frequency generation techniques permits determining the order of the alkyl chains, as well as the average orientation of the terminal methyl group. A novel -- and extremely sharp -- phase transition is observed at low compression, which is attributed to a curling of the alkyl chains due to increased exposure of the (hydrophobic) alkyl chains to the water surface.
As ultrafast multiphoton microscopes become more useful for biological imaging, a major challenge for researchers is to determine the exposure conditions that provide the best combination of image resolution, contrast and specimen viability. To do this requires an accurate understanding of the spatial and temporal evolution of ultrashort pulses at the focus produced by a microscope objective. The objective itself, however, can significantly alter the pulses. Some effects, such as the broadening of pulses due to group delay dispersion in materials along the path, are understood and partial compensation for them can be made. Other effects, such as radial variations in the propagation time and variations in the pulse width, are less well understood. In this work, we investigate the radially dependent propagation and focusing of ultrashort pulses through a Zeiss CP- Achromat 100X, 1.25 NA, infinity-corrected, oil immersion microscope objective. We also extend to this high numerical aperture case the technique of collinear type II second harmonic generation frequency-resolved optical grating which has previously been used to measure the temporal intensity and phase of ultrashort pulses at the focus of air objectives with lower numerical aperture.
It is shown that based on spectrally selective excitation of individual molecules in the focus of a high NA lens together with position sensitive imaging sub-resolution imaging of three-dimensional structures can be realized. The feasibility of the idea is demonstrated with NA equals 0.55 optics on a model system of pentacene molecules in p-terphenal host matrix.
High intensity chirped pulses can be used for probing microscopic chemical environments through the use of a particular choice of dye, for instance SNAFL2. The basis for this technique is that the excited state populations can be manipulated through control over the temporal order of the excitation frequencies in the excitation pulse -- i.e. chirp - - with the outcoming fluorescence as the reporting parameter. A chirp dependent fluorescence response can also be observed in larger molecular systems with more degrees of freedom like for instance green fluorescent proteins. In preparation for application of the technique to microscopy we use a facility permitting observation of this phenomenon in various dyes with high sensitivity. High power, 30 fs pulses from an OPA, tunable from 400 nm to 1.5 micron are used. These pulses with a repetition rate of 1 kHz are sufficiently intense that a relatively large sample region can be excited to saturation from which then a sub-region with uniform excitation conditions can be selected for signal collection.
Recently a novel imaging technique based on third-harmonic generation (THG) was introduced. This technique relies on a third-order non-linear interaction to generate a coherent signal response on the third-harmonic frequency with respect to the fundamental input radiation. Here we report on the input NA dependence of the THG signal and examine the resulting imaging characteristics of this novel technique in terms of resolution and contrast generation. We'll demonstrate the potential of the technique through a number of imaging examples, with special emphasis on in vivo applications. The latter illustrates the non-invasive character of the technique.
This contribution discusses some biological applications of ultrashort laser pulses. Some examples are given of recently developed techniques that exploit the special features offered by ultrashort laser pulses: real time two-photon microscopy with multipoint excitation, fluorescence lifetime measurement by double pulse saturation excitation and pH-sensing by multiphoton quantum control.
Pulse broadening of ultrashort optical pulses, as short as 15 femtoseconds, due to the propagation through high- numerical-aperture microscope objectives can be pre- compensated to ensure temporal pulse integrity at the focal point. The predictions from dispersive ray-tracing calculations show excellent agreement with the experimental results from two-photon absorption autocorrelation for the Zeiss CP-Achromat 100X/1,25 oil microscope objective. From this, general predictions can be inferred for dispersion in most types of microscope objectives. Key element to the work is a carefully designed dispersion pre- compensation configuration, which minimizes pulse broadening due to residual third order dispersion. The capability to focus these ultrashort pulses with control of the pulse definition at the focal point is important for two-photon absorption and time-resolved microscopy.
Over the last few years a number of microscopical techniques have been developed that take advantage of ultrashort optical pulses. All these techniques rely on temporal pulse integrity at the focal point of a high-numerical aperture (NA) focusing system. We have investigated the dispersion induced broadening for pulses on the optical axis, using the two-photon absorption autocorrelation (TPAA) technique. We demonstrate that the induced broadening can be pre- compensated for by a properly designed dispersion pre- compensation unit for pulses as short as 15 femtosecond. Another source of pulse broadening in high-NA focusing systems is due to radial variations in the dispersion over the pupil of the objective. This may cause differences in the group delay between on-axis and outer ray wave packets, as well as differences in the broadening of the wave packets themselves. In this paper we present experimental results on the measurement of these radial variations in the dispersion characteristics over the aperture of high-NA microscope objectives, using a slightly modified TPAA technique.
