Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two‐photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy‐to‐operate platform capable to perform two‐photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012. © 2012 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.     

Nonlinear bio-photonic crystal effects revealed with multimodal nonlinear microscopy

Highly optically active nonlinear bio‐photonic crystalline and semicrystalline structures in living cells were studied by a novel multimodal nonlinear microscopy. Numerous biological structures, including stacked membranes and aligned protein structures are highly organized on a nanoscale and have been found to exhibit strong optical activities through second‐harmonic generation (SHG) interactions, behaving similarly to man‐made nonlinear photonic crystals. The microscopic technology used in this study is based on a combination of different imaging modes including SHG, third‐harmonic generation, and multiphoton‐induced fluorescence. With no energy release during harmonic generation processes, the nonlinear‐photonic‐crystal‐like SHG activity is useful for investigating the dynamics of structure–function relationships at subcellular levels and is ideal for studying living cells, as minimal or no preparation is required.     

Third‐harmonic generation imaging of breast tissue biopsies

We demonstrate for the first time the imaging of unstained breast tissue biopsies using third‐harmonic generation (THG) microscopy. As a label‐free imaging technique, THG microscopy is compared to phase contrast and polarized light microscopy which are standard imaging methods for breast tissues. A simple feature detection algorithm is applied to detect tumour‐associated lymphocyte rich regions in unstained breast biopsy tissue and compared with corresponding regions identified by a pathologist from bright‐field images of hematoxylin and eosin stained breast tissue. Our results suggest that THG imaging holds potential as a complementary technique for analysing breast tissue biopsies.     

Fibre‐optical microendoscopy

Microendoscopy has been an essential tool in exploring micro/nano mechanisms in vivo due to high‐quality imaging performance, compact size and flexible movement. The investigations into optical fibres, micro‐scanners and miniature lens have boosted efficiencies of remote light delivery to sample site and signal collection. Given the light interaction with materials in the fluorescence imaging regime, this paper reviews two classes of compact microendoscopy based on a single fibre: linear optical microendoscopy and nonlinear optical microendoscopy. Due to the fact that fluorescence occurs only in the focal volume, nonlinear optical microendoscopy can provide stronger optical sectioning ability than linear optical microendoscopy, and is a good candidate for deep tissue imaging. Moreover, one‐photon excited fluorescence microendoscopy as the linear optical microendoscopy suffers from severe photobleaching owing to the linear dependence of photobleaching rate on excitation laser power. On the contrary, nonlinear optical microendoscopy, including two‐photon excited fluorescence microendoscopy and second harmonic generation microendoscopy, has the capability to minimize or avoid the photobleaching effect at a high excitation power and generate high image contrast. The combination of various nonlinear signals gained by the nonlinear optical microendoscopy provides a comprehensive insight into biophenomena in internal organs. Fibre‐optical microendoscopy overcomes physical limitations of traditional microscopy and opens up a new path to achieve early cancer diagnosis and microsurgery in a minimally invasive and localized manner.     

Fluorescent resolution target for super-resolution microscopy

We report methods of near‐field infrared microscopy with transient optically induced probes. The first technique – a transient aperture (TA) – uses photoinduced reflectivity in semiconductors to generate a relatively large transient mirror (TM) with a small aperture at its centre. We report the optical properties of the TM and TA and experiments performed on near‐field imaging with the TA. The second technique is based on solid immersion microscopy, in which high resolution is achieved when light is focused inside a solid with a high refractive index. By creating a transient Fresnel lens on the surface of a semiconductor wafer via photoinduction, we were able to form a solid immersion lens (SIL) for use as a near‐field probe. The use of transient probes eliminates the need for mechanical scanning of the lens or sample, and thus provides a much faster scanning rate and the possibility to work with soft and liquid objects.     

Optical microscopy with flexible axial capabilities using a vari‐focus liquid lens

The axial imaging range of optical microscopy is restricted by its fixed working plane and limited depth of field. In this paper, the axial capabilities of an off‐the‐shelf microscope is improved by inserting a liquid lens, which can be controlled by a driving electrical voltage, into the optical path of the microscope. First, the numerical formulas of the working distance and the magnification with the variation of the focus of the liquid lens are inferred using a ray tracing method and conclusion is obtained that the best position for inserting a liquid lens with consistent magnification is the aperture plane and the rear focal plane of the objective lens. Second, with the liquid lens embedded in the microscope, the numerical relationship between the magnification and the working distance of the proposed flexible‐axial‐capability microscope and the liquid lens driving voltage is calibrated and fitted using the inferred numerical formulas. Third, techniques including autofocus, extending depth of field and three‐dimensional imaging are researched and applied, improving the designed microscope to not only flexibly control its working distance, but also to extend the depth of field near the variable working plane. Experiments show that the presented flexible‐axial‐capability microscope has a long working distance range of 8 mm, and by calibrating the magnification curve within the working distance range, samples can be observed and measured precisely. The depth of field can be extended to 400 μm from the variable working plane and is 20 times that of the off‐the‐shelf microscope.     

