text:
University of California, Beckman Laser Institute, Irvine, California 92697
Center for Nano-Bio Technology, Division of Convergence Technology, Korea Research Institute of Standards and Science, 1 Doryong-Dong, Yuseong-Gu, Daejeon 305-340, Republic of Korea
Coherent Raman scattering (CRS) microscopy is gaining acceptance as a valuable addition to the imaging toolset of biological researchers. Optimal use of this label-free imaging technique benefits from a basic understanding of the physical principles and technical merits of the CRS microscope. This tutorial offers qualitative explanations of the principles behind CRS microscopy and provides information about the applicability of this nonlinear optical imaging approach for biological research.
Coherent Raman scattering (CRS) techniques are sensitive to the same molecular vibrations that are probed in spontaneous Raman spectroscopy. Unlike linear Raman spectroscopy, however, CRS techniques exhibit a nonlinear dependence on the incoming light fields and produce coherent radiation. It is this latter property that has popularized CRS as a microscopy modality, as it is intimately related to the technique's strong optical signals that enable fast imaging applications.
CRS microscopy makes it possible to generate images based on vibrational Raman contrast at imaging speeds much faster than attained with conventional Raman microscopes. Clearly, this attribute is very attractive for biological imaging, where imaging speed is an important experimental parameter. Nonetheless, the qualities of CRS in the area of optical imaging were not immediately evident when the phenomenon was first demonstrated in 1962.1,2 Its use as a contrast mechanism in optical microscopy was still 20 years ahead.3 Early applications of CRS included frequency conversion of laser light and gas phase spectroscopy measurements.4–6 Driven by the development of pulsed lasers with increasingly shorter temporal pulse widths, the technique was later used as a time-resolved spectroscopy tool for precision measurements of molecular vibrations in the condensed phase.7–9
Intrinsic to condensed phase CRS measurements were the relatively long lengths over which the incident pulses interact with the material to generate the signal, a condition that needed special beam geometries to avoid phase mismatching of the light fields, most notably in coherent anti-Stokes Raman scattering (CARS).10 Interestingly, it is the notion of phase mismatching that held back a practical implementation of CRS in an optical microscope. It was believed that the complex beam geometries associated with conventional CRS spectroscopy were difficult to combine with the use of high numerical aperture lenses. In hindsight, this notion was wrong, as beautifully demonstrated in 1999:11 due to the much shorter interaction lengths, phase mismatching is much less of a concern in microscopy, especially when the signal is detected in the forward-propagating direction.12 This realization simplified the beam configuration and turned CRS into a workable tool, triggering the exponential growth of the coherent Raman microscopy field.
More than a decade later, the CRS microscope has matured into a user-friendly instrument that is now firmly part of the tool set available for biological research.13 Similar to other nonlinear optical imaging techniques that came before it, CRS imaging systems can be purchased commercially from leading microscope manufacturers.14,15 Consequently, the focus has shifted from technique development to biological applications. Indeed, the availability of label-free vibrational contrast at realistic image acquisition rates represents a major step forward in dynamic imaging of tissues and cells, as shown in numerous studies. Examples include studying the health of the nervous system through CRS imaging of myelin,16–20 following lipid metabolism in living organisms,21,22 mapping cholesterol content in atherosclerotic plaques,23–27 and recording water diffusion in cells and tissues.28,29
In this tutorial, we highlight some of the most important imaging properties of CRS microscopy. We explain the basics of CRS in a simple yet intuitive way to emphasize the unique character of this technique compared to related imaging methods. We also discuss the experimental advantages and disadvantages of this imaging approach and illustrate its utility with several biomedical imaging applications.
We emphasize that what follows is a tutorial, which does not attempt to provide the most comprehensive overview of what has been achieved in the field of CRS microscopy to date. Technological developments and applications of CRS microscopy have been vast over the last decade, yet it is not the purpose of this contribution to broadly cover these achievements. Instead, this tutorial focuses on the principles of CRS and provides student-level explanations of basic features of the CRS microscope. For more inclusive reviews on technical advances and biological imaging applications, the reader is referred elsewhere.13,30–33
The vibrationally sensitive CRS signal relies on the Raman effect. Spontaneous Raman scattering involves two photons, an incident photon with frequency ωp, called pump, and a scattered photon with frequency ωs, called Stokes. Assuming that the molecule is initially in its ground state, the Raman scattered photon is of lower frequency than the incident photon. The energy difference between the photons, E=ℏ(ωp−ωs), corresponds to the vibrational energy of the molecule, which is left in a vibrationally excited state after the light-matter interaction (see Fig. 1).
Spontaneous and coherent Raman scattering (CRS). (a) In spontaneous Raman scattering, the presence of Raman active molecular mode ωυ dresses the incoming light ω1 with red- and blue-shifted frequency components. (b) In coherent Raman scattering, two incident fields (ω1,ω2) drive the molecule at Ω=ω1−ω2, producing coherent Raman signals at ω1±Ω and ω2±Ω.
Classically, the Raman effect can be understood as follows. The radiation produced by the molecule can be modeled as a radiating dipole positioned at r. In the presence of a classical field with frequency ωp, the dipole will oscillate at ωp as well. This motion physically corresponds to the adiabatic motion of the molecule's most moveable electrons, typically its valence electrons. However, the electron cloud, and thus the effective dipole, is capable of oscillating at different frequencies as well. This happens when electron motion is coupled to motional degrees of freedom of the nuclei.34 We can model nuclear motion along a generalized coordinate q in terms of vibrational motion with a characteristic frequency ωυ. When coupling exists, the electronic polarizability α can change with the nuclear displacement, i.e., δα/δq≠0, a property that is intimately related to the symmetry of the molecule. The component of the dipole that is driven at ωp and radiating at ωs is written as Display Formula
CRS senses the same molecular property. The radiating dipole in CRS can be summarized as Display Formula
This discussion above underlines the similarity between CRS and spontaneous Raman as they both depend on the molecular property (δα/δq)0. But there are also important differences, which we highlight next.
The detected signal resulting from the single radiating dipole described above is given as Display Formula
Upon inserting the expressions of Eqs. (1) and (2) in Eq. (3), it is clear that the spontaneous Raman scattering and CRS signals exhibit different dependencies on the intensity of the incident fields. The spontaneous Raman signal scales as Ip, the intensity of the incoming field. It is, therefore, classified as a linear optical signal. The CRS signals, on the other hand, are nonlinear, as they depend nonlinearly on the intensity of the incoming fields.
Another important difference is the presence of the phase φ in the oscillating dipole in Eq. (1), which is absent in the CRS expression in Eq. (2). In spontaneous Raman, the phase of the nuclear mode at equilibrium is different for each molecule. As a consequence, the phase of the radiating field from a given molecule is uncorrelated with the phase of the field from a different molecule. The total Raman scattered signal is a sum of the intensities of the individual dipole radiators. Display Formula
The situation is different in CRS. Here the incident fields dictate the phase of the oscillatory electron motion, a direct consequence of the active driving of the molecule. The random phase of the ground-state nuclear mode is no longer a factor. Because all molecules in the interaction volume see the same driving fields, their electron motions are now phase correlated as they all move in step. The total signal is a coherent sum of the dipole contributions: Display Formula
The coherence of the CRS signal is one of its most celebrated features. It is partly responsible for the inherently much higher signal yields in CRS compared to spontaneous Raman scattering. In the next subsection, we will illuminate this aspect for both CARS and SRL. Another important practical implication of coherence is that the signal propagates in a well-defined direction in a manner that is similar to the propagation of a laser beam (see Fig. 2). This implies that the signal can be very efficiently collected with a collimating lens, which yields high detection efficiencies of the CRS signal.12 This is particularly relevant when imaging transparent samples, in which case the detector can be placed in the forward-propagation direction—the direction in which the CRS signals are commonly phase-matched. The situation in spontaneous Raman is different: incoherent radiation does not exhibit a well-defined propagation direction. Hence, the signal collection efficiency is generally less in spontaneous Raman microscopy.
Sketch depicting that spatial coherence in CRS leads to strong and directional radiation. (a) The waves resulting from spontaneous Raman emission by separate dipoles (black dots) in the focal volume are not phase coherent, and no constructive interference occurs in a particular direction. (b) The CRS radiation from different dipole emitters constructively interferes in the forward-propagating direction, indicated by the red dotted lines.
The CRS techniques can be roughly divided into two classes: signals that are measured at a new frequency, i.e., color, and signals that are measured at a frequency similar to that of the incident light fields (ωp and ωs). CSRS and CARS fall into the first category, while both SRG and SRL fall into the second. Although all CRS signals are derived from the same Raman interaction with the molecule, the two classes display different dependencies on such factors as excitation intensity and concentration of molecules. We will illustrate some of these differences for the cases of CARS and SRL.
