Application of a high power Yb fiber‐based laser compatible with commercial optical parametric oscillator for coherent anti‐stokes raman scattering microscopy

Coherent anti‐Stokes Raman scattering (CARS) microscopy is a powerful tool for chemical analysis at a subcellular level, frequently used for imaging lipid dynamics in living cells. We report a high‐power picosecond fiber‐based laser and its application for optical parametric oscillator (OPO) pumping and CARS microscopy. This fiber‐based laser has been carefully characterized. It produces 5 ps pulses with 0.8 nm spectral width at a 1,030 nm wavelength with more than 10 W of average power at 80 MHz repetition rate; these spectral and temporal properties can be slightly modified. We then study the influence of these modifications on the spectral and temporal properties of the OPO. We find that the OPO system generates a weakly spectrally chirped signal beam constituted of 3 ps pulses with 0.4 nm spectral width tunable from 790 to 930 nm optimal for CARS imaging. The frequency doubling unconverted part is composed of 7–8 ps pulses with 0.75 nm spectral width compatible with CARS imaging. We also study the influence of the fiber laser properties on the CARS signal generated by distilled water. In agreement with theory, we find that shorter temporal pulses allow higher peak powers and thus higher CARS signal, if the spectral widths are less than 10 cm^?1. We demonstrate that this source is suitable for performing CARS imaging of living cells during several hours without photodamages. We finally demonstrate CARS imaging on more complex aquatic organisms called copepods (micro‐crustaceans), on which we distinguish morphological details and lipid reserves. Microsc. Res. Tech. 77:422–430, 2014. © 2014 Wiley Periodicals, Inc.     

The use of optical parametric oscillator for harmonic generation and two-photon UV fluorescence microscopy

Ultrafast lasers have found increasing use in scanning optical microscopy due to their very high peak power in generating multiphoton excitations. A mode-locked Ti:sapphire laser is often employed for such purposes. Together with a synchronously pumped optical parametric oscillator (OPO), the spectral range available can be extended to 1,050-1,300 nm. This broader range available greatly facilitates the excitation of second harmonic generation (SHG) and third harmonic generation (THG) due to better satisfaction of phase matching condition that is achieved with a longer excitation wavelength. Dental sections are then investigated with the contrasts from harmonic generation. In addition, through intra-cavity doubling wavelengths from 525-650 nm are made available for effective two-photon (2-p) excitation with the equivalent photon energy in the UVB range (290-320 nm) and beyond. This new capacity allows UV (auto-) fluorescence excitation and imaging, for example, from some amino acids, such as tyrosine, tryptophan, and glycine.     

Continuously wavelength tuned optical parametric oscillator based on fan-out periodically poled lithium niobate

A PC-controlled optical parametric oscillator (OPO) based on fan-out periodically poled lithium niobate (MgO : PPLN) structures was developed. Continuous wavelength tuning (2.40–3.85 μm) was realized via linear displacements of fan-out MgO : PPLN structures using a PC-controlled precision motorized translation stage. The total wavelength scanning time in the range of 2.40–3.85 μm was ≤1 min. The OPO was developed as a source of tunable radiation for use in a laser photo-acoustic gas analyzer. Studies of the methane absorption spectrum showed a good coincidence of the experimental and theoretical data.     

Autofluorescence spectroscopy and imaging of Platymonas subcordiformis irradiated by diode laser based on LSCM

