Enabling the detection of UV signal in multimodal nonlinear microscopy with catalogue lens components

Using an optical system made from fused silica catalogue optical components, third‐order nonlinear microscopy has been enabled on conventional Ti:sapphire laser‐based multiphoton microscopy setups. The optical system is designed using two lens groups with straightforward adaptation to other microscope stands when one of the lens groups is exchanged. Within the theoretical design, the optical system collects and transmits light with wavelengths between the near ultraviolet and the near infrared from an object field of at least 1 mm in diameter within a resulting numerical aperture of up to 0.56. The numerical aperture can be controlled with a variable aperture stop between the two lens groups of the condenser. We demonstrate this new detection capability in third harmonic generation imaging experiments at the harmonic wavelength of ～300 nm and in multimodal nonlinear optical imaging experiments using third‐order sum frequency generation and coherent anti‐Stokes Raman scattering microscopy so that the wavelengths of the detected signals range from ～300 nm to ～660 nm.     

Quantitative theory of ideal phase-contrast microscopy,taking object width into account

In ‘ideal’ phase-contrast microscopy all the direct light and none of the diffracted light is influenced by the phase plate in the back focal plane of the objective. Contrary to almost all previous work, it appears that the intensity of an ideal phase-contrast image is affected not only by the transmittance and retardation of the object and of the phase plate, but also by the width of the specimen (or total width of multiple specimens) relative to the microscopic field. Equations and computer code are presented with which the intensity of such images can be calculated. Previously published equations are special cases, and implicitly or explictly assume either that the object is of negligible width, or occupies precisely half the microscopic field. The absolute brightness of an image in ideal central dark-field microscopy is a function of the object retardation, but the intensity of the image relative to the background is a function only of the width of the object(s) relative to the field. The equations give results for ideal phase-contrast microscopy identical with those of a computer program simulating microscopic imaging. The program can in addition take into account non-ideal factors including a finite width of phase plate, finite objective aperture, deviations from best focus, glare, primary spherical aberration and obliquity of the coherent illumination.     

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.     

两路固体激光相干合成的实验研究

相干合成技术是获得高功率、高亮度激光输出的有效途径之一。现提出了基于角锥棱镜的两路固体激光相干合成方案;在理论分析的基础上,开展了两路固体激光器相干合成的实验研究;得到了不同占空比和出射光束直径条件下的远场光强分布。远场光强分布和条纹可见度主要受到占空比和出射光束直径的影响。随着激光光束间距的减小,相干合成后的光斑数量减少,光斑的宽度增大,相干度明显提高;出射光束直径增大时,高阶模增多,光束质量变差但合成功率增大。     

Ultrafast optical pulse delivery with fibers for nonlinear microscopy

Nonlinear microscopies including multiphoton excitation fluorescence microscopy and multiple-harmonic generation microscopy have recently gained popularity for cellular and tissue imaging. The optimization of these imaging methods for minimally invasive use requires optical fibers to conduct light into tight space, where free-space delivery is difficult. The delivery of high-peak power laser pulses with optical fibers is limited by dispersion resulting from nonlinear refractive index responses. In this article, we characterize a variety of commonly used optical fibers in terms of how they affect pulse profile and imaging performance of nonlinear microscopy; the following parameters are quantified: spectral bandwidth and temporal pulse width, two-photon excitation efficiency, and optical resolution. A theoretical explanation for the measured performance of these fibers is also provided.     

Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging

The aim of this work is to demonstrate that multiphoton microscopy is a preferred technique to investigate intact cornea structure without slicing and staining. At the micron resolution, multiphoton imaging can provide both large morphological features and detailed structure of epithelium, corneal collagen fibril bundles and keratocytes. A large area multiphoton cross-section across an intact eye excised from a GFP mouse was obtained by a homebuilt multiphoton microscope. The broadband multiphoton fluorescence (435-700 nm) and second harmonic generation (SHG, 360-400 nm) signals were generated by the 760 nm output of a femtosecond titanium-sapphire laser. A water immersion objective (Fluor, 40X, NA 0.8; Nikon) was used to facilitate imaging the curve ocular surface. The multiphoton image over entire cornea provides morphological information of epithelial cells, keratocytes, and global collagen orientation. Specifically, our planar, large area multiphoton image reveals a concentric pattern of the stroma collagen, indicative of the laminar collagen organization throughout the stroma. In addition, the green fluorescence protein (GFP) labeling contributed to fluorescence contrast of cellular area and facilitated visualizing of inactive keratocytes. Our results show that multiphoton imaging of GFP labeled mouse cornea manifests both morphological significance and structural details. The second harmonic generation imaging reveals the collagen orientation, while the multiphoton fluorescence imaging indicates morphology and distribution of cells in cornea. Our results support that multiphoton microscopy is an appropriate technology for further in vivo investigation and diagnosis of cornea.     