We summarize recent progress aimed at observing biochemical and biological dynamics using confocal microscopy with 3D spatial resolution down to a few hundred nm and temporal resolution to 15 fs. We also review recent control of population dynamics using tailored ultrafast pulses, i.e. quantum control. Progress is described for i) feedback control, ii) multiphoton control, and iii) molecular (pi) pulse. Finally, using ultrafast light pulses, we combine confocal and quantum control techniques to produce a new way to measure the microscopy chemical environment, int his case pH, potentially with a spatial resolution of a few hundred nanometers.
It is shown that nanosecond to picosecond fluorescence relaxation phenomena can be accessed for imaging after double pulse saturation excitation. This new technique has been introduced before as fluorescence lifetime imaging (DPFLIm) (Mueller et al, 1995). An OPA laser system generating ultra short, widely tunable, high power optical pulses provides the means for the selective excitation of specific fluorophores at sufficient excitation levels to obtain the necessary (partial) saturation of the optical transition. A key element in the developed method is that the correct determination of fluorescence relaxation times does allow for non-uniform saturation conditions over the observation area. This is true for the validation demonstration experiments reported here as well as for imaging applications at a later stage. Measurements on bulk solutions of Rhodamine B and Rhodamine 6G in different solvents confirm the experimental feasibility of accessing short fluorescence lifetimes with this technique. As only integrated signal detection is required no fast electronics are needed, making the technique suitable for fluorescence lifetime imaging in confocal microscopy, especially when used in combination with bilateral scanning and cooled CCD detection.
In PSAF (point spread autocorrelation function) imaging a fluorescence signal is generated from the interference response in the overlap region of two spatially shifted point spread functions (PSFs). It is experimentally demonstrated that a resolution improvement of approximately 30% can be realized in the case of axially shifted PSFs under high numerical-aperture (NA) conditions. A similar improvement in resolution is expected from numerical modeling for the lateral case. The presented technique can also be applied to the measurement of the effective point spread function itself in all three dimensions. It is found that the technique -- which in the latter case uses a bulk fluorescing solution -- is an excellent tool to access the apodization conditions of a practical optical system, such as a high-numerical aperture objective.
We present a new technique that provides a functional response similar--but not identical--to the point spread function which can be used for (i) characterizing the apodization conditions of a high numerical aperture (NA) lends, or (ii) imaging with an improved resolution over confocal. This measurement technique employs a spatial autocorrelation of the focal field, absorption and subsequent fluorescence in a sample containing a suitable fluorophore. The experimental results presented show the sensitivity of the new technique to different apodization conditions of the lens. Theoretically the experiments are modeled from the first Rayleigh-Sommerfeld integral of scalar diffraction theory in the Kirchhoff approximation. Without any fitting parameters, the calculated curves show excellent agreement with the experimental results. Based on the same principle, the technique can be used to obtain improved resolution over that obtainable in confocal microscopy. The imaging method is presented and the imaging resolution is analyzed for two model objects. The method employs only one high NA lens for the potentially robust common path delivery of the autocorrelated excitation beams.
A novel technique that provides a functional response, similar to the point spread function, for the characterization of high NA lenses in real time is presented. This measurement technique employs absorption and subsequent fluorescence in a bulk solution of a suitable fluorophore. Both the dye and the solvent can be chosen to match the experimental conditions best for which the lens is tested, especially with respect to the refractive index of the solvent and the wavelengths of excitation and emission. The experimental results presented show the sensitivity of the new technique to different apodization conditions of the lens. Theoretically the experiments are modeled from the Rayleigh-Sommerfeld integral of scalar diffraction theory in the Kirchhoff approximation. Applying the shift-invariance approximation to save computing time, theoretical curves are calculated without any fitting parameters that show excellent agreement with the experimental results.
Based on the principle of spatial autocorrelation of the focal field, improved resolution can be obtained over that obtainable in confocal microscopy. The method is presented and the imaging resolution is analyzed for two model objects. The method employs only one high NA lens for the, potentially robust, common path delivery of the autocorrelated excitation beams.
A new technique for the measurement of fluorescence lifetimes relies on the (near steady state) excitation with short optical pulses. The novel technique has the potentiality to provide high time resolution--since it relies on the laser pulse duration, rather than on electronic gating techniques--and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. Combined with confocal microscopy it enables the spatial determination of the fluorescence lifetimes, the value of which is influenced by the local environment of fluorescent probe molecules in biological samples. The principle of the technique is discussed within a theoretical framework taking into account various secondary effects.
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