The total or integrated fluorescence intensity of a through-focus series of a thin standardized uniform fluorescent or
calibration layer is shown to be suitable for image intensity correction and calibration in sectioning microscopy. This
integrated intensity can be derived from the earlier introduced SectionedImagingProperty or SIPcharts, derived from the
3D layer datasets.
By correcting the 3D image of an object with the 3D image of the standardized uniform fluorescent layer obtained under
identical conditions one is able to express the object fluorescence in units fluorescence of the calibration layer. With
object fluorescence intensities in fluorescence layer unit's or FLU's the object image intensities becomes independent of
microscope system and imaging conditions.
A direct result is that the often-appreciable lateral intensity variations present in confocal microscopy are eliminated
(shading correction). Of more general value is that images obtained with different objectives, magnifications or from
different microscope systems can be quantitatively related to each other.
The effectiveness of shading correction and relating images obtained under various microscope conditions is
demonstrated on images of standard fluorocent beads.
Expressing the object fluorescence in FLU units seems to be a promising approach for general quantification of
sectioning imaging enabling cross-correlation of imaging results over time and between imaging systems.
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.
Layer-by-Layer or self-assembly techniques can be used to prepare Fluorescent polymer samples on glass coverslips
serving as benchmark for two-photon excitation microscopy from conventional to 4Pi set-up, or more in general
for sectioning microscopy. Layers can be realized as ultra-thin (<< 100 nm) or thin (approx. 100 nm)
characteristics coupled to different fluorescent molecules to be used for different microscopy applications. As well, stacks hosting different fluorescent molecules can be also produce. Thanks to their controllable thickness, uniformity and fluorescence properties, these polymer layers may serve as a simple and applicable standard to
directly measure the z-response of different scanning optical microscopes. In two-photon excitation microscopy z-sectioning plays a central role and uniformity of illumination is crucial due to the non-linear behaviour of emission. Since the main characteristics of a particular image formation situation can be efficiently summarized
in a Sectioned Imaging property chart (SIPchart), we think that coupling this calibration sample with SIPchart is a very important step towards quantitative microscopy. In this work we use these polymer layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes, selecting properly ultra-thin and thin layers. Due to their uniformity over a wide region, i.e. coverslip surface, it is possible to quantify the z-response of the system over a full field of view area. These samples are also useful for monitoring photobleaching
behavior as function of the illumination intensity. Ultrathin layers are also useful to supersede the conventional
technique of calculating the derivative of the axial edges of a thick fluorescent layer. Polymer layers can be
effciently used for real time alignment of the microscope.
In this paper we study the spatial distribution and wavefront characteristics of third harmonic generation in relation to some material and interface conditions over the focal region of the fundamental beam. We investigate, mostly from an experimental point of view, the implications the physics of the THG generation process has in situations where THG may be employed for 3D imaging. Due to the non- linear character of the THG generation process it is inherently suitable for this application. For the first time images of the distribution of the THG radiation, as the interface is moved through focus, are shown. Experiments on closely spaced interfaces or bilayers confirm unambiguously the correctness of the vector model for THG generation (Ward et al. 1969) in uniform media. In view of these and other data the image formation, especially for biological objects, with THG radiation will be discussed.
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.
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.
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.
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.
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.
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.
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.
Real-time confocal imaging is subject to a number of constraints connected with the emission capabilities of (especially) fluorescent specimen and the particular confocal imaging technique employed. We will see that there are from the confocal image collection techniques no basic impediments towards real-time 3D imaging. The limitations sooner lie in the specimen fluorescence emission capabilities--both with respect to emission rate and total emission and of course biological tolerance limits to the exciting radiation. The various factors are examined and it is found that parallel confocal illumination and detection approaches especially via techniques of direct field (also known as direct view) confocal imaging. These combined with detection on CCD detectors offer probably the optimal and most convenient way to realize real-time imaging.
The high quantum efficiency and high red sensitivity of a charge coupled device (CCD) make it a very suitable detector for confocal fluorescence imaging, especially for applications in biology, where short wavelength illumination may cause undesirable radiation damage. In addition, its high dynamic range matches the high contrast imaging inherent in confocal microscopy, and enables one to record small intensity differences if this dynamic range is well exploited. Applications shown include imaging with bleaching rate and non-linear fluorescence excitation as parameters. The bilateral scan technique permits effective confocal imaging using this type of device. CCDs can be used for confocal imaging both in high speed (up to real- time) applications as well as in the integration mode, where the high sensitivity application areas for this confocal technique are present.
In the confocal scanning light microscope a specific volume is sampled during the imaging process. The physical process is explained, together with how the size of the pinholes used affect the actual size of this volume. The thus produced 3-D imaging is of high quality but subject to a number of limitations. A novel (bilateral scanning) arrangement is presented that may relieve some of these. In this approach, a double-sided scanning mirror element and a charge coupled device (CCD for image collection) are used.
In the confocal scanning light microscope a specific volume is sampled during the imaging process. The physical process is explained, together with how the size of the pinholes used affects the actual size of this volume. The thus produced 3-dimensional imaging is of high quality but subject to a number of limitations. A novel (bilateral scanning) arrangement is presented which may relieve some of these. Use is made in this approach of a double sided scanning mirror element and a charge coupled device (CCD) for image collection.
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