The CARS signal has the frequency ωp+Ω. This is a new optical frequency that can be detected with a photodetector equipped with spectral bandpass filters. In this detection mode, the CARS intensity can be written as Display Formula
The SRL signal is detected at the optical frequency ωp. This frequency is the same as the frequency of the pump light. Since CRS is coherent, there is a phase relation between this CRS radiation and the incident light. Consequently, the SRL signal is detected as the interference between the pump beam and the radiated CRS response. Display Formula
Using high-frequency modulation techniques,35–37 the first two terms on the right-hand side can be suppressed and only the latter term is detected. Although the signal depends on the same molecular property χNL as measured in the CARS experiment, the detected SRL signal, −2IpIs Im{χNL}, shows a different dependence on the nonlinear susceptibility and the incident intensities. The signal scales linearly with χNL and depends on the intensities of two fields rather than three. Like spontaneous Raman, the SRL signal is linearly dependent on the molecular number density. Unlike incoherent Raman, however, the radiation is coherent and is interferometrically enhanced. The efficiency of interferometric enhancement scales with the intensity Is of the Stokes beam, yielding higher signals than what can be obtained with the noninterferometric, spontaneous Raman process. We, thus, see that the coherent character of the CRS interaction in both CARS and SRL boosts the overall signal yield relative to spontaneous Raman scattering, albeit in slightly different ways. However, the coherent character of CRS alone is not enough to produce the strong signals necessary for high-speed microscopy applications. The use of pulsed excitation also plays an important role, which we will discuss in Sec. 3.2.
Whether the CRS response is best detected through a CARS or an SRL detection scheme depends on the nature of the experiment. For detection of compounds with high local concentrations in the sample, such as the detection of lipid molecules in lipid droplets, both CARS and SRL provide excellent chemically selective signals with high signal-to-noise-ratios (SNR). When the concentration of the target compound is low, SRL oftentimes offers better SNR in imaging mode compared to CARS. The main reason for this up to 10 times better sensitivity is the lack of background contributions in SRL that can otherwise be prevalent in CARS. Since the total nonlinear susceptibility is a sum of both vibrationally resonant and nonresonant contributions, the CARS image can be overwhelmed by nonresonant contributions, which appear as an interfering background. SRL, on the other hand, detects the imaginary part of χNL, which contains only vibrationally resonant contributions. For high-speed imaging applications beyond the visualization of lipids, SRL has proven to be often a better choice, although it requires a slightly more complicated setup that includes a high-frequency amplitude modulator plus demodulation detection electronics.
Because CRS uses visible or near-infrared excitation light, the standard optics of a laser-scanning multiphoton microscope are compatible with CRS modalities. In particular, CRS uses the same high numerical aperture (NA) lenses that are employed in multiphoton microscopes. In fact, a CRS imaging modality shares many of the imaging properties of a multiphoton microscope, including fast image acquisition, submicrometer resolution, and ease-of-use (see Table 2). Commercial laser-scanning microscopes can be upgraded with a CRS module with only minimal modification. In its simplest form, a CRS microscope can be constructed from a fluorescence laser-scanning microscope by equipping an epi-detector with proper bandpass filters and interfacing the system with a CRS light source. The epi-CRS signal from lipids in tissues is generally strong and comparable with multiphoton fluorescence signals. Because a CRS modality can be added to a standard microscope platform multimodal imaging is possible, whereby the CRS contrast is one of many optical contrast mechanisms available on the imaging system (Fig. 3).23,38,39
Multimodal imaging with CRS microscopy. (a) Stimulated Raman loss (SRL) image of a mouse meibomian gland, visualizing the lipid-rich meibum at 2850 cm−1. (b) SRL image (green) overlaid with fluorescence images from 4',6-diamidino-2-phenylindole (DAPI) (red), showing cell nuclei, and phalloidin (blue), highlighting the extracellular matrix. Image adapted with permission from 40.
Perhaps the most relevant configuration difference with a conventional laser-scanning microscope system is the frequent use of a forward detector in CRS, which is uncommon in fluorescence microscopes (Fig. 4). Since the CRS signals are phase-matched in the forward-propagation direction, strongest signals are expected in that direction when thin samples are used.12 Although standard condensers, which have a NA of ∼0.55, suffice to capture a significant portion of the forward-propagating signal, better collection efficiencies are obtained with higher-NA condensers. This is especially important for the stimulated Raman scattering (SRS, which includes both SRL and SRG) techniques, where photothermal and position-dependent interference effects may introduce artifacts in the image.41,42 Such effects can be mitigated by choosing a high-NA condenser.
Basic layout of a CRS microscope based on a standard inverted microscope. The microscope is equipped with non-descanned detectors in the forward and epi-detection direction. CRS can be performed on most laser-scanning multiphoton microscopes with minimal modifications.
The choice of light source has important consequences for the strength of the signal. To illustrate this, let us consider the CARS signal generated by the two frequencies ωp and ωs. We can write Eq. (6) more explicitly as Display Formula
In terms of practical experimental parameters, the magnitude of the CRS signal can be written as follows: Display Formula
Using a 5-ps, 800-nm pulse with a pulse energy of 0.1 nJ, a peak intensity of more than 1010 W/cm2 is obtained when focusing the light pulse by a 1.2 NA water immersion lens. At such excitation intensities, CARS signals of ∼0.1 photon per shot are regularly detected from a single lipid bilayer, which contains ∼106CH2 oscillators in the interaction volume. In the case where a pulse repetition rate of 80 MHz is used, ∼10 detected photons per microsecond can be registered, which is sufficient to visualize the lipid membrane at high scanning speeds (∼1 s per frame for a 512×512 image). Note that at 80 MHz, the average power of a pulse train with 0.1-nJ pulses is only 8 mW. Such average powers are generally tolerable by biological samples, especially under fast imaging conditions and with excitation wavelengths in the near-infrared range.
The shorter the pulse, the stronger the CRS signal. However, by shortening the pulse, the spectral bandwidth of the excitation light increases, which implies that the spectral resolution of the experiment is affected. For a bandwidth limited pulse of ∼100 fs around 800 nm, the spectral width is ∼150 cm−1. Broad Raman features, such as the OH-stretching band of water28 or the complete CH-stretching band of lipids,44 are suitable targets for femtosecond-excitation sources. Nonetheless, many individual Raman lines in the condensed phase have much narrower widths (∼10 cm−1), and the use of femtosecond pulses reduces the ability to resolve individual signatures. The most direct solution to improve spectral resolution is the use of picosecond pulses with a width comparable to the width of the Raman lines. An added benefit of picosecond pulses is the greatly improved resonant-to-nonresonant signal ratio in CARS relative to shorter pulses.45 Picosecond light sources have been used with great success for biological imaging applications where speed is important. To achieve comparable spectral resolution with femtosecond sources, advanced spectral shaping of the excitation pulses can be employed, including the use of phase-only masks46–48 and combined phase and amplitude shaping.49
Equation (9) also suggests that reducing the repetition rate under constant average power increases the CRS signal. As long as the dominant mechanism of photodamaging in the sample scales linearly with intensity, lowering the repetition rate while keeping the average power at a fixed level is a good strategy. However, nonlinear photodamaging mechanisms put a limit to the maximum peak power that can be tolerated by the sample and, thus, regulate the range of practical pulse repetition rates for imaging applications. The combination of nonlinear photodamaging and the requirement for short pixel dwell times (∼μs) puts the useable range of repetition rates for the CRS light source between 1 and 100 MHz.50
All CRS techniques need coherent incident radiation at ωp and ωs. This condition is typically satisfied by using two laser beams of different color, either by synchronizing two independent lasers51 or by utilizing synchronously pumped optical parametric oscillator systems.52 Similar conditions can be achieved with an ultrafast oscillator in combination with a fiber-based frequency conversion device.53,54 An alternative approach is to use broadband light sources, which span a spectral range that includes both ωp and ωs, and is often used in combination with spectral shapers to obtain sufficient spectral resolution.46 An important recent development is the design of all-fiber-based light sources for CRS microscopy, offering a more compact and often cheaper alternative to solid-state laser light sources.55 Fiber sources have been successfully demonstrated for CARS microscopy56–58 and, in combination with balanced detection techniques, have also signaled their potential for SRS imaging.59
Fast image acquisition rates are the prime advantage of CRS imaging over spontaneous Raman microscopy.60 As stated above, using high repetition rate picosecond pulse trains with average powers in the 10 mW range routinely produces CRS signals with acceptable SNR in as little as a few microsecond per pixel. For an image with 512×512 pixels, this translates in acquisition rates of less than a frame per second. Such imaging speeds are on par with those of linear and multiphoton fluorescence microscopy techniques. When the number of Raman oscillators in the focal volume is relatively high, such as the number of CH2 oscillators in a micrometer-sized lipid droplet, the resulting signals are correspondingly strong and permit even shorter pixel dwell times well below a microsecond. These conditions enable video-rate scanning speeds of >20 frames per second (see Fig. 5). Both video-rate CARS and SRS have been successfully utilized for visualization of live tissues.20,61,62 This form of real-time imaging with label-free vibrational contrast is where the imaging capabilities of CRS microscopy are most evident: it is currently impossible to accomplish such dynamic imaging studies with any other vibrational microscopy technique.
High-speed CRS imaging of animal tissue in vivo. (a) Coherent anti-Stokes Raman scattering (CARS) image from mouse skin, showing lipid-rich sebaceous glands when visualized at a Raman shift of 2845 cm−1. Image acquired at 20 frames/s. (b) Three-dimensional reconstruction of mouse skin in vivo.