Acousto-optic modulator system for femtosecond laser pulses

Femtosecond laser pulses have made a revolution in multiphoton excitation microscopy, micromachining, and optical storage for their unprecedented high peak power. However, modulation of their intensity with acousto-optic modulator (AOM) is frustrated by dispersion which results in a significant stretch in pulse width. Here we report a scheme composed of two acousto-optic deflectors (AODs) to modulate the intensity of the femtosecond laser pulses with simultaneous compensation for the temporal dispersion. With commercial AODs, we demonstrated such an AOM system for the femtosecond laser pulses with overall transmission efficiency of around 80%. The pulse width of the exit beam is 115-177 fs for an input pulse of 110 fs, across the wavelength range of 720-920 nm when the temporal dispersion compensation is optimally tuned at 800 nm. The fluorescence intensity in a two-photon microscopy experiment performed using this system increased 5.5-fold over that of the uncompensated AOM.     

Multiphoton versus confocal high resolution z-sectioning of enhanced green fluorescent microtubules: increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors

Multiphoton excitation was originally projected to improve live cell fluorescence imaging by minimizing photobleaching effects outside the focal plane, yet reports suggest that photobleaching within the focal plane is actually worse than with one photon excitation. We confirm that when imaging enhanced green fluorescent protein, photobleaching is indeed more acute within the multiphoton excitation volume, so that whilst fluorescence increases as predicted with the square of the excitation power, photobleaching rates increase with a higher order relationship. Crucially however, multiphoton excitation also affords unique opportunities for substantial improvements to fluorescence detection. By using a Pockels cell to minimize exposure of the specimen together with multiple nondescanned detectors we show quantitatively that for any particular bleach rate multiphoton excitation produces significantly more signal than one photon excitation confocal microscopy in high resolution Z‐axis sectioning of thin samples. Both modifications are readily implemented on a commercial multiphoton microscope system.     

Potential of ultraviolet wide-field imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes

Dehydroergosterol (DHE) is an intrinsically fluorescent sterol with absorption/emission in the ultraviolet (UV) region and biophysical properties similar to those of cholesterol. We compared the potential of UV-sensitive low-light-level wide-field (UV-WF) imaging with that of multiphoton (MP) excitation microscopy to monitor DHE in living cells. Significantly reduced photobleaching in MP microscopy of DHE enabled us to acquire three-dimensional z-stacks of DHE-stained cells and to obtain high-resolution maps of DHE in surface ruffles, nanotubes, and the apical membrane of epithelial cells. We found that the lateral resolution of MP microscopy is ～1.5-fold higher than that of UV-WF deconvolution microscopy, allowing for improved spatiotemporal analysis of plasma membrane sterol distribution. Surface intensity patterns of DHE with a diameter of 0.2 μm persisting over several minutes could be resolved by MP time-lapse microscopy. Diffusion coefficients of 0.25-μm-diameter endocytic vesicles containing DHE were determined by MP spatiotemporal image correlation spectroscopy. The requirement of extremely high laser power for visualization of DHE by MP microscopy made this method less potent for multicolor applications with organelle markers like green fluorescent protein-tagged proteins. The signal-to-noise ratio obtainable by UV-WF imaging could be significantly improved by pixelwise bleach rate fitting and calculation of an amplitude image from the decay model and by frame averaging after pixelwise bleaching correction of the image stacks. We conclude that UV-WF imaging and MP microscopy of DHE provide complementary information regarding membrane distribution and intracellular targeting of sterols.     

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.     