Autofluorescence spectra and optical imaging of Platymonas subcordiformis after irradiation of diode laser were observed via laser scanning confocal microscopy (LSCM). With 488 nm Ar(+) laser excitation, the horizontal and vertical dimensions of a cup-shaped chloroplast of the irradiation group increased about 10% compared with the control group. The fluorescence spectra were similar between irradiation group and control group with a maximum fluorescence band around 682 nm, whereas the former has a higher intensity. Image of a small circular substance with stronger two-photon autofluorescence (TPA) was obtained when using two-photon excitation wavelength of 800 nm in single-channel mode. Further analysis by the 800 nm excitation based on two independent-channels mode showed an emission band of the small circular substance around 376-505 nm, which corresponded to the eyespot of P. subcordiformis. In lambda scanning mode, with two-photon wavelength of 800 nm excitation, six fluorescence peaks that are located at 465, 520, 560, 617, 660 and 680 nm were observed; the fluorescence intensity of the irradiation group was higher than that of the control group, especially at 520, 560 and 617 nm. As a conclusion, diode laser irradiation can promote chloroplast growth of P. subcordiformis cells in the form of expanding area and the increasing content of protein, phospholipids and chlorophyll. LSCM, especially TPA imaging based on femtosecond laser excitation, provides a nondestructive, real-time and accurate method to study changes of living algal cells under laser irradiation and other environmental factors.     

三镜直腔结构MgO∶PPLN高效连续光参量振荡器

为了优化MgO:PPLN连续光参量振荡器(OPO)的输出特性,对三镜直腔结构的内腔式OPO系统进行腔结构设计,对其同时输出高效的信号光和闲频光进行研究。采用半导体激光端面抽运Nd:YVO4晶体实现连续的1064 nm激光为基频光。对比分析了基频激光腔和OPO腔各腔镜分别采用平面镜或平凹镜的三种腔型结构的激光输出特性。基于30.5 μm的极化周期和12.4 W入射抽运功率时,获得了最高输出功率3.92 W（信号光2.6 W和闲频光1.32 W）,转化效率31.6%的激光输出,对应的信号光和闲频光的中心波长分别为1549 nm和3394 nm。结果表明三个腔镜均采用平凹镜时,可有效的压缩基频激光腔在MgO:PPLN晶体上的光斑,提升基频激光的功率密度,而且基频激光腔和OPO腔的基模光斑在MgO:PPLN晶体上更好的匹配,从而提升变频效率。     

A novel light source for SICM–SNOM of living cells

We have developed a novel light source for use in a scanning near‐field optical microscope (SNOM or NSOM) based on a nanopipette whose distance from the sample surface is controlled using scanning ion conductance microscopy. The light source is based on the general principle of the chemical reaction between a fluorophore in the pipette and ligand in the bath, to produce a highly fluorescent complex that is continually renewed at the pipette tip. In these experiments we used fluo‐3 and calcium, respectively. This complex is then excited with an Ar⁺ laser, focused on the pipette tip, to produce the light source. This method overcomes the transmission problem of more traditional SNOM probes and has been used to acquire simultaneous high‐resolution topographic and optical images of biological samples in physiological buffer. A resolution of ～220 nm topographic and ～190 nm optical was determined through imaging fixed sea‐urchin sperm flagella. Live A6 cells were also imaged, demonstrating the potential of this system for SNOM imaging of living cells.     

Flexible and stable optical parametric oscillator based laser system for coherent anti‐Stokes Raman scattering microscopy

The characteristics of a stable and flexible laser system based on a synchronously pumped optical parametric oscillator (OPO) is presented. This OPO can offer very stable operation with both ～1 ps and ～300 fs outputs over a broad wavelength range, i.e., 920–1200 nm. Combining the pump tuning with the OPO tuning, a total Raman range of 1900–5500 cm^?1 is accessible. For maximum spectral sensitivity, the CARS microsope based on the ps laser system is presented in detail. The lateral resolution of the microscope is diffraction limited to be about 390 nm. Fast wavelength switching (sub‐second) between two Raman vibrational frequencies, i.e., 2848 cm^?1 for C? H aliphatic vibrations and 3035 cm^?1 for C? H aromatic vibrations is presented as an example, although this also extends to other Raman frequencies. The possibility of obtaining a multimodal imaging system based on the fs laser system is also discussed. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

High-resolution compact X-ray microscopy

We demonstrate compact full‐field soft X‐ray transmission microscopy with sub 60‐nm resolution operating at λ= 2.48 nm. The microscope is based on a 100‐Hz regenerative liquid‐nitrogen‐jet laser‐plasma source in combination with a condenser zone plate and a micro‐zone plate objective for high‐resolution imaging onto a 2048 × 2048 pixel CCD detector. The sample holder is mounted in a helium atmosphere and allows imaging of both dry and wet specimens. The microscope design enables fast sample switching and the sample can be pre‐aligned using a visible‐light microscope. High‐quality images can be acquired with exposure times of less than 5 min. We demonstrate the performance of the microscope using both dry and wet samples.     