The fastest CRS imaging studies so far have been performed in the single-frequency limit: a relatively narrow region of the spectrum is probed in which the spectral information is integrated. However, important information contained in the vibrational spectrum is not utilized in this mode of operation, thus limiting the analytical capabilities of the technique. In some studies, such as the quantitative mapping of lipid distributions in cells and tissues, operating CRS in the fast-scanning single-frequency mode is very useful.32 Yet, in other biological imaging applications, the separation of multiple spectral signatures is essential for identifying target compounds.63,64
Improving the analytical capabilities of the CRS microscope is a subtle balancing act between fast image acquisition and expanding the instrument's sensitivity along the spectral dimension. Some of the many possible methods are sketched in Fig. 6. A straightforward approach is to broaden the spectral range in a picosecond-CRS microscope by sweeping the optical frequency of either the pump or Stokes beam, while collecting an image consecutively for each setting of the frequency.65–67 This procedure yields a spectral stack of images that can be used for further multivariate analysis (see Fig. 7).68–70 Effective spectral acquisition times are typically in the millisecond range, which is much faster than a spontaneous Raman microscope, but much slower than the microsecond dwell times of the single-frequency mode. Alternative methods include clever use of broadband sources and single photodetectors, such as sweeping a narrow spectral segment of a broader pulse spectrum,71 rapidly scanning the Raman shift in the spectral focusing technique,72,73 or collecting temporal interferograms followed by a Fourier transformation.74–76 Relative to the single-frequency mode, all these methods involve additional time to acquire the spectral information.
Several hyperspectral CRS schemes, here shown for CARS. (a) Spectral sweeping of ωs with narrow band pump and Stokes pulse spectra. A single point detector is used. (b) Temporal sweeping of spectrally chirped pump and Stokes pulses, also called spectral focusing. Temporal scan results in a spectral scan of ωp−ωs with narrowband resolution while using a single point detector. (c) Temporal sweeping of two replicas of a broadband pulse, resulting in temporal interferences. A Fourier transform of the interferogram yields the spectral content. Spectral resolution is determined by the Nyquist sampling of the temporal scan. A single point detector is used. (d) Multiplex CARS with a narrowband pump pulse and a broadband Stokes pulse. The spectral resolution is determined by the width of the pump pulse. The signal is spectrally dispersed and detected by a detector array.
Hyperspectral SRL imaging of C. elegans. (a) SRL image at 2850 cm−1. Scale bar is 25 μm. (b) Hyperspectral image taken in the 2800 to 3000 cm−1 range with 6 cm−1 spectral resolution. Colors are the RGB representation based on the weights of the three largest components from a vertex component analysis. (c) Hyperspectral image represented by the retrieved Raman spectra from a principal component analysis. Each color corresponds to a distinct Raman spectrum.
Rather than collecting the spectral dimension in a sequential manner, spectra can be acquired in a single shot by the use of spectral dispersion in combination with detector arrays. Multiplex CARS, for instance, uses a spectrometer equipped with a CCD camera for rapid acquisition of CARS spectra.77 In this fashion, broadband CARS hyperspectral imaging has been achieved in tissues and cells with effective spectral acquisition times in the tens of milliseconds range.78–80 This broadband modality is most similar to spontaneous Raman scattering, where virtually the entire vibrational spectrum is captured, which allows discovery-based spectral imaging. Note that in the complete spectral imaging mode, multiplex CARS is still more than an order of magnitude faster than the fastest spontaneous Raman microscope. As camera technology continues to improve, even faster acquisition times can be expected, taking full advantage of stronger signal levels in CRS relative to incoherent Raman.
Although recording vibrational spectra is the most intuitive approach for strengthening the analytical capabilities of the CRS microscope, the coherent character of the technique allows for elegant excitation schemes that can be used to identify specific chemicals among compounds with relatively similar Raman spectra. Spectral tailoring of the excitation pulses in both amplitude and phase to optimally drive the CRS response of one particular compound, while coherently suppressing the response from others, has proven a useful strategy.49,81,82 This method takes advantage of a single photodetector, which enables data acquisition that is currently many orders of magnitude faster than what can be achieved with array-based detectors. Multifrequency CRS microscopy can, thus, be performed at the high acquisition speeds that have popularized the single-frequency approach.
The imaging properties of CRS microscopy are ideally suited for generating chemical maps of tissues: submicrometer resolution, fast image acquisition, and label-free chemical contrast are essential for successful tissue imaging. The applicability of CRS is limited, however, by linear interactions of photons with tissue components: absorption and scattering of light. These processes are effectively described by the absorption coefficient μa and the scattering coefficient μs.83 At near-infrared wavelengths, μs≫μa in most tissue types, which makes the process of light scattering the dominant limiting factor in tissue imaging studies with focused beam geometries.
Light scattering affects CRS imaging in several ways. First, scattering of incident radiation distorts the wavefront and compromises the quality of the focal volume. Due to scattering, the average shape of the focal volume increases in all dimensions, most notably along the axial coordinate. In addition to a decrease in spatial resolution, the enlarged focal spot is accompanied by a reduced density of the excitation field and, thus, a lower CRS signal. Since CRS signal generation depends on both the amplitude and phase of the focal excitation fields, spatial distortions of the focus affect CRS signals more prominently than fluorescence signals, which only scale with the focal intensity. Second, scattering can alter the propagation direction of the incident photon waves, lowering the total amount of photons that constructively interfere at the location of the focal spot. Assuming that light scattering in a given optical tissue can be described by a single scattering coefficient, the intensity I in the focus at depth d in the tissue is roughly I0e−μsd for each beam, where I0 is the incident intensity before entering the specimen. This effect alone results in an exponential decay of the CRS excitation efficiency as the probing volume is focused deeper into the tissue. Third, after the CRS signal is generated, light scattering impacts the propagation of the signal in the tissue and, thus, influences the amount of signal that can be retrieved at the photodetector.
In general, the effect of light scattering on CRS imaging is akin to the problems encountered in virtually all forms of focused beam microscopy, including confocal microscopy and multiphoton fluorescence microscopy. In addition, the enhanced sensitivity of CRS to focal field distortions due to the coherent nature of the signal is very similar to the situation in SHG microscopy. Nonetheless, because CRS relies on focusing different color incident fields to the same focal spot, chromatic aberrations related to the dispersive properties of the sample can reduce the depth-dependent image quality in CRS microscopy relative to SHG and multiphoton fluorescence microscopy. The combined effects of light scattering compromise the ability to acquire images deeper in the tissue.84 In CRS, depending on the tissue type and excitation wavelength, typical penetration depths are between 0.15 and 0.50 mm when high-NA lenses are used.
The shallow imaging depth of CRS microscopy is a serious limitation for tissue imaging applications, especially in vivo. Many tissue abnormalities manifest themselves at tissue layers deeper than 0.5 mm, emphasizing the need to push the technical capabilities beyond the current numbers. Research in the area of adaptive optics85 and the development of modulation techniques86 for improving the SNR deeper in the tissue are examples of efforts in this direction. An alternative strategy is the use of excitation frequencies in a spectral window where tissue scattering is inherently small. This approach has been successfully used in multiphoton fluorescence and third-harmonic generation (THG) microscopy, where imaging depths down to 1 mm were achieved with excitation wavelengths in the 1.2 to 1.7 μm range.87,88
Despite the deleterious effects of light scattering, the redirection of light in the tissue is nonetheless an essential mechanism that enables epi-detection of CRS signals from thick tissue samples. CRS radiation in thick scattering samples is generally attenuated to undetectable levels in the forward-propagating direction, rendering epi-detection the only feasible option for intercepting the CRS signal in, for instance, tissue imaging of animals in vivo.61 Because the CRS signal is phase-matched in the forward direction, most of the signal would go undetected in the absence of light scattering, as signal photons would not be redirected toward the epi-detector. The principle of backscattering of CRS signals is sketched in Fig. 8. Up to 40 to 45% of the forward-propagating CRS signal is backscattered, and a significant portion of this light can be detected, especially when the aperture of the detector is large.62 Note that although most of the signal detected in the epi-direction is backscattered, the spatial resolution of the image is not compromised by the detection configuration. This is because the resolution is defined by the size of the probing volume, and not by scattering that occurs after generation of the CRS radiation. Similarly, postgeneration scattering also does not affect the interference between the signal field and incident field that underlies the SRS process [Eq. (7)]. Because the fields have the same optical frequency and are phase coherent in the interaction volume, subsequent light scattering in the tissue, which is deterministic, affects both fields in exactly the same manner and, thus, preserves coherence among them.89
Backscattering of CRS radiation in turbid media. Although most CRS signal is initially propagating in the phase-matched forward direction, linear scattering at tissue structures redirects a large portion of the CRS signal in the backward direction, enabling epi-detection with acceptable collection efficiency.