Second harmonic generation imaging of the deep shade plant Selaginella erythropus using multifunctional two‐photon laser scanning microscopy

Background : Multifunctional two‐photon laser scanning microscopy provides attractive advantages over conventional two‐photon laser scanning microscopy. For the first time, simultaneous measurement of the second harmonic generation (SHG) signals in the forward and backward directions and two photon excitation fluorescence were achieved from the deep shade plant Selaginella erythropus. Results : These measurements show that the S. erythropus leaves produce high SHG signals in both directions and the SHG signals strongly depend on the laser's status of polarization and the orientation of the dipole moment in the molecules that interact with the laser light. The novelty of this work is (1) uncovering the unusual structure of S. erythropus leaves, including diverse chloroplasts, various cell types and micromophology, which are consistent with observations from general electron microscopy; and (2) using the multifunctional two‐photon laser scanning microscopy by combining three platforms of laser scanning microscopy, fluorescence microscopy, harmonic generation microscopy and polarizing microscopy for detecting the SHG signals in the forward and backward directions, as well as two photon excitation fluorescence. Conclusions : With the multifunctional two‐photon laser scanning microscopy, one can use noninvasive SHG imaging to reveal the true architecture of the sample, without photodamage or photobleaching, by utilizing the fact that the SHG is known to leave no energy deposition on the interacting matter because of the SHG virtual energy conservation characteristic.     

Study of the focused laser spots generated by various polarized laser beam conditions

In this work, three‐dimensional near‐field imaging of the focused laser spot was studied theoretically and experimentally. In the theoretical simulation, we use the electromagnetic equivalent of the vectorial Kirchhoff diffraction integral to calculate the intensity distribution of the focal region, and a high depolarization is found in high numerical aperture systems (NA = 0.85). The experimental set‐up is based on a near‐field scanning optical microscope (NSOM) system. A high‐NA objective lens is used to focus incident light of various polarizations, and a tapered near‐field optical fibre probe of the NSOM system is used to determine the intensity of the focal field. The results show an asymmetric distribution of the focused intensity with the linear polarized laser beam.     

Near-field second harmonic imaging of the c/a/c/a polydomain structure of epitaxial PbZrxTi1–xO3 thin films

Near-field optical second harmonic microscopy has been applied to imaging of the c/a/c/a polydomain structure of epitaxial PbZr _x Ti_{1– x} O₃ thin films in the 0 <  x  < 0.4 range. Comparison of the near-field optical images and the results of atomic force microscopy and X-ray diffraction studies show that an optical resolution of the order of 100 nm is achieved. Symmetry properties of the near-field second harmonic signal allow us to obtain good optical contrast between the local second harmonic generation in c- and a-domains. Experimentally measured near-field second harmonic images have been compared with the results of theoretical calculations. Good agreement between theory and experiment is demonstrated.     

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.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

3D microscopy of transparent objects using third-harmonic generation

It is demonstrated that third-harmonic generation (THG) near interfaces in the refractive index or the third-order nonlinear susceptibility (χ⁽³⁾) permits three-dimensional imaging of transparent objects. The nonlinear dependence of THG on the excitation power provides inherent optical sectioning. At the same time, the nonresonant nature of THG, in combination with the near-IR excitation wavelengths used (1–2 μm), render this technique potentially (biologically) nondamaging and nonbleaching. A specific property of THG imaging is its sensitivity to — and potential use for imaging of — the relative orientation of interfaces with respect to the axis of propagation of the excitation radiation.     