Analogous on‐axis interference topographic phase microscopy (AOITPM)

The refractive index (RI) of a sample as an endogenous contrast agent plays an important role in transparent live cell imaging. In tomographic phase microscopy (TPM), 3D quantitative RI maps can be reconstructed based on the measured projections of the RI in multiple directions. The resolution of the RI maps not only depends on the numerical aperture of the employed objective lens, but also is determined by the accuracy of the quantitative phase of the sample measured at multiple scanning illumination angles. This paper reports an analogous on‐axis interference TPM, where the interference angle between the sample and reference beams is kept constant for projections in multiple directions to improve the accuracy of the phase maps and the resolution of RI tomograms. The system has been validated with both silica beads and red blood cells. Compared with conventional TPM, the proposed system acquires quantitative RI maps with higher resolution (420 nm @λ = 633 nm) and signal‐to‐noise ratio that can be beneficial for live cell imaging in biomedical applications.     

Live cell ultraviolet microscopy: a comparison between two- and three-photon excitation

We compare conventional infrared laser based three-photon excitation with a visible laser based two-photon excitation scheme for imaging the ultraviolet fluorophore serotonin in solution and in live cells. To obtain a signal level of 1000 photons per second per mM serotonin solution, we need a back aperture power of 5 mW at 550 nm (for two-photon excitation) and 33 mW at 740 nm (for three-photon excitation). The detectivity of serotonin (defined as the concentration of serotonin that yields a signal equivalent to three times the standard deviation of the signal obtained from the buffer alone) is 12 microM for two-photon, and 220 microM for three-photon excitation. Surprisingly, for live cell imaging of vesicular serotonin in serotonergic cells, three-photon excitation appears to provide better image contrast than two-photon excitation. The origin of this is traced to the concentration-dependent shift of the serotonin emission spectrum.     

Characterization of microscope objective lenses from 1,400 to 1,650 nm to evaluate performance for long-wavelength nonlinear microscopy applications

We have demonstrated a simple method for characterization of objective lens performance at longer wavelengths for 3PLSM and THG imaging. We investigated a range of air and oil-immersion objective lenses across a wavelength range of 1,400-1,650 nm using a synchronously pumped optical parametric oscillator laser source. In the first instance, we investigated the percentage light transmission across this spectral range. Second, we used a simple second harmonic autocorrelation pulse measurement technique to study the dispersion properties of these lenses at the range of input wavelengths. For the objective lenses investigated, we observed pulse broadening on the order of around 4%-7% for air immersion lenses and 9%-12% for oil immersion lenses. Even for the greater dispersion incurred by the application of the oil immersion lenses, these objectives are suitable for longer wavelength application in conjunction with a suitable light source. The same techniques could easily be applied for a larger range of objective lenses and adapted for alternative spectral windows and pulse durations.     

An optical spectrometer for a confocal scanning laser microscope

An optical spectrometer for the visible range has been developed for the confocal scanning laser microscope (CSLM) Phoibos 1000. The spectrometer records information from a single point or a user-defined region within the microscope specimen. A prism disperses the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit cools the diode array, thereby reducing the detector dark current to a level, which allows integration times of up to 60 s. The spectral resolving power, λ/Δλ, ranges from 400 at λ = 375 nm to 100 at λ = 700 nm. Since the entrance aperture of the spectrometer has the same diameter as the detector aperture of the CSLM, the three-dimensional spatial resolution for spectrometer readings is equivalent to that of conventional confocal scanning, that is, down to 0.2 μm lateral and 0.8 μm axial resolution with an N.A.=1.3 objective.     