Equation (9) suggests that the CRS signal rapidly grows when the average excitation intensity is increased. Nonetheless, photodamaging of the sample prevents a continual increase of the signal with incident power as higher powers inevitably lead to damage of the specimen. Photodamage may result from a plethora of mechanisms, which are all highly dependent on the excitation conditions and the material properties of the specimen. General rules that predict photodamage are, therefore, difficult to synthesize. A useful categorization of photodamaging mechanisms takes into consideration the intensity dependence of the damaging process. As indicated above, both linear and nonlinear photodamage may occur to the sample. Linear photodamage is the result of linear absorption and subsequent heating of the sample. For instance, local increases in the temperature of a few degrees or more can negatively impact cell functions and may result in cell abnormalities or trigger apoptosis. Substantial rises in local temperature may occur when strong absorbers are present in the sample at relatively high concentrations. Examples include chromophores such as carotenoids and chlorophylls in plant tissues and melanin in human skin. When the tissue is devoid of strongly absorbing chromophores, linear heating in aqueous environments is generally small, much less than a degree in fast imaging applications, for near-infrared pulsed excitation and average powers <10 mW.90,91 Nonlinear photodamage includes heating of UV chromophores after multiphoton absorption, Raman heating, and the excitation of high-energy molecular states that are reactive. The latter process can generate photoproducts that are toxic, such as highly reactive radicals.92 In the high intensity limit, nonlinear processes can also ionize the sample and generate local plasmas.93 Several experiments indicate that under typical CRS conditions, using high-repetition rate, near-infrared picosecond pulses at average powers of 10 mW or more, both linear and nonlinear effects contribute to photodamaging in live cells.50,94 Although the effects of laser illumination on biological samples can never be fully avoided, keeping average powers <10 mW and pulse energies <1 nJ has been shown to minimize the impact of light-induced changes to cell cultures under fast CRS imaging conditions.
Many biological microscopy studies rely on the use of fluorescent markers to achieve molecular selectivity. There is no doubt that labeling with fluorescent agents is a highly successful strategy in cellular biology, yet there are many applications in which label-free imaging is essential. Fluorescent labeling is not without artifacts. First, labeling target compounds can be incomplete or nonuniform. An example is found in visualizing lipids with established fluorophores, such as Oil Red O and Nile Red. It has been reported that these agents label lipid distributions unevenly and sometimes color nonlipid structures.95,96 Labeling artifacts complicate quantitative analysis of lipids in cells and tissues considerably. Second, some fluorescent agents interfere with cellular function, especially at higher concentrations. Putative toxicity of labeling agents is a serious issue that may affect, implicitly and/or explicitly, the interpretation of fluorescence microscopy measurements. Third, virtually all fluorophores photobleach over time, which limits the observation time window of the experiment. Fourth, not all molecular compounds can be associated with a fluorescent label. This shortcoming is particularly relevant to small molecules, such as water, inorganic polyatomic ions, and small molecular metabolites, for which fluorescent probes currently do not exist. Label-free imaging avoids all of these issues. CRS microscopy fills in an important void in the field biological imaging by offering label-free molecular imaging at speeds that are relevant to cell and tissue biology.
However, CRS microscopy is not a cure-all solution for biological problems where fluorescence imaging fails. The molecular selectivity of CRS is linked to information contained in the sample's Raman spectrum. The Raman effect is sensitive to particular chemical bonds and chemical groups. Many biomolecular compounds are composed of a limited set of chemical motifs, most notably motifs formed from chemical bonds among carbon, hydrogen, oxygen, and nitrogen atoms. This implies that the number of distinct Raman resonances is finite and that many molecules that share similar motifs also exhibit comparable Raman signatures. A case in point is the Raman spectrum of protein structures. Although proteins are functionally incredibly diverse, chemically they display limited variation: all proteins are built from polypeptide chains, which contain only a limited set of chemical groups. Hence, differences in the vibrational spectra of different proteins are either negligible or extraordinarily subtle, rendering vibrational imaging virtually inadequate for discriminating proteins.
CRS microscopy is a good choice for visualizing biomolecular compounds in biological samples when the molecular target (1) is present at locally high concentrations such that it is spatially separated from other sample components or (2) exhibits unique Raman signatures that can be discriminated from other compounds in the sample. The first situation is relevant when imaging lipids, water, or the overall protein distribution in biological materials. These molecular classes are present at high local concentrations in cells and tissues and can be identified with confidence in a CRS microscope. Using hyperspectral CRS, lipid pools can be analytically examined and information on degree of saturation, lipid packing density, and lipid class (sterols versus aliphatic lipids) can be retrieved from the vibrational spectra. Discrimination of these molecular classes is important in tissue imaging, where lipid and protein densities demarcate cells and tissue components, generating tissue maps with essential diagnostic contrast akin to what can be obtained with traditional hematoxylin/eosin stains.97,98
Many successful CRS studies have used the strong signatures of methyl, methylene, and methine stretching modes in the Raman spectrum (2800 to 3050 cm−1) as the contrast mechanism for fast imaging. However, the application of CRS is by no means limited to the CH-stretching range of the vibrational spectrum. Many CRS studies have focused on the fingerprint region (∼800 to 1800 cm−1), which harbors a rich set of molecular vibrations that enable a deeper level of chemical analysis than what can be attained with the modes in the CH-stretching range alone. Broadband CARS studies are a good example in which the full range of fingerprint signatures are used to discriminate cellular compartments.78,80 In addition, the number of single-frequency CRS studies that use fingerprint contrast for biological imaging is steadily growing.40,64
Although many molecular compounds in biology exhibit similar chemical groups, there are many others that can be uniquely identified by means of their vibrational signatures. Many exogenous molecules, such as pharmaceuticals and their carrier materials, have spectral features that are notably different from the spectral properties of tissues. For instance, the strong aromatic ring-breathing mode (∼1600 cm−1) of a drug like ketoprofen is not very abundant in endogenous compounds of the tissue matrix, enabling a reliable identification of the drug when dialing in at the Raman frequency of this band.99,100 Another excellent example is the use of deuterated analogs of the targeted biomolecule. When replacing hydrogen with deuterium, vibrational mode frequencies shift often to a quiet range of the vibrational spectrum where bands of endogenous constituents are scarce (see Fig. 9). This approach has been widely used in high-speed CRS microscopy to rapidly identify the compound of interest in tissues and cells, including flowing water,28 phospholipids,101,102 neutral lipids,103 and drug compounds.100,104 Recently, the unique vibrational contrast of isotope-labeled compounds has also been employed to visualize de novo synthesis of macromolecules in cells by feeding them small deuterated precursor molecules that become permanently incorporated in newly synthesized biomolecular components.105
Improving selectivity in CRS imaging with isotope labeling. Shown here is the Raman spectrum of partially deuterated (d-) cholesterol, introducing C-D specific bands in the 2000 to 2400 cm−1 range. Insets show SRL images of d-cholesterol crystals, demonstrating the good spectral selectivity that can be obtained with isotope labeling. Scale bar is 100 μm.
The use of isotope labels is part of a wider strategy that aims to develop efficient Raman labels.106 In contrast to fluorescence labeling, Raman labels can be very small. In the case of isotope labeling, a single element in the molecule is replaced with its isotope, which has minimal chemical impact, yielding a probe with virtually the same size as the original molecule. The advantage of Raman labels over fluorescent labels is clear: the probes minimally disrupt regular cell function and do not photobleach, allowing persistent imaging over long timescales.
The discussion above underlines that CRS microscopy is an excellent choice for biological imaging when molecular compounds with strong and accessible vibrational signatures are targeted. In these cases, fast imaging of cells and tissue can be performed. The enhanced speed of CRS imaging over spontaneous Raman scattering microscopy not only enables dynamic measurements of biological samples in vivo, but also facilitates studies that necessitate examination of larger tissue areas. CRS microscopy makes it possible to quantitatively analyze mesoscale tissue samples without compromising spatial resolution. In next subsections, we illustrate the utility of CRS microscopy by discussing two typical applications.
Lipids have been the most successful molecular targets in CRS microscopy applications. The reason for strong and distinct lipid CRS signals is the high local concentration of carbon-hydrogen bonds that can be found in lipid deposits, producing bright CARS and SRS signals when tuned to the C-H stretching range of the spectrum. Submicrometer-sized lipid droplets give rise to CRS signals that are many times higher than other resonant and nonresonant contributions from the cell, allowing their rapid identification in live cells. Although other label-free techniques, such as THG, have been shown to display sensitivity to lipid droplets as well, CRS is superior in terms of chemical selectivity and contrast.107
Lipid droplets are the cell's prime reservoirs of neutral lipids and they are implicated in many key cellular processes. CRS microscopy is fast enough to track the motions of such intracellular droplets. But CRS is also the method of choice for quantitative analysis of lipid quantities in cell cultures. Such quantitative studies require sampling of many cells, and speed is of essence to cover significant parts of the cell population within a realistic time frame. An example is shown in Fig. 10, where the lipid content in macrophages is determined as a function of external signaling molecules (cytokines) and the presence of endotoxins. CRS enables rapid imaging of hundreds of cells, and in combination with automated lipid droplet counting algorithms,108 it facilitates the discovery of statistically significant trends among cell populations.
Statistics of lipid droplets with CRS imaging. (a) CARS image of lipid droplets in macrophage cell. Scale bar is 25 μm. (b) Automatic recognition of lipid droplets enables rapid counting of the number of lipid droplets per cell (light blue indicates a recognized droplet). (c) Fast CRS imaging makes it possible to examine lipid droplet contents in hundreds of cells, giving rise to reliable statistics. Here the number of lipid droplets per cell is determined for macrophage cells exposed to different cytokines and endotoxin, from a total of 1200 cells.
The desired imaging properties for interrogating cell cultures versus three-dimensional tissues are not exactly the same. Although imaging of individual cells requires high spatial resolution and contrast to visualize intracellular details, tissue-level imaging is more forgiving in terms of the ultimate spatial resolution but requires fast acquisition of large volumetric datasets with sustained signal quality. CRS microscopy is well suited for the task of generating mesoscale tissue maps with micrometer resolution. In addition, the CRS chemical contrast from molecular classes in the tissue, such as lipids, protein distributions, and water, are meaningful and can be routinely achieved at high imaging speeds.
