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.     

Frequency domain lifetime and spectral imaging microscopy

In the femtoliter observation volume of a two-photon microscope, multiple fluorophores can be present and complex photophysics can take place. Combined detection of the fluorescence emission spectra and lifetimes can provide deeper insight into specimen properties than these two imaging modalities taken separately. Therefore, we have developed a detection scheme based on a frequency-modulated multichannel photomultiplier, which measures simultaneously the spectrum and the lifetime of the emitted fluorescence. Experimentally, the efficiency of the frequency domain lifetime measurement was compared to a time domain set-up. The performance of this spectrally and lifetime-resolved microscope was evaluated on reference specimens and living cells labeled with three different stains targeting the membrane, the mitochondria, and the nucleus.     

Fluorescence intensity is a poor predictor of saturation effects in two-photon microscopy: artifacts in fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has become an increasingly important measurement tool for biological and biomedical investigations, with the capability to assay molecular dynamics and interactions both in vitro and within living cells. Information recovery in FCS requires an accurate characterization and calibration of the observation volume. A number of recent reports have demonstrated that the calibration of the observation volume is excitation power dependent, a complication that arises due to excitation saturation. While quantitative models are now available to account for these volume variations, many researchers attempt to avoid saturation issues by working with low nonsaturating excitation intensities. For two-photon excited fluorescence, this is typically thought to be achievable by working with excitation powers for which the total measured fluorescence signal maintains its quadratic dependence on excitation intensity. We demonstrate that observing only the power dependence of the fluorescence intensity will tend to underestimate the importance of saturation, and explain these findings in terms of basic physical models.     

Compact non‐contact total emission detection for in vivo multiphoton excitation microscopy

We describe a compact, non‐contact design for a total emission detection (c‐TED) system for intra‐vital multiphoton imaging. To conform to a standard upright two‐photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non‐contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c‐TED device compared to heavily optimized objective‐based emission collection. The best light collection enhancement was seen from murine fat (5×–2× gains as a function of depth), whereas murine skeletal muscle and rat kidney showed gains of over two and just under twofold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a twofold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers, enabled by greater light collection efficiency, yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo‐damage, as well as the applicability of this device to other multiphoton imaging methods is discussed.     

Measurement of two‐photon‐absorption spectra through nonlinear fluorescence produced by a line‐shaped excitation beam

We propose an innovative experimental approach to estimate the two‐photon absorption (TPA) spectrum of a fluorescent material. Our method develops the standard indirect fluorescence‐based method for the TPA measurement by employing a line‐shaped excitation beam, generating a line‐shaped fluorescence emission. Such a configuration, which requires a relatively high amount of optical power, permits to have a greatly increased fluorescence signal, thus avoiding the photon counterdetection devices usually used in these measurements, and allowing to employ detectors such as charge‐coupled device (CCD) cameras. The method is finally tested on a fluorescent isothiocyanate sample, whose TPA spectrum, which is measured with the proposed technique, is compared with the TPA spectra reported in the literature, confirming the validity of our experimental approach.     

Two‐photon microscopy assessment of the overall energy metabolism alteration of amoeba in hypertonic environment

In this study, a two‐photon fluorescence microscopic imaging technique is reported for assessment the effect of dynamic hypertonic environment on the overall energy metabolism alteration and adaptation of soil‐living amoeba Dictyostelium discoideum. For that purpose the fluorescence intensity of mitochondrial reduced nicotinamide adenine dinucleotide (NADH) was monitored and quantified in order to evaluate the corresponded metabolic state of monolayer cultured cells. The two‐photon excitation of NADH with 720 nm near infrared irradiation produced blue fluorescence emission with maximum wavelength centered at 460 nm. The benefits of reported noninvasive microscopic technique are the significantly less cellular damage and avoiding the excitation of other biomolecules except of NADH. It enabled to acquire data for NADH levels of the observed cells on agar plate specimen and hypertonic nutrition media in a Petri dish. The method demonstrated also good sensitivity, reproducibility and the obtained results revealed that D. discoideum species form aggregation in hypertonic environment within several minutes with aim to survive. The formed aggregate had amorphous shape and it consisted from dozen amoeba cells, which kept their NADH amount in constant level for few hours. The reported imaging method might be applicable in various studies for characterization of metabolic events and assessment of the cell energy balance in hypertonic environment.     

