High-power picosecond fiber source for coherent Raman microscopy

We report a high-power picosecond fiber pump laser system for coherent Raman microscopy (CRM). The fiber laser system generates 3.5ps pulses with 6W average power at 1030nm. Frequency doubling yields more than 2W of green light, which can be used to pump an optical parametric oscillator to produce the pump and the Stokes beams for CRM. Detailed performance data on the laser and the various wavelength conversion steps are discussed, together with representative CRM images of fresh animal tissue obtained with the new source.Chemistry and Chemical Biolog

Multicolor Stimulated Raman Scattering Microscopy with a Rapidly Tunable Optical Parametric Oscillator

Stimulated Raman scattering (SRS) microscopy allows label-free chemical imaging based on vibrational spectroscopy. Narrowband excitation with picosecond lasers creates the highest signal levels and enables imaging speeds up to video-rate, but it sacrifices chemical specificity in samples with overlapping bands compared to broadband (multiplex) excitation. We develop a rapidly tunable picosecond optical parametric oscillator with an electro-optical tunable Lyot filter, and demonstrate multicolor SRS microscopy with synchronized line-by-line wavelength tuning to avoid spectral artifacts due to sample movement. We show sensitive imaging of three different kinds of polymer beads and live HeLa cells with moving intracellular lipid droplets.Chemistry and Chemical Biolog

Quantitative Chemical Imaging with Multiplex Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a newly developed label-free chemical imaging technique that overcomes the speed limitation of confocal Raman microscopy while avoiding the nonresonant background problem of coherent anti-Stokes Raman scattering (CARS) microscopy. Previous demonstrations have been limited to single Raman band measurements. We present a novel modulation multiplexing approach that allows real-time detection of multiple species using the fast Fourier transform. We demonstrate the quantitative determination of chemical concentrations in a ternary mixture. Furthermore, two imaging applications are pursued: (1) quantitative determination of oil content as well as pigment and protein concentration in microalgae cultures; and (2) 3D high-resolution imaging of blood, lipids, and protein distribution in ex vivo mouse skin tissue. We believe that quantitative multiplex SRS uniquely combines the advantage of fast label-free imaging with the fingerprinting capability of Raman spectroscopy and enables numerous applications in lipid biology as well as biomedical imaging.Chemistry and Chemical BiologyEngineering and Applied Science

Label-Free Live-Cell Imaging of Nucleic Acids Using Stimulated Raman Scattering Microscopy

Imaging of nucleic acids is important for studying cellular processes such as cell division and apoptosis. A noninvasive label-free technique is attractive. Raman spectroscopy provides rich chemical information based on specific vibrational peaks. However, the signal from spontaneous Raman scattering is weak and long integration times are required, which drastically limits the imaging speed when used for microscopy. Coherent Raman scattering techniques, comprising coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, overcome this problem by enhancing the signal level by up to five orders of magnitude. CARS microscopy suffers from a nonresonant background signal, which distorts Raman spectra and limits sensitivity. This makes CARS imaging of weak transitions in spectrally congested regions challenging. This is especially the case in the fingerprint region, where nucleic acids show characteristic peaks. The recently developed SRS microscopy is free from these limitations; excitation spectra are identical to those of spontaneous Raman and sensitivity is close to shot-noise limited. Herein we demonstrate the use of SRS imaging in the fingerprint region to map the distribution of nucleic acids in addition to proteins and lipids in single salivary gland cells of Drosophila larvae, and in single mammalian cells. This allows the imaging of DNA condensation associated with cell division and opens up possibilities of imaging such processes in vivo.Chemistry and Chemical Biolog

LETTERS Imaging chromophores with undetectable fluorescence by stimulated emission microscopy

Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging 1,2 . However, many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay The phenomenon of stimulated emission was first described by Einstein in 1917 (ref. 4). An atom or molecule in its excited state can be stimulated down to the ground state by an incident light field, resulting in the creation of a new coherent photon identical to those in the original incident field. This process only occurs when the frequency of the incident field matches the transition energy. Stimulated emission was later used as a fundamental principle for light amplification in the laser 5 . The depopulation aspect of stimulated emission has been used for population dumping from excited states 6 , super-resolution fluorescence microscopy 7 , and fluorescence lifetime imaging 8 . Here we use the light-amplification aspect of stimulated emission as a contrast mechanism for highly sensitive imaging of chromophores that have undetectable fluorescence. Such chromophores have very short-lived excited states with much faster non-radiative decay rates than their spontaneous emission rates. As a result, their feeble fluorescence is overwhelmed by backgrounds, such as stray light, solvent Raman scattering, and detector dark counts. Our solution to this problem is to conduct a dual-beam experiment to interrogate the short-lived excited state by stimulated emission, which can compete with the non-radiative decay under a strong enough stimulating field Considering the optical excitation at frequency v 01 where N 0 is the number of molecules in the ground state. For a single chromophore, DI E /I E is of the order of 10 27 . Such small attenuation cannot be detected by conventional absorption microscopy. We note that single-molecule absorption was previously achieved in cryogenic temperatures using a frequency-modulation method 10 , which is, however, difficult to implement at room temperature because of the broad molecular absorption linewidth. Moreover, absorption measurement is often complicated by scattering when imaging biological specimens. Instead of detecting direct absorption, here we detect the stimulated emission followed by the excitation of the molecule. According to Einstein 4 , the molecular cross-section s stim for stimulated emission is comparable to s abs , because of microscopic reversibility. Unlike the absorption that results in attenuation, the stimulation beam will experience an intensity gain after interacting with the molecules

Fiber Four-Wave Mixing Source for Coherent Anti-Stokes Raman Scattering Microscopy

We present a fiber-format picosecond light source for coherent anti-Stokes Raman scattering microscopy. Pulses from an Yb-doped fiber amplifier are frequency-converted by four-wave mixing in normal dispersion photonic crystal fiber to produce a synchronized two-color picosecond pulse train. We show that seeding the four-wave mixing process overcomes the deleterious effects of group-velocity mismatch and allows efficient conversion into narrow frequency bands. The source generates more than 160 mW of nearly-transform-limited pulses tunable from 775 to 815 nm. High-quality coherent Raman images of animal tissues and cells acquired with this source are presented.Chemistry and Chemical Biolog

Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy

Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and readily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis.Chemistry and Chemical Biolog

Organizational Support and Contract Fulfillment as Moderators of the Relationship Between Preferred Work Status and Performance

Purpose The purpose of this study was to examine organizational context variables as moderators of the relationship between preferred work status and job performance. The moderators were perceived organizational support (POS) and psychological contract fulfillment. Design/Methodology/Approach Survey data was collected from 164 participants working in a health and fitness organization. These participants ranged in age from 18 to 79 years old (M = 40, SD = 12.5) and held various positions including middle managers, clerical workers, maintenance workers, and sports trainers. Findings The relationship between preferred work status and extra-role performance was negative when POS was higher but not when POS was lower. Also, the relationship between preferred work status and extra-role performance was positive when contract fulfillment was lower but not when it was higher. No moderating effects were found when examining in-role performance. Implications Given the large and growing use of part-time workers it is important to understand differences across various subgroups of them in order to better inform human resource policies and practices. Specifically, the results highlight a key role for the management of reciprocity perceptions. Originality/Value The literature on part-time workers suggests there are important differences between employees who work part-time because they prefer it and those who work part-time but prefer to work full-time. Research regarding the relationship between preferred work status and performance has produced mixed results. This study helps reconcile conflicting results regarding the relationship between preferred work status and performance by examining the moderating effects of theoretically relevant variables

Imaging Chromophores With Undetectable Fluorescence by Stimulated Emission Microscopy

Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging. However, many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. Here we use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, and report a new contrast mechanism for optical microscopy. In a pump-probe experiment, on photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, the intensity of which is concurrently increased. We extract the miniscule intensity increase with shot-noise-limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz frequency. The signal is generated only at the laser foci owing to the nonlinear dependence on the input intensities, providing intrinsic three-dimensional optical sectioning capability. In contrast, conventional one-beam absorption measurement exhibits low sensitivity, lack of three-dimensional sectioning capability, and complication by linear scattering of heterogeneous samples. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging.Chemistry and Chemical Biolog