In many cases, sufficient information about proteins and lipids can be obtained by examining a very limited region of the vibrational spectrum. Relative to collecting broad spectra, limiting the spectral range increases acquisition rates, which is essential for mapping tissues over larger areas. Hyperspectral datasets from a limited spectral range, such as the CH-stretching range of the spectrum, may yield enough discriminatory spectral features to identify tissue components.109Figure 11 shows an example of a narrow hyperspectral stack acquired from muscle tissue. The protein matrix, which displays a low concentration of aliphatic molecules, can be readily separated from areas with higher concentrations of lipid-like compounds, which in muscle tissue most likely correspond to mitochondrial tubules.110 Note that the differences in the spectra are subtle, yet are sufficient for a reliable mapping of functional units in the tissue. These are the types of applications where the imaging properties of CRS are in full effect: label-free chemical contrast, fast image acquisition, and (sub)micrometer resolution.
Hyperspectral SRL imaging of mouse muscle tissue. (a) RGB image representation of the dataset based on the three largest components from a vertex component analysis (VCA). (b) VCA retrieved spectra showing a protein-rich spectrum (green), a protein spectrum mixed with lipids (red), and a water-dominated spectrum (blue). Image size is 71×71 μm.
In this tutorial, we have discussed the physical origin of CRS signals, its implementation in microscopy, and its usefulness for biological research. CRS contrast is now a trusted complement to existing contrast mechanisms in optical microscopy. Its uses are rapidly expanding, as witnessed by the explosive rise in the number of publications that use CRS as a tool for solving biological problems. With the availability of commercial CRS systems and modules, its impact in biology is likely to grow even more significant. Nonetheless, as CRS microscopy gradually finds its way into the mainstream, the quality of future imaging studies remains contingent on a basic understanding of the principles of the technique, its technical merits, and the scope of its applicability. These relevant issues formed the basis of this tutorial and were touched on herein.
We acknowledge support from NSF grant CHE-0847097, NIH grant P41-RR01192 (Laser Microbeam and Medical Program, LAMMP), the Balsells Fellowship Program, and the Agilent University Research Grant. E.S.L. thanks the support from Bio-signal Analysis Technology Innovation Program funded by the Ministry of Science, ICT and Future Planning, Republic of Korea.
Alba Alfonso-García obtained a BS in physics from University of Barcelona, Spain, and a MSc in optics and photonics from the Karlsruhe Institute of Technology, Germany. She is currently a PhD student in the biomedical engineering program at the University of California, Irvine. She is interested in applying nonlinear optical techniques to image biological processes, with a particular focus on cholesterol metabolism.
Richa Mittal is a PhD graduate student in the Chemical Engineering and Material Science Department at University of California-Irvine. She works at Beckman Laser Institute at UCI on miniaturizing nonlinear optical microscopy techniques for in vivo skin imaging.
Eun Seong Lee is a principal research scientist in Korea Research Institute of Standards and Science in South Korea. His research interests include nonlinear optical imaging of biological samples and developing spectrometers for material study.
Eric O. Potma is an associate professor in the Department of Chemistry at the University of California, Irvine (UCI). He holds an adjunct position in the Beckman Laser Institute and Medical Clinic at UCI. His research group is active in developing nonlinear optical imaging techniques for the purpose of interrogating biological tissues and nanostructured materials.
Alba Alfonso-García ; Richa Mittal ; Eun Seong Lee and Eric O. Potma"Biological imaging with coherent Raman scattering microscopy: a tutorial", J. Biomed. Opt. 19(7), 071407 (Mar 10, 2014). ; http://dx.doi.org/10.1117/1.JBO.19.7.071407
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Amazon opens art marketplace with works of over 4,500 artists
Amazon has launched Amazon Art, a marketplace that includes more than 40,000 works of art by the likes of Norman Rockwell and Claude Monet from over 150 galleries and dealers.
Amazon has launched Amazon Art, a marketplace that includes more than 40,000 works of art by the likes of Norman Rockwell and Claude Monet from over 150 galleries and dealers.
Amazon.com founder and CEO Jeff Bezos has announced a surprise plan to buy The Washington Post for US$250 million in cash.
Apple is allowing children under 13 to have their own iTunes accounts as part of a new push coming later this year to increase the use of its iPad in education.
Apple should end its existing e-book agreements with five major publishers and sign no new price-setting distribution contracts for five years under remedies for e-book price fixing proposed by the U.S. Department of Justice and 33 state attorneys ge...
China Mobile, the country's largest mobile carrier, introduced two own-brand handsets on Friday, as a way to bring more affordable smartphones to its 740 million customers.
A proposed code of conduct for mobile app developers intended to make them explain how user data is collected and used does not have a clear enforcement mechanism, one privacy advocate said.
A project underway in Kenya is linking Bitcoin with M-Pesa, a popular mobile payments system, in an experiment designed to spur innovative payments in Africa.
A lawsuit filed by Apple over Amazon.com's use of the term "Appstore" has ended after Apple decided to withdraw the case.
A simple piece of malicious software is circulating that steals Litecoins, a virtual currency based on the Bitcoin system.
A U.S. regulatory filing for a Bitcoin investment trust by the Winklevoss twins said they will protect the virtual currency like gold bars -- in vaults.
The largest Bitcoin exchange has filed key paperwork with the US Treasury's anti-money laundering agency, but it may have come too late.
PayPal, which claims more than 128 million active accounts in 193 markets and 25 currencies around the globe, is now looking at outer space.
While mobile payments transform financial life in many less developed countries, retailers and payment providers in the U.S. and Europe are still trying to find the right incentives to drive adoption.
A foundation created last year to shepherd Bitcoin's software development has received a stern warning from a California financial regulator.
Apple Senior Vice President Eddy Cue offered only short answers in testimony Thursday in federal court when questioned by U.S. Justice Department prosecutors trying to solidify their case that Apple, along with five of the largest book publishers, wo...
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HP OfficeJet 250 Mobile Printer
The HP OfficeJet 250 Mobile Printer is a great device that fits perfectly into my fast paced and mobile lifestyle. My first impression of the printer itself was how incredibly compact and sleek the device was.
HP OfficeJet 250 Mobile Printer
Wireless printing from my iPhone was also a handy feature, the whole experience was quick and seamless with no setup requirements - accessed through the default iOS printing menu options.
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HP OfficeJet Pro 8730
As a freelance writer who is always on the go, I like my technology to be both efficient and effective so I can do my job well. The HP OfficeJet Pro 8730 Inkjet Printer ticks all the boxes in terms of form factor, performance and user interface.
Windows 10 for Business / Dell XPS 13
I'd happily recommend this touchscreen laptop and Windows 10 as a great way to get serious work done at a desk or on the road.

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Business, Education, Health, Self-HelpFollow
Awarded 'Best of iTunes', EOFire is a 7-day a week Podcast where John Lee Dumas chats with todays most successful Entrepreneurs. Pat Flynn, Seth Godin, Tim Ferriss, Tony Robbins and Gary Vaynerchuk are just 5 of the over 1400 interviews to date. EOFire was created for YOU, the Entrepreneur, Side-Preneur, Solo-Preneur and Small Business Owner. If you're looking for ACTIONABLE advice during your daily commute, workout, or 'me' time, JLD brings the HEAT. Each episode details the journey of a successful Entrepreneur who shares their WORST Entrepreneurial moment and lessons learned, an AH-HA moment and how they turned that idea into success, and much more. Each episode ends with THE LIGHTNING ROUND where JLD extracts golden nuggets, Internet resources and action steps for you, FIRE NATION!
July 5th, 2015
Episode 1034 of 1817 episodes
Ken hosts The EntreLeadership Podcastand is theAuthor ofOne Question:Life Changing Answers from Today'sLeading Voices. Ken'sinterviews are with heads of state, champions of sport,captains of industry, cultural icons, and thought leaders.Legendary Duke Basketball Coach Mike Krzyzewski called Ken "a young CharlieRose". Press and hold link to visit the page Show Notes Page Resource:Voxer Book:One Question,Steal Like an Artist Ken's Page Below are two free resources to IGNITE your Entrepreneurial journey! FreePodcastCourse.com: A free 15-day course that will teach you how to create, grow, and monetize YOUR Podcast! TheWebinarCourse.com: A free 10-day course that will teach you how to create and present Webinars that convert! Show Sponsors XERO: Xero is THE online accounting software for your small business. Sign up for a free 30 day trial at Xero.com/podcasts. Zip Recruiter: With Zip Recruiter you can post to fifty plus job sites, including social networks like LinkedIn, Facebook and Twitter, all with a single click. Post a job free at ZipRecruiter.com/fire
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Accent Novotechnik Position Transducers Contactless Potentiometric Rotary
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accent, novotechnik, position, transducers, contactless, potentiometric, rotary
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3001 Kaiser Drive UNIT A, Santa Clara CA, 95051 (Residential)
Sold Price:
2 beds , 1 Full/1 Half baths
992 sqft
Status: Sold
Last Updated: 6/23/2017 10:39 PM
Description for 3001 Kaiser Drive UNIT A Santa Clara, CA 95051
upgraded home, bright and clean, perfect for first time buyer or investor. close to central park, shopping , new apple campus...