Intravital dual-colored visualization of colorectal liver metastasis in living mice using two photon laser scanning microscopy

A major challenge of cancer biology is to visualize the dynamics of the metastatic process in secondary organs at high optical resolution in vivo real-time. Here, we presented intravital, dual-colored imaging of liver metastasis formation from a single cancer cell to metastatic colonies in the living liver of living mice using two photon laser scanning microscopy (TPLSM). Red fluorescent protein expressing murine (SL4) or human (HT29) colorectal cancer cell lines were inoculated to the spleen of green fluorescent protein expressing mice. Intravital TPLSM was performed by exteriorizing and fixing the liver lobe of living mice. This was repeated several times for the long-term imaging of the same mouse. Viable cancer cells in the living liver of living mice were visualized intravitally at a magnification of over 600×. Single cancer cells were arrested within hepatic sinusoids 2 h after injection. Platelet aggregation surrounding a cancer cell was observed, indicating a phenomenon of tumor-cell induced platelet aggregation. Cancer cells were extravasated from hepatic sinusoids to the space of Disse. Protrusions of Kupffer cells surrounding a cancer cell were observed, indicating that Kupffer cells appear to phagocytose cancer cells. SL4 cells formed liver metastatic colonies with extensive stromal reaction. Liver metastases by HT29 cells were observed as a cluster of micrometastatic nodules. High-resolution, dual-colored, real-time visualization of cancer metastasis using intravital TLPSM can help to understand spatiotemporal tumor-host interactions during metastatic processes in the living organs of living animals.     

一种新的高分子聚合物光限幅特性的实验研究

报道了一种具有σ-π电子共轭轨道的硅聚合物的光限幅特性.在532nm波长,测量了不同浓度下样品的透射率与光强的关系.实验结果指出该聚合物具有较好的光限幅特性.利用Z扫描方法测定了该样品的有效双光子吸收系数β=3.5×10^-9cm/W.     

Quasi-spherical focal spot in two-photon scanning microscopy by three-ring apodization

We present a beam-shaping technique for two-photon excitation (TPE) fluorescence microscopy. We show that by inserting a properly designed three-ring pupil filter in the illumination beam of the microscope, the effective optical sectioning capacity of such a system improves so that the point spread function gets a quasi-spherical shape. Such an improvement, which allows the acquisition of 3D images with isotropic quality, is obtained at the expense of only a small increase of the overall energy in the axial sidelobes. The performance of this technique is illustrated with a scanning TPE microscopy experiment in which the image of small beads is obtained. We demonstrate an effective narrowing of 12.5% in the axial extent of the point spread function, while keeping the 82% of the spot-fluorescence efficiency.     

Spectra and lifetimes of fluorescence resonance energy transfer fluorophores under two-photon excitation

We show two-photon spectra and lifetimes acquired using conventional confocal microscopes equipped with an ultra-short pulsed laser and a time-gated intensified charge coupled device. We report on the two-photon spectra and lifetimes of Alexa350, enhanced green fluorescent protein (EGFP), EGFP-CD46, and Cy3 labelled antibodies. Cellular and extracellular EGFP two-photon spectra and lifetimes are compared.     

双光子三维微细加工技术及实验系统的开发

介绍了一种新型的三维微细加工技术,描述了自行开发的双光子微细加工实验系统.双光子三维微细加工技术利用材料与飞秒激光束在焦点局域发生的双光子激发,通过逐点扫描,实现微器件的三维成型.由于材料发生双光子激发的几率与激发光强的平方成正比,所以具有极高瞬时光强的飞秒激光器和可以对光束进行强聚焦的显微镜装置成为系统的关键部件,对此进行了详细的说明.最后,给出了部分利用上述加工系统所获得的初步实验结果.     