Multiphoton confocal microscopy using a femtosecond Cr:forsterite laser

With its output wavelength covering the infrared penetrating window of most biological tissues at 1,200-1,250 nm, the femtosecond Cr:forsterite laser shows high potential to serve as an excellent excitation source for the multiphoton fluorescence microscope. Its high output power, short optical pulse width, high stability, and low dispersion in fibers make it a perfect replacement for the currently widely used Ti:sapphire laser. In this paper, we study the capability of using a femtosecond Cr:forsterite laser in multiphoton scanning microscopy. We have performed the multiphoton excited photoluminescence spectrum measurement on several commonly used bioprobes using the 1,230 nm femtosecond pulses from a Cr:forsterite laser. Efficient fluorescence can be easily observed in these bioprobes through two-photon or three-photon excitation processes. These results will assist in the selection of dichroic beam splitter and band pass filters in a multiphoton microscopic system. We have also performed the autofluorescence spectrum measurement from chlorophylls in live leaves of the plant Arabidopsis thaliana excited by 1,230 nm femtosecond pulses from the Cr:forsterite laser. Bright luminescence from chlorophyll, centered at 673 and 728 nm, respectively, can be easily observed. Taking advantage of the bright two-photon photoluminescence from chlorophyll, we demonstrated the two-photon scanning paradermal and cross-sectional images of palisade mesophyll cells in live leaves of Arabidopsis thaliana.     

Measurement of glutathione levels in intact roots of Arabidopsis

Levels of glutathione were measured for different cell types in roots of intact Arabidopsis seedlings after labelling with monochlorobimane to give fluorescent glutathione S‐bimane (GSB) and imaging using confocal laser scanning microscopy with excitation at 442 nm. Labelling increased to a plateau in most cell types after about 15–20 min and the GSB accumulated rapidly in the vacuole. Formation of GSB in the cytoplasm was not affected by treatment with sodium azide; however, vacuolar transport of GSB was substantially inhibited under these conditions. We infer that vacuolar sequestration was mediated by a tonoplast glutathione S‐conjugate pump. Quantitative estimates of the cytoplasmic glutathione concentration involved correction for the loss in fluorescence signal with depth into the specimen using an empirically determined model derived in situ from a permeabilized root. Correction for the dilution experienced on transport into the vacuole also required an estimate of the amount of cytoplasm present in each cell type. This was achieved in two stages: first, the levels of protein were mapped after fixation, permeabilization and labelling with fluroescein isothiocyanate. Second, the corresponding cytoplasmic volume was determined as 40% for epidermal cells in the elongation zone by manual segmentation of the cytoplasm in serial optical sections. Values of relative cytoplasmic volume for other cells were extrapolated in proportion to their protein content. Using this approach, cytoplasmic glutathione concentrations were found to be 2–3 mm in most cell types. There was a marked difference between the central cells and the neighbouring, rapidly dividing initials, and between the columella cells and the outermost cells of the root cap. In the latter case, the difference was equalized in the presence of azide. This might indicate that additional cell–cell movement and preferential sequestration of GSB can occur during the detoxification process in an intact system.     

An engineered microenvironment for multidimensional microscopy of live cells

Multidimensional imaging (MD) of live cells is gaining importance in biomedical research as the commercial availability of confocal, nonlinear optical microscopes, environmental chambers, and specific fluorescence probes grows. One crucial aspect of the MD live cell imaging involves the proper immobilization of cells, which refers to the rapid and sufficient immobilization of cells on the microscope stage, neither disrupting the cellular structure and functions nor affecting the optical properties of the cells and the environments. Conventional cell immobilization methods glue the anchoring cells to coated surfaces, but such methods require centrifugation or extended incubation and are not suitable for cells in suspension. Most of the current three-dimensional (3-D) gels either exhibit unsatisfactory optical properties or have adverse effects on cell functions in culture. Recently, an engineered 3-D microcapsule has been developed that involves the complex coacervation of a positively charged collagen and a negatively charged polymer of 2-hydroxyethyl methacrylate--methacrylic acid--methyl methacrylate (HEMA-MMA-MAA). Hence, confocal imaging of live cells in this engineered 3-D microenvironment was investigated for its optical properties and cellular function compatibility. We report here that this microenvironment facilitates efficient cell immobilization, exhibits good optical properties, and can preserve cellular structures and functions, which will be useful in MD imaging of live cells for various applications.     

Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging

Two‐photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580–1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two‐photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two‐photon and the one‐photon emission spectra. However, a blue‐shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non‐linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles.     

Laser scanning gradient index optics microscope: submicrometre scale spectroscopy and imaging at cryogenic temperatures

A laser scanning far-field optical microscope for low-temperature imaging and spectroscopy based on gradient index optics is presented. A rod-shaped gradient index microlens is used as a zero-working-distance solid immersion objective lens. The obtained lateral resolution is 310 nm of the FWHM at a wavelength of 545 nm. A laser scanning mechanism located outside an optical cryostat enables one to achieve large scanning ranges independent of temperature. The use of the microscope for submicrometre-scale spectroscopy and low-temperature photochemistry performed on molecular J aggregates in thin polymer films is presented.     

High-resolution simultaneous three-photon fluorescence and third-harmonic-generation microscopy

In recent years, nonlinear laser scanning microscopy has gained much attention due to its unique ability of deep optical sectioning. Based on our previous studies, a 1,200-1,300-nm femtosecond laser can provide superior penetration capability with minimized photodamage possibility. However, with the longer wavelength excitation, three-photon-fluorescence (3PF) would be necessary for efficient use of intrinsic and extrinsic visible fluorophores. The three-photon process can provide much better spatial resolution than two-photon-fluorescence due to the cubic power dependency. On the other hand, third-harmonic-generation (THG), another intrinsic three-photon process, is interface-sensitive and can be used as a general structural imaging modality to show the exact location of cellular membranes. The virtual-transition characteristic of THG prevents any excess energy from releasing in bio-tissues and, thus, THG acts as a truly noninvasive imaging tool. Here we demonstrated the first combined 3PF and THG microscopy, which can provide three-dimensional high-resolution images with both functional molecule specificity and sub-micrometer structural mapping capability. The simultaneously acquired 3PF and THG images based on a 1,230-nm Cr:forsterite femtosecond laser are shown with a Hoechst-labeled hepatic cell sample. Strong 3PF around 450 nm from DNA-bounded Hoechst-33258 can be observed inside each nucleus while THG reveals the location of plasma membranes and other membrane-based organelles such as mitochondria. Considering that the maximum-allowable laser power in common nonlinear laser microscopy is less than 10 mW at 800 nm, it is remarkable that even with a 100-mW 1,230-nm incident power, there is no observable photo damage on the cells, demonstrating the noninvasiveness of this novel microscopy technique.     

Multiphoton microscopy in life sciences

Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three‐dimensional fluorescence imaging based on non‐resonant two‐photon or three‐photon fluorophor excitation requires light intensities in the range of MW cm^?2 to GW cm^?2, which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi‐gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non‐invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth‐resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non‐invasive fluorophore loading into single living plant cells. Non‐destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis‐like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two‐photon excitation process rather than a one‐photon or three‐photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two‐photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid‐induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm^?2 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non‐invasive NIR laser surgery within a living cell or within an organelle is therefore possible.     

Laser scanning gradient index optics microscope: submicrometre scale spectroscopy and imaging at cryogenic temperatures

A laser scanning far-field optical microscope for low-temperature imaging and spectroscopy based on gradient index optics is presented. A rod-shaped gradient index microlens is used as a zero-working-distance solid immersion objective lens. The obtained lateral resolution is 310 nm of the FWHM at a wavelength of 545 nm. A laser scanning mechanism located outside an optical cryostat enables one to achieve large scanning ranges independent of temperature. The use of the microscope for submicrometre-scale spectroscopy and low-temperature photochemistry performed on molecular J aggregates in thin polymer films is presented.