Sold Price:
Sold Date:07/29/2016
Santa Clara
1 Full/1 Half
992 (approx)
upgraded home, bright and clean, perfect for first time buyer or investor. close to central park, shopping , new apple campus...
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Santa Clara Real Estate
Santa Clara is located in California. Santa Clara, California 95051 has a population of 126,215.
The median household income in Santa Clara, California 95051 is $93,840. The median household income for the surrounding county is $93,854 compared to the national median of $53,482. The median age of people living in Santa Clara 95051 is 34.3 years.
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/// Alibaba Says Founder Jack Ma's Tiananmen Square Quotes Were Misinterpreted
July 21, 2013 | All Things Digital
Alibaba Group Holding Ltd. is disputing the veracity of an interview with its founder, Jack Ma, which was published in a Hong Kong newspaper and has triggered a fiery debate in the Chinese blogosphere. Regarding the interview with the South China Morning Post, in which Mr. Ma refers to the 1989 crackdown on Tiananmen Square protesters as the "most correct decision" at the time, Alibaba is saying that Mr. Ma's quotes were misinterpreted because some words were omitted from the newspaper's published transcript. Read the rest of this post on the original site »
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Beautiful secluded and secure campsite in the heart of the North Wales countryside surrounded by the Clwyd mountains. Panoramic views. Perfectly placed for exploring North Wales coast and hills. Local country walks.
Toilet, shower and washing up facilities. Large campfire pit. Friendly local pubs serving food nearby.
Children and pets welcome.
Only 15 miles from Chester with nearby market towns of Mold (market Saturday & Wednesday) and Holywell ( Sunday).
Large well drained rally field now available. Seasonal pitches available.
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3.00pm – Saturday Cinema
"Cattle Queen Of Montana" (1954) starring Barbara Stanwyck and Ronald Reagan.
Followed by Closedown
7.20pm – News and Sport
7.30pm – Rugby Special
Pontypool v Cross Keys introduced by Cliff Morgan
8.15 – Resurrection
"Temptation" starring Alan Dobie
9.00 – Great Gambling Britain
Trevor Philpott looks at the growth of gambling in this country.
9.45 – Once More With Felix
Tonight, Julie's guests are Lee Hazlewood and Oscar Ghiglia
10.15 – Release
"Octagon" – A look at the unique Octagon Theatre in Bolton, Lancs.
"One Plus One" – A profile of French film producer Jean Luc Godard.
10.55 – News Summary
11.00 – Colour Me Pop
11.30 – Midnight Movie
"Behind The Mask" starring Michael Redgrave and Tony Britton. Also in colour.


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Top 10 Ways to Get the Most Out of Convergence 2010
published March 30, 2010
The economy is probably as good as it's going to be for a good while, which means we all have to figure out how to do more with our existing Microsoft Dynamics applications. The best way to do so is to add to our knowledge, and Microsoft Convergence 2010 (April 24-27 in Atlanta) is a great place to learn more about Microsoft Dynamics, lots more.
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How Microsoft Dynamics CRM 5 Will Transform the User Interface, Ease Customization, and Improve Integration March 29, 2010 How ERP Systems Are Turning Retailer Customers into Retailer Buddies August 15, 2010


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Diesel Weekend - Sunday 24th September 2017
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Diesel Weekend - Saturday 23rd & Sunday 24th September 2017
All available diesel locos will be out over this BRAND NEW Diesel Weekend.
Class 26 D5310 (subject to repairs), Class 37 6940 and Class 47 1566, which is due to return to traffic after a 5 year overhaul. There will also be a Heritage Railcar in service.
All services and locomotives are subject to availability.
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24th September 2017
24th September 2017
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Carnival Conquest
Sail Date: June 01, 2013
Reviewed: 4 years ago
Traveled As: Couple
Room Type: Inside
Pros: last minute getaway great price
Cons: not enough to do on sea days
Recommends Carnival Conquest? Yes!
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Reliance Industries hiring Desktop Support Engineer
Company Name: Reliance Industries Ltd
Position: Desktop Support Engineer
Education: 0 – 3 yrs
Experience: BE/B.Tech, B Sc
Salary: Not specified
Location: Navi Mumbai
Good communications skills and aptitude. Knowledge of desktop, laptop, printers.
Windows servers administration. Basic networking knowledge.
DNS, DHCP, Active Directory
Knowledge of desktop and server technologies
Configuring, installing and troubleshooting of PC systems.
Hardware and software installation.
If you are interested in exploring this role, please respond to this mail with you contact coordinates. Post your resume is shortlisted we would then get in touch with you.
Company Profile:
Reliance Industries Ltd (RIL) is India`s largest private sector company on all major financial parameters with turnover of Rs. 2,58,651 crore ($ 58,000 million), cash profit of Rs. 34,530 crore ($ 7,743 million), net profit of Rs. 20,286 Crore ($ 4,549 million) and total assets of Rs. 2,84,719 Crore ($ 63,846 million). RIL is the first private sector company from India to feature in the Fortune Global 500 list of 'World's Largest Corporations' and ranks 119th amongst the world's top companies in terms of profits.
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|2004-05-03, 00:24
|Link #204
Join Date: May 2004
Age: 33
I don't think she is really their mother... i think it was revealed in episode 2 that the reason they called her mother was because of her being their previous ruler... and i think that it was also revealed that they are all not real siblings... i'll have to rewatch and check to make sure
|2004-05-03, 00:44
|Link #205
Weapon of Mass Discussion
Join Date: Feb 2003
Location: New York, USA
I just had the strangest thought about Wolf... I think I know the answer and the way I got there is actually interesting. I notice that the beliefs about Wolf being a girl are split soundly into two camps. People who know anime best are insisting that Wolf is a boy because they have seen plenty of his type. The other camp is generally less well experienced with anime and associates his mannerisms with feminity. With that said, here is my answer.
I think it's a colossal bait and switch! Since everyone who knows his type, knows that Wolf has to be a girl, they are going to make Wolf a boy. That would actually be a clever surprise to make Wolf a girl in disguise instead of the pretty boy that everyone expects. They are dropping just a few clues such as Wolf's refusal to strip to the waist to play fair.
|2004-05-03, 01:11
|Link #207
Join Date: Apr 2004
I totally agree with you... there are too many things that leave questions... like as you said the fact that he didn't strip and his eyes (long lashes) and the comment his mum made "So thats how boys should act?" in ep 2... I also think he is in disguise and the all know that apart from Yuri.......hehehe .... hmm or maybe I am getting ready for a let down if it turns out to be a boy.. oh well
I hope there are going to be more than 10 episodes to see what embarassing situations they will get up to...^______________^
|2004-05-03, 01:19
|Link #208
Join Date: May 2004
Age: 33
|2004-05-03, 02:48
|Link #209
Senior Member
Join Date: Nov 2003
::sigh:: There is no proof that he's a girl. On all the Japanese sites I've been to, novel or otherwise, they have ALL referred to him as a boy. Unless this is such a well kept secret that not even fans of the novel that have read the entire series thus far have been able to figure it out--since every single fanpage I've been to also refers to him as "male"--I think you're going to have to admit that he's just a pretty boy. One of the summaries for the book even noted Wolfram's gender like this: ♂
I really don't know why this is so hard for people to swallow. He's not a girl disguising "herself" as a boy. There is no point for him to do so. What purpose would it serve in the plot? It's not like he would get anything out of pretending he was a boy.
|2004-05-03, 03:10
|Link #211
Join Date: May 2004
Age: 33
Yep, it does settle it... but ruined our fun!!
I just hope they bring more serious scenes in the future... the light-hearted scenes are cool and all, but I dont like it when it totally dominates the anime... i need a dosage of seriousness from time to time... like if his life is threatened i want the mood to get serious...
|2004-05-03, 03:21
|Link #212
Join Date: Nov 2003
Location: California
I didn't want to know that... actually I'm kind of glad I know... hehe... It's for the best... don't wanna have to go kick my own ass for... uhh...
Anyways, I didn't wanna download this anime at first from the post about it being a guy/guy show. But I got bored and downloaded it... LOL I don't really care about the guy/guy deal anymore, this is just one funny anime.
|2004-05-03, 09:22
|Link #215
Senior member
Join Date: Jul 2003
I also love the end theme and chibi!Gunther running after chibi!Yuuri with hearts above his head
I think the seiyuu cast is having fun too... I know I am!
|2004-05-03, 09:26
|Link #216
Join Date: Feb 2004
Spoiler for episode 3:
Last edited by Secca; 2004-05-03 at 09:54.
|2004-05-03, 10:11
|Link #217
Join Date: Mar 2004
It's kinda like when yaoi fangirls insisist a character is gay because he stands next to another bishounen ^__^;;
guys come on admit he's a boy or you are being stupid like those girls.
|2004-05-03, 10:21
|Link #218
Senior member
Join Date: Jul 2003
But he sure has a long list of roles at his credit:
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"Girls" Review: 18 Years Later And Reality Still Bites
By Joanna Robinson | TV | April 20, 2012 | Comments ()
By Joanna Robinson | TV | April 20, 2012 |
Oh, alright internet, bravo, well done. Really, "Artist Laurie Simmons' Daughter"? How many of you (other than my mom's next door neighbor. . .shut it, Mrs. Baumgarten) know who Laurie Simmons is? How many countless actors and actresses were born to privilege? Do you really think HBO was so bowled over by Dunham's impressive credentials as heir to the Simmons family fortune that they bent over to give her a TV show?! Did they really refer to Judd Apatow as ADAM SANDLER'S OLD ROOMMATE?!? Ugh, I tell you what, hipsters, I liked "Girls" better when it was called Reality Bites and starred Jerry Stiller's son. Think about it, who better to skewer the hypocrisies of white privilege than someone who was born and raised in it? Dunham's upbringing is the exact reason why her show rings so true. Bite me with your nepotism angle, haters. Next?