Multifunctional imaging of endogenous contrast by simultaneous nonlinear and optical coherence microscopy of thick tissues

A variety of high resolution optical microscopy techniques have been developed in recent years for basic and clinical studies of biological systems. We demonstrate a trimodal microscope combining optical coherence microscopy (OCM) with two forms of nonlinear microscopy, namely two-photon excited fluorescence (2PF) and second harmonic generation (SHG), for imaging turbid media. OCM combines the advantages of confocal detection and coherence gating for structural imaging in highly scattering tissues. Nonlinear microscopy enables the detection of biochemical species, such as elastin, NAD(P)H, and collagen. While 2PF arises from nonlinear excitation of fluorescent species, SHG is a form of nonlinear scattering observed in materials that lack a center of inversion symmetry, such as type I collagen. Characterization of the microscope showed nearly diffraction-limited spatial resolution in all modalities. Images were obtained in fish scales and excised human skin samples. The primary endogenous sources of contrast in the dermis were due to elastin autofluorescence and collagen SHG. Multimodal microscopy allows the simultaneous visualization of structural and functional information of biological systems.     

Second‐harmonic microscopy of ex vivo porcine corneas

The ex vivo cornea of porcine eyes has been studied with second‐harmonic microscopy with a laboratory‐built system to examine the structure of collagen fibrils at different length scales, as well as the image dependence on polarization and wavelength of the illumination source. We found that collagen fibrils can effectively be visualized with second‐harmonic microscopy, in agreement with previous findings, at different wavelengths of the illumination. The same laser source used for imaging may also be used to induce changes to the corneal tissues that are observable both in the linear and second‐harmonic imaging channels. Such studies are essential first steps towards a future high‐resolution optical characterization technique for simultaneous corneal surgery and wound healing of the human eye.     

In vivo coherent anti-Stokes Raman scattering imaging of sciatic nerve tissue

We report in vivo nonlinear optical imaging of mouse sciatic nerve tissue by epidetected coherent anti‐Stokes Raman scattering and second harmonic generation microscopy. Following a minimally invasive surgery to open the skin, coherent anti‐Stokes Raman scattering imaging of myelinated axons and second harmonic generation imaging of the surrounding collagen fibres were demonstrated with high signal‐to‐background ratio, three‐dimensional spatial resolution, and no need for labelling. The underlying contrast mechanisms of in vivo coherent anti‐Stokes Raman scattering were explored by three‐dimensional imaging of fat cells that surround the nerve. The epidetected coherent anti‐Stokes Raman scattering signals from the nerve tissues were found to arise from interfaces as well as back reflection of forward coherent anti‐Stokes Raman scattering.     

Scanning near-field optical microscopy of a cell membrane in liquid

The applications of scanning near‐field optical microscopy to biological specimens under physiological conditions have so far been very rare since common techniques for a probe–sample distance control are not as well suited for operation in liquid as under ambient conditions. We have shown previously that our own approach for a distance control, based on a short aperture fibre probe and a tuning fork as force sensor in a tapping mode, works well even on soft material in water. By means of an electronic self‐excitation circuit, which compensates for changes of the resonance frequency due to evaporation of liquid, the stability of the force feedback has now been further improved. We present further evidence for the excellent suitability of the tapping‐mode‐like distance control to an operation in liquid, for example, by force‐imaging of double‐stranded DNA. Moreover, we demonstrate that a nuclear envelope in liquid can be imaged with a high optical resolution of ～70 nm without affecting its structural integrity. Thereby, single nuclear pores in the nuclear envelope with a nearest neighbour distance of ～120 nm have been optically resolved for the first time.     

A series of flexible design adaptations to the Nikon E‐C1 and E‐C2 confocal microscope systems for UV,multiphoton and FLIM imaging

Multiphoton microscopy is widely employed in the life sciences using extrinsic fluorescence of low‐ and high‐molecular weight labels with excitation and emission spectra in the visible and near infrared regions. For imaging of intrinsic and extrinsic fluorophores with excitation spectra in the ultraviolet region, multiphoton excitation with one‐ or two‐colour lasers avoids the need for ultraviolet‐transmitting excitation optics and has advantages in terms of optical penetration in the sample and reduced phototoxicity. Excitation and detection of ultraviolet emission around 300 nm and below in a typical inverted confocal microscope is more difficult and requires the use of expensive quartz optics including the objective. In this technical note we describe the adaptation of a commercial confocal microscope (Nikon, Japan E‐C1 or E‐C2) for versatile use with Ti‐sapphire and OPO laser sources and the addition of a second detection channel that enables detection of ultraviolet fluorescence and increases detection sensitivity in a typical fluorescence lifetime imaging microscopy experiment. Results from some experiments with this setup illustrate the resulting capabilities.     