A deep tissue fluorescence imaging system with enhanced SHG detection capabilities

We describe a novel two‐photon fluorescence microscopy system capable of producing high‐quality second harmonic generation (SHG) images in thick turbid media by using an innovative detection system. This novel detection system is capable of detecting photons from a very large surface area. This system has proven effective in providing images of thick turbid samples, both biological and artificial. Due to its transmission detection geometry, the system is particularly suitable for detecting SHG signals, which are generally forward directed. In this article, we present comparative data acquired simultaneously on the same sample with the forward and epidetection schemes. Microsc. Res. Tech. 77:368–373, 2014. © 2014 Wiley Periodicals, Inc.     

Calcium silicate cement‐induced remineralisation of totally demineralised dentine in comparison with glass ionomer cement: tetracycline labelling and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy, in combination with tetracycline labelling, was used to observe the remineralising potentials of a calcium silicate‐based restorative material (Biodentine^TM) and a glass ionomer cement (GIC:?Fuji?IX) on totally demineralised dentine. Forty demineralised dentine discs were stored with either cement in three different solutions: phosphate buffered saline (PBS) with tetracycline, phosphate‐free tetracycline, and tetracycline‐free PBS. Additional samples of demineralised dentine were stored alone in the first solution. After 8‐week storage at 37 °C, dentine samples were imaged using two‐photon fluorescence microscopy and Raman spectroscopy. Samples were later embedded in PMMA and polished block surfaces studied by 20 kV BSE imaging in an SEM to study variations in mineral concentration. The highest fluorescence intensity was exhibited by the dentine stored with Biodentine^TM in the PBS/tetracycline solution. These samples also showed microscopic features of matrix remineralisation including a mineralisation front and intra‐ and intertubular mineralisation. In the other solutions, dentine exhibited much weaker fluorescence with none of these features detectable. Raman spectra confirmed the formation of calcium phosphate mineral with Raman peaks similar to apatite, while no mineral formation was detected in the dentine stored in cement‐free or PBS‐free media, or with GIC. It could therefore be concluded that Biodentine^TM induced calcium phosphate mineral formation within the dentine matrix when stored in phosphate‐rich media, which was selectively detectable using the tetracycline labelling.     

Effects of aberrations and specimen structure in conventional, confocal and two-photon fluorescence microscopy

Specimen-induced aberrations cause a reduction in signal levels and resolution in fluorescence microscopy. Aberrations also affect the image contrast achieved by these microscopes. We model the effects of aberrations on the fluorescence signals acquired from different specimen structures, such as point-like, linear, planar and volume structures, when imaged by conventional, confocal and two-photon microscopes. From this we derive the image contrast obtained when observing combinations of such structures. We show that the effect of aberrations on the visibility of fine features depends upon the specimen morphology and that the contrast is less significantly affected in microscopes exhibiting optical sectioning. For example, we show that point objects become indistinguishable from background fluorescence in the presence of aberrations, particularly when imaged in a conventional fluorescence microscope. This demonstrates the significant advantage of using confocal or two-photon microscopes over conventional instruments when aberrations are present.     

Sequential multitrack nonlinear ex vivo imaging of esophageal stroma based on backscattered second-harmonic generation and two-photon autofluorescence

We demonstrate the technique of subsequent multitrack nonlinear imaging based on backscattered second-harmonic generation (B-SHG) and two-photon autofluorescence (TPA) to obtain large-area, high-contrast, submicron-resolution image ex vivo of esophageal stroma. Our findings show that this technique is effective in improving the B-SHG/TPA image contrast. It was found that the method can quantitatively obtain microscopic structural and biochemical information on stroma. Our work suggests that the technique has the potential to provide accurate and comprehensive information in determining the physiological and pathological states of the esophagus.     

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.     

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.     

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.     

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.