Ahhh, race. We're upset because this particular show about four young women is a depiction of four young white women. Um, well, to be perfectly honest here, I think that's actually part of the damned point. Given that I was raised with the liberal guilt that comes hand in hand with white privilege, I'm appreciative of the increasingly colorblind casting that populates both network and cable television. And there's a part of me that's glad for the hue and cry that has accompanied the casting for "Girls" simply because it did not plague the "Sex And The City" cast 14 years ago. The conversation about race has progressed, and that's great. But if Dunham is cleaving fairly close to her own life story with the show, then who's to say this isn't an accurate portrayal of her social circle? Is it accurate of all of New York City? Hell no. Of Oberlin college where Dunham went to school? Not really. But if it's true to Dunham's life, then what the f*ck ever. Go bother the milquetoasts over on "How I Met Your Mother."
Finally, the body thing. As I mentioned earlier, Dunham is on the frump end of the spectrum when it comes to very telegenic TV actresses. Listen, at least she located her hairbrush somewhere between this show and Tiny Furniture. But one of my favorite things about that movie (and this show to be honest) is that yeah, homegirl has cellulite. At one point during the pilot she shame-eats a cupcake for breakfast. So the f*ck what? You've never had a cupcake for breakfast? Fuck you, Andrea Peyser of the NY Post. That "dimpled ugliness" comment is appalling. As I've mentioned over and over, this show is, above all else, a realistic look at a young woman's life. She's holding a mirror to herself and the rest of the nation as well. If you choose not to see your own ugliness (dimpled or otherwise) reflected there, well, that's up to you.
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3/219 Harcourt Street New Farm 4005 QLD
Two-bedroom, older style apartment in centrally located block of only 4, mere moments away from the trendy James Street shopping, dining and entertainment precinct, close to public transport with parking for two cars.
Features include:
- Large lounge room
- Eat in kitchen with servery to lounge
- Lots of storage space
- Large single garage with plenty of room for storage
- Additional car park in driveway
- Lots of Natural Light
- Built in robes
- Internal Laundry
Centrally located, this home is within easy reach of every amenity you could need including shopping and dining precincts (New Farm, the Valley, Gas Works and James Street), entertainment (Judith Wright Centre, the Powerhouse & New Farm Six Cinemas), public transport options and easy access to the CBD via the RiverWalk.
Click on the "Book Inspection" or "Request an Inspection" Button under the open for inspections times - alternatively you can click on the 'Virtual Tour" link & follow the prompts.
*** By registering for an inspection you will be INSTANTLY informed of any updates, changes or cancellations for your property appointment. If no one registers for an inspection time - then that inspection may not proceed. If there are no times set for this property yet, STILL REGISTER and as soon as times are set, you will be advised of the day time.
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Agent Information
LJ Hooker New Farm
New Farm QLD, 4005
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Real Estate For Rent in New Farm, QLD 4005 priced between $200 and $380


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Sail Date: October 06, 2016
Reviewed: 8 months ago
Traveled As: Couple
Room Type: Balcony
The staff was friendly, courteous and helpful. Food at Specialty restaurants and at the Grande restaurant was excellent, the others average, service at all restaurants was also very good.
Completing web site to choose restaurants and shows was too convoluted I spent hours trying to figure out time to dine and time to attend shows.
I processed all pre-embarkation requirements, including downloading photos. At the port, I spent what I feel was a long time to board ship including a problem where a bar code did not print in a document given to me at the port, so I had to go back to a service desk to get one printed with the bar code to be able to board. Disembarkation was a lot better than expected although my flight was early, I got to airport with plenty of time
Category Ratings
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|Cabin / Stateroom
|Cabin had a good size and closet space, bed was comfortable and bathroom facilities were very good
|Embarkation and Disembarkation
|Embarkation was a problem for me, already explained earlier in this survey.
|Ship Quality
|Destinations and Excursions
|Onboard Activities
|Children's Programs
|Did not check out
|Entertainment was good but not what was expected. We Will Rock You was built too high for the type of music it developed. We thought the Gift was a sad story when we were there to have fun.
|Service and Staff
|Roberto our cabin steward was exponentially superb. Others in restaurants Ronnie in Chick. Kacan in Chops Grille and my server in Jamies Italian restaurant was outstanding, he was from Ukraine but I cannot remember his name.
|Food and Dining
|I explained before that specialty restaurants and the Grande were Grand. Others were average. The staff in all restaurants were excellent
Port Ratings
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|Cape Liberty, New Jersey
|Boston, Massachusetts
|We took Trolley Citi Tour then walked along the Market Place had lunch listened to music, went to the Granary Cemetery to see tombs of Paul Revere and John Hancock. Lets face it, Boston is a rich tradition and historical city
|At Sea
|We relaxed this last day and went shopping in ship picked up photos had a nice lunch. Played Bingo and went to activities in Casino. I think the last day at sea prior to embarkation is a great idea
Even booking in advance, you still had to wait a few minutes to dine in regular restaurants, but not in the specialty restaurants. I logged in for the first time on July 11 and could not find any restaurants offering dinner between 7:30 and 8:00 PM. I did not like process of so many restaurants


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- 1 used to say 'yes' to somebody 'Will you open the wine?' 'Sure, where is it?' Did it hurt? Sure it hurt. Sure, no problem.
- 2used to emphasize something that you are saying Boy, it sure is hot. 'Amazing view'. 'Sure is.' That song sure as hell sounds familiar. He sure looked unhappy.
- 3used to reply to somebody who has just thanked you for something 'Thanks for the ride.' 'Sure—anytime.' Word OriginMiddle English: from Old French sur, from Latin securus 'free from care'.Idioms
adverbjump to other results
BrE BrE//ʃʊə(r)//, BrE//ʃɔː(r)//; NAmE NAmE//ʃʊr//(informal, especially North American English)
(old-fashioned, British English, informal) used to say that something is definitely true
(as) sure as eggs is eggsjump to other results
used to say that something happened as expected I said he'd forget, and sure enough he did. 'Stop,' she said, 'I can hear something.' Sure enough, in the distance we could hear the sound of a car approaching.
sure enoughjump to other results
Check pronunciation: sure


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The National Gallery was established in 1996 with the hopes of promoting local and international artwork as well as infused the community with knowledge on this important subject through educational and outreach programs. Tourists will find this is a great place to see pieces created by regional artists including those on other islands.
The gallery has a permanent exhibit of local artists on display, but also has several rotating exhibit each year. Additionally, they set up galleries throughout the rest of the Cayman Islands in order to expand the reach of art in the community.
Cayman Islands National Gallery is located in Seven Mile Beach, an area on Grand Cayman.
Aside from Cayman Islands National Gallery, the opportunities to go shopping in the vicinity are scarce.
The National Gallery is closed on all public holidays.
Regular operating hours are 10:00 a.m. to 5:00 p.m. Monday through Friday and 10:00 a.m. to 3:00 p.m. on Saturday. They are closed on Sundays.
Location: Esterley Tibbetts Highway, Seven Mile Beach, Grand Cayman
Phone: (345) 945-8111
Website: https://www.nationalgallery.org.ky/
Help us improve! We welcome your corrections and suggestions.


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Jensen- Mark A MD telephone number is (574) 267-8940.
Telefax: No fax number available
Website: No web address available
Biz owner? Moderate reviews or edit listing.
Search entire category for Arthritis Specialists
'Arthritis Specialists' in Warsaw are medical experts and doctors who are trained and qualified to treat any type of arthritis. Post detection, a specialist would ideally put his patient on suitable medication and physical therapy.


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All reader reviews by Fidel
Villandry (Great Portland St)
I had dinner with a group of friends and we really regreted the choice of venue. The service was SO poor we refused to pay the service charge when they "finally" brought the bill.
The food was totally unmemorable and we all felt the place lacks ambiance.
I've heard it isn'tso dreadful at lunch times but I doubt I'll be willing to take the risk.
Wednesday, March 22, 2006
Overall rating
Food 2 | Service 0 | Atmosphere 0 | Value for money 1


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Every year we run a photography contest with people affected by homelessness. The theme is My London.
We handed our 105 single-use cameras on 1 July 2016 and the participants had 5 days to take photos. The resulting exhibition is chosen by the participants - they choose five photos each.
A panel of professional photographers from The Royal Photographic Society, FujiFilm, Amateur Photographer magazine, The London Photo Festival, and members of organisations with an understanding or homelessness including Homeless Link chose the 20 main exhibition photos which were voted on by the public in the first week of August.