Mapping piezoelectric-field distribution in gallium nitride with scanning second-harmonic generation microscopy

Taking advantage of the electric field-enhanced second-harmonic generation effect in bulk gallium nitride (GaN) and indium gallium nitride (InGaN) quantum wells, we demonstrated the piezoelectric field distribution mapping in bulk GaN and InGaN multiple-quantum-well (MQW) samples using scanning second-harmonic generation (SHG) microscopy. Scanning SHG microscopy and the accompanying third-harmonic generation (THG) microscopy of the bulk GaN sample were demonstrated using a femtosecond Cr:forsterite laser at a wavelength of 1230 nm. Taking advantage of the off-resonant electric field-enhanced SHG effect and the bandtail state-resonance THG effect, the second- and third-harmonic generation microscopic images obtained revealed the piezoelectric field and bandtail state distributions in a GaN sample. Combined with 720 nm wavelength excited two-photon fluorescence microscopy in the same sample, the increased defect density around the defect area was found to suppress bandedge photoluminescence, to increase yellow luminescence, to increase bandtail state density, and to decrease residue piezoelectric field intensity. Scanning SHG microscopy of the InGaN MQW sample was resonant excited with 800 nm femtosecond pulses from a Ti:sapphire laser in order to suppress SHG contribution from the bulk GaN substrate. Taking advantage of the strong piezoelectric field inside the InGaN quantum well, the wavelength resonant effect, and the electric field-enhanced SHG effect of InGaN quantum wells, resonant scanning SHG microscopy revealed the piezoelectric field distribution inside the wells. Combined with accompanying three-photon fluorescence microscopy from the bulk GaN substrate underneath the quantum wells, the direct correspondence between the piezoelectric field strength inside the quantum well and the substrate quality can be obtained. According to our study, the GaN substrate area with bright bandedge luminescence corresponds to the area with strong SHG signals indicating a higher stained-induced piezoelectric field. These scanning harmonic generation microscopies exhibit superior images of the piezoelectric field and defect state distributions in GaN and InGaN MQWs not available before. Combining with scanning multiphoton fluorescence microscopy, these techniques open new ways for the physical property study of this important material system and can provide interesting details that are not readily available by other microscopic techniques.     

Three‐dimensional structural imaging of starch granules by second‐harmonic generation circular dichroism

Chirality is one of the most fundamental and essential structural properties of biological molecules. Many important biological molecules including amino acids and polysaccharides are intrinsically chiral. Conventionally, chiral species can be distinguished by interaction with circularly polarized light, and circular dichroism is one of the best‐known approaches for chirality detection. As a linear optical process, circular dichroism suffers from very low signal contrast and lack of spatial resolution in the axial direction. It has been demonstrated that by incorporating nonlinear interaction with circularly polarized excitation, second‐harmonic generation circular dichroism can provide much higher signal contrast. However, previous circular dichroism and second‐harmonic generation circular dichroism studies are mostly limited to probe chiralities at surfaces and interfaces. It is known that second‐harmonic generation, as a second‐order nonlinear optical effect, provides excellent optical sectioning capability when combined with a laser‐scanning microscope. In this work, we combine the axial resolving power of second‐harmonic generation and chiral sensitivity of second‐harmonic generation circular dichroism to realize three‐dimensional chiral detection in biological tissues. Within the point spread function of a tight focus, second‐harmonic generation circular dichroism could arise from the macroscopic supramolecular packing as well as the microscopic intramolecular chirality, so our aim is to clarify the origins of second‐harmonic generation circular dichroism response in complicated three‐dimensional biological systems. The sample we use is starch granules whose second‐harmonic generation‐active molecules are amylopectin with both microscopic chirality due to its helical structure and macroscopic chirality due to its crystallized packing. We found that in a starch granule, the second‐harmonic generation for right‐handed circularly polarized excitation is significantly different from second‐harmonic generation for left‐handed one, offering excellent second‐harmonic generation circular dichroism contrast that approaches 100%. In addition, three‐dimensional visualization of second‐harmonic generation circular dichroism distribution with sub‐micrometer spatial resolution is realized. We observed second‐harmonic generation circular dichroism sign change across the starch granules, and the result suggests that in thick biological tissue, second‐harmonic generation circular dichroism arises from macroscopic molecular packing. Our result provides a new method to visualize the organization of three‐dimensional structures of starch granules. The second‐harmonic generation circular dichroism imaging method expands the horizon of nonlinear chiroptical studies from simplified surface/solution environments to complicated biological tissues.