Optimizing multiphoton fluorescence microscopy light collection from living tissue by noncontact total emission detection (epiTED)

A benefit of multiphoton fluorescence microscopy is the inherent optical sectioning that occurs during excitation at the diffraction-limited spot. The scanned collection of fluorescence emission is incoherent; that is, no real image needs to be formed on the detector plane. The nearly isotropic emission of fluorescence excited at the focal spot allows for new detection schemes that efficiently funnel all attainable photons to detector(s). We previously showed [Combs, C.A., et al. (2007) Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector. J. Microsc. 228, 330-337] that parabolic mirrors and condensers could be combined to collect the totality of solid angle around the excitation spot for tissue blocks, leading to ～8-fold signal gain. Using a similar approach, we have developed an in vivo total emission detection (epiTED) instrument modified to make noncontact images from outside of living tissue. Simulations suggest that a ～4-fold enhancement may be possible (much larger with lower NA objectives than the 0.95 NA used here) with this approach, depending on objective characteristics, imaging depth and the characteristics of the sample being imaged. In our initial prototype, 2-fold improvements were demonstrated in the mouse brain and skeletal muscle as well as the rat kidney, using a variety of fluorophores and no compromise of spatial resolution. These results show this epiTED prototype effectively doubles emission signal in vivo; thus, it will maintain the image signal-to-noise ratio at two times the scan rate or enable full scan rate at approximately 30% reduced laser power (to minimize photo-damage).     

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.     

Ultracompact autocorrelator for multiphoton microscopy

Pulse temporal characterization is a fundamental task when operating a Ti:Sapphire ultrafast laser system for multiphoton microscopy applications. In the present report, an ultracompact autocorrelator setup and a simple procedure is reported to perform pulse width measurements at the focal plane of the microscope objective without the need of any further instrumentation, aside from a few optical elements, since the confocal microscope, detection, data acquisition, processing, and displaying capabilities are used.     

Multimodal and non‐linear optical microscopy applications in reproductive biology

A plethora of optical techniques is currently available to obtain non‐destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four‐dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567–582, 2016. © 2016 Wiley Periodicals, Inc.     

Optical diagnostics of mercury jet for an intense proton target

An optical diagnostic system is designed and constructed for imaging a free mercury jet interacting with a high intensity proton beam in a pulsed high-field solenoid magnet. The optical imaging system employs a backilluminated, laser shadow photography technique. Object illumination and image capture are transmitted through radiation-hard multimode optical fibers and flexible coherent imaging fibers. A retroreflected illumination design allows the entire passive imaging system to fit inside the bore of the solenoid magnet. A sequence of synchronized short laser light pulses are used to freeze the transient events, and the images are recorded by several high speed charge coupled devices. Quantitative and qualitative data analysis using image processing based on probability approach is described. The characteristics of free mercury jet as a high power target for beam-jet interaction at various levels of the magnetic induction field is reported in this paper.     

Resolution in nonlinear laser scanning microscopy

The lateral and depth resolution of nonlinear microscopy was studied systematically. Nonlinear microscopy can be classified into several categories depending on the coherence properties of the process that generates the imaging signal from the illuminating light, on whether a single- or a two-beam geometry is used, and whether the optical setup is Type I or Type II. An evaluation of the imaging equations shows that (i) lateral and depth resolution improve with increasing nonlinearity, (ii) the differences between coherent and incoherent imaging diminish, and (iii) nonlinear imaging allows depth discrimination in Type I microscopy.     

A compact instrument for adjusting laser beams to be accurately coincident and coaxial and its use in biomedical imaging using wave-mixed laser sources

Biomedical imaging applications that involve nonlinear optical processes such as sum-frequency generation (SFG) and four-wave mixing require that the pulses are synchronized in time and the beams are coaxial to better than 400 μrad. For this reason, folding mirrors are normally used to extend the beam path over a few meters so that detectors can be put into the beams to check their overlap at the start of a long path and also at the end of it. We have made a portable instrument with a footprint of only 22 cm × 11 cm × 16 cm that uses a short focal length lens and a telephoto combination for viewing the near-field and far-field simultaneously. Our instrument is simple to build and use, and we show its application in coherent anti-Stokes Raman scattering microscopy and SFG-based two-photon fluorescence microscopy.     

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.     

A simple introduction to multiphoton microscopy

Multiphoton microscopy is a powerful technique based on complex quantum mechanical effects. Thanks to the development of turnkey mode-locked laser systems, multiphoton microscopy is now available for everyone to use without extreme complexity. In this short introduction, we describe qualitatively the important concepts underlying the most commonly used type of multiphoton microscopy (two-photon excitation). We elucidate how those properties lead to the powerful results that have been achieved using this technique. As with any technique, two-photon excitation microscopy has limitations that we describe, and we provide examples of particular classes of experiments where two-photon excitation microscopy is advantageous over other approaches. Finally, we briefly describe other useful multiphoton microscopy approaches, such as three-photon excitation and second harmonic generation imaging.     

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.