The calendar was sold in Spitalfields Arts Market from 10-16 October during the annual Cafe Art Market at Spitalfields, and then in the main Spitalfields E1 market until early January.
We still have a few left, but have almost sold all the 9,000 printed. You can order the calendar here
have I still got time to enter the next competition let me know please mary
Can I still enter the photography contest? I hope I can
I'm a member of Crisis and providence row, but just came across this now.
Thank you
Hi I would like to enter the competition this year I'm a member of SHP
Which is it July 2nd or July 1st look at you 2 postings as it is a bit of a confusing.
My mum and daughter would love to enter the competition and get the camera Linda Hawkins and katie peck


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10 Instances Where We Could All Use A Little More Space
These days, we pretty much need all the space we can get. Fortunately, we can make room for all the other things in our lives with 50% off new smartphones from Verizon — America's Largest and Most Reliable Network.
1. Trying to parallel park when there is just no way.
Yet we still try.
2. When only one elevator works in the entire building.
It's still not worth taking the stairs.
3. Public transportation of any kind.
Sometimes you wonder if there is a global deodorant shortage.
4. When your overbearing boss micromanages every little thing you do.
A reminder to fill out your expense report was exactly what you were missing.
5. When you attempt to maneuver through rush hour traffic.
Only a few more inches and I would have made it! I swear!
6. When your dad thinks he is cool and wants to hang out in public.
To be fair, having to deal with "cool" parents is a rite of passage.
7. Living in a dorm.
Ahh the good ol' days. Never before has so much been squeezed into so little.
8. When your co-worker is sick but still insists on coming in.
But strangely enough, insists on not covering his sneezes.
9. When it's raining out and the bus stop isn't big enough.
Chances are it's a Monday.
10. When you are relaxing at the beach, and someone decides to sit RIGHT next to you.
It's as if they are trying to annoy you. Unbelievable.


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Does God Punish Disobedience?
November 13, 2012
Tullian Tchividjian is the pastor of Coral Ridge Presbyterian Church, a visiting professor at Reformed Theological Seminary, and grandson of Billy and Ruth Graham. He is a bestselling author, most rec... Read More
Good people get good stuff. Bad people get bad stuff. Or as the Beatles sang with their last gasp on Abbey Road, "In the end, the love you take is equal to the love you make."
Now I love John, Paul, George, and Ringo, but I take issue with them here, and I know I am in the minority. After all, the world runs on retribution. "This for that" comes as naturally to us as breathing. While no one can deny that our actions have consequences—that if you put your finger in a light socket you will "reap" a shock—we do God (and ourselves!) a great disservice when we project this false schema onto Him.
when you and I insist on that all-too-comfortable paradigm of cosmic score keeping, we stop talking about Christianity and in fact adopt a Westernized form of Hinduism.
That is, when we moralize our suffering and that of others. The lab test results come back positive, and we interpret it as some sort of punishment. Or your loved ones interpret it that way. Your marriage falls apart, and you assume God is meting out His judgment on your indiscretions. Most of us—not all, I'm afraid—would stop short of blaming the citizens of New Orleans for the devastation of Hurricane Katrina, but that doesn't mean we don't moralize our suffering in other, more subtle ways.
Christians believe that Jesus severed the link between suffering and deserving once for all on Calvary. God put the ledgers away and settled the accounts. But when you and I insist on that all-too-comfortable paradigm of cosmic score keeping, we stop talking about Christianity and in fact adopt a Westernized form of Hinduism.
We are talking about karma. If you are a bad person and things are going well for you, it is only a matter of time before karma catches up with you and "you get yours." If you are good person, the inverse is true: just be patient and your good deeds will come back to you. This is a simplification of the complex Hindu understanding of history as determined by the past lives of others: that we are all stuck in an eternal cycle of suffering perpetuated by reincarnation.
The problem in this worldview comes, as it always does, when we flip it around. If you are suffering, you have done something to merit it. Pain is proof.
These ideas are echoed in the "prosperity gospel"—characterized by the belief that God wants to bless His people, that He wants to give us tangible rewards such as riches and success and that we claim these rewards by exercising our faith in obedience. The side effect of this belief is that it implies if you experience suffering in some way, it's a result of either a lack of faith or some hidden sin.
Perhaps you erected some barrier that keeps God from working in your life. You do not believe enough, or you do not live well enough. In all cases, the emphasis is firmly on the believer and his or her abilities (or lack thereof).
Whenever our ears pick up an "if-then" statement, we can reliably assume we are in the arena of the law. "If you want to live, then you will obey the Lord's commands." "If you meet my standard of (beauty, wealth, intelligence), then I'll love you." The Gospel, on the other hand, says, "I'll love you despite the fact that you may not meet My standards or obey My commands."
The Christian tradition is full of those who have suffered death and persecution as a result of their faith and, in some cases, for no discernible reason whatsoever.
Grace fundamentally rejects the circularity of karma.
Sadly, this "new evangelical theology"—the prosperity gospel—has left a legacy of spiritual wreckage in the lives of countless believers who simply desired to love and obey God. The prosperity gospel does not answer why bad things happen; it merely encourages us to focus on how to live in the present. It tells us that God is a good God, who gives only good things to His children. Which is about as classic a half-truth as you could possibly find.
Unfortunately, Christians are never, ever promised in the Bible that God will rescue us from our suffering—never. It is extremely difficult to study the book of Job or the letters of Paul and still believe in the teachings of the prosperity theology.
In fact, the Bible is replete with examples of faithful men and women who suffer, Jesus being the most obvious example. The Christian tradition is full of those who have suffered death and persecution as a result of their faith and, in some cases, for no discernible reason whatsoever. Roman Catholics call them saints!
C. S. Lewis put it more eloquently than I ever could: "We were … promised sufferings. They were part of the program. We were even told 'Blessed are they that mourn.'"
As much as we might wish it weren't so, biblical faith ultimately eschews such conditionality. You simply cannot look at the cross and see the suffering of Jesus on behalf of rebellious sinners like you and me without also accepting that God sometimes ordains pain. This is not to say that suffering is never the result of something you or I have done, that it can't be related to some form of deserving. Rather, it just means that there is no longer a one-to-one correlation. In fact, there may not be any correlation at all!
So while the prosperity gospel pays lip service to the God of the Bible, it worships a God who waits for the suffering person to snap out of it and claim victory. In other words, it posits a God who is powerless to save sinners.
Jesus Himself tackles the topic of karma head-on. In the ninth chapter of John, Jesus and His disciples come across a blind man. His disciples asked him,
"Rabbi, who sinned, this man or his parents, that he was born blind?" "Neither this man nor his parents sinned," said Jesus, "but this happened so that the works of God might be displayed in his life" (John 9:2–3).
I'm not sure how Jesus could be any clearer! Something else was going on in this man's suffering than a this-for-that retributive exchange. The man's affliction was not evidence of a curse or punishment. In fact, God was somehow present in it. The works of God displayed in infirmity and weakness, rather than health
and strength? Perhaps it is no coincidence that the man who spoke these words would do more than voice them. He would live and die them.
Excerpted from chapter 4 of Glorious Ruin (David C. Cook, October 2012) by Tullian Tchividjian.
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Top Comments
Jesus's Commandments were not "the law". They were: Love. Not, "be a good person," but be an extraordinary lover. Holiness needs to be redefined to those who pursue Christ in love and grace, not those who are able to actually pull off being a good Christian.
Peter said we should expect suffering. But we really expect blessings and thus have a hard time when suffering comes to us. As Christians we should count it an opportunity to reveal that our relationship with God, our friendship with Him is not about what He is DOING for us but the fact that He is WITH us. It was Job's privilege to suffer and reveal that God could trust him; that he wouldn't let God down. It is our privilege to suffer and say "Thank you God for being with me through this experience" rather than asking Him for the things we think are best for us. He ALWAYS does the right thing by us - especially in suffering.
33 Comments
Friends My name is Franck
I was born Christian with a father Pastor. I was a good singer ,prayer and I enjoyed living in church. In my teen ages I started drinking alcohol with friends. Sometimes I came back to god and ask for Forgiveness. When I get here in U.S I kept drinking with friends but knowing the power of God and Believing Him. I decided many times to stop everything and dedicate all my time to my God but after few weeks I returned in the sins. I had planned to change completely. On November 8, 2012 my friend invited me to his house and He offered me the Alcohol. On my way back the police stopped me and charged me as Operating under influence (OUI).Yesterday (Nov 23) I received a letter from Motor vehicle that my License is suspended for 90 days. I have risks to lose my job. My first born goes to school everyday and I was the one who take him. My second son was born premature and have a lot coming appointments .The record is destroyed. Since Nov 8 I started asking my God to forgive me and give and help me in this situation.
Do you believe that my God still listening to me?
Can MY God still help me in this problem? Please pray for me and Encourage me on my email adresse: email@example.com
Thank you
This is an incorrect statement.
"Unfortunately, Christians are never, ever promised in the Bible that God will rescue us from our suffering—never. It is extremely difficult to study the book of Job or the letters of Paul and still believe in the teachings of the prosperity theology."
God says....
Psalm 50:15 "And call me in the day of trouble;
I will deliver you, and you will glorify me."
If you want to claim that this Psalm was not directly written to Christians because it is in the Old Testament, then you must also remove the mentioning of the book of Job as support for your argument.
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