Multimodal Nonlinear Optical Microscopy.

3.

Experimental realization of multimodal

NLO imaging on a CARS microscope 3.1 CARS microscope

The development of CARS microscopy has been extensively reviewed [ 5 – 8 , 10 , 31 , 33 , 72 – 75 ]. Here we focus on the developments towards multimodal imaging. The block diagram of a modern high-speed, multifunctional CARS microscope is shown in Fig. 2 . The key technological steps, which opened CARS microscopy for biomedical applications over the past decade, include: (1) Collinear beam geometry, which significantly simplifies the beam configuration; (2) Laser scanning on a confocal platform, which allows fast CARS imaging with speed as high as video rate; (3) Backward (epi) detection, which enables CARS imaging of sub-wavelength features and tissues in live animals; (4) Development of robust laser sources. Over the past decade, several generations of laser systems have been developed for CARS microscopy. It is important to note that the choice of pulse duration for CARS microscopy is a compromise between CARS signal strength and specificity as first shown analytically by Cheng et al. [ 33 ]. Shorter broadband pulses produce stronger signal but also stronger non-resonant background, while longer pulses generate lower signal but better spectral resolution and specificity. High-speed CARS imaging was first demonstrated with ps lasers [ 76 , 77 ]. Although ps pulse excitation gives better contrast, the femtosecond (fs) laser sources have higher peak power that is required to generate strong NLO signals for one-beam modalities [ 78 ]. Towards the goal of coupling CARS with widely used multiphoton fluorescence microscopes, Chen et al. [ 78 ] and Pegoraro et al. [ 73 ] demonstrated multimodal NLO imaging on a CARS microscope with fs laser pulses. Rocha-Mendoza et al. [ 79 ] applied spectral focusing of broadband fs pulses [ 80 ] to achieve high spectral resolution in CARS imaging. More recently, Lu et al. demonstrated that a unique dual 4-f paired-gratings spectral filtering of a fs laser could easily switch between ps and fs pulse duration, which rendered high contrast CARS imaging and high quality multiphoton imaging, respectively [ 81 ]. Performances of CARS microscopy using ps and fs laser systems were recently compared by Svedberg et al. [ 82 ]. In general, fs system can provide the same CARS intensity with about 60 times lower excitation powers compared to the ps system [ 82 ]. Meanwhile, it should be emphasized that the spectral resolution in fs CARS is much reduced and this method is suitable for isolated Raman bands. A modern CARS microscope can be operated using either ps or fs laser systems, including two synchronized Ti:sapphire lasers [ 33 , 83 ], optical parametric oscillators (OPOs) pumped with solid state lasers [ 77 , 78 , 84 , 85 ] or with fiber lasers [ 86 ], several non-linear fiber based systems [ 87 – 92 ] and an Er:fiber based all fiber system [ 93 ]. The Ti:sapphire lasers provide the capability of wide tuning and availability of ps and fs configuration in the same cavity. The drawback of this system is the timing jitter of electronic synchronization. The OPO-based laser systems completely resolve the timing jitter problem, because the OPO pulse train is inherently synchronized with pump laser pulse train. A remaining drawback of the free space lasers and OPOs is their high cost and large volume. Therefore, fiber-based solutions are attractive because of less expensive and more compact laser sources. Currently, the average power of the Stokes beam for fiber-based systems is limited to about 10 mW. A relatively large power for the pump beam of shorter wavelength is then necessary to achieve high speed CARS imaging. Such configurations however may increase the possibility of tissue damage.

Coupling CARS with TPEF and SHG

A challenge for multimodal imaging is simultaneous acquisition of various NLO signals in a convenient way. Such challenge can be dealt with by utilizing the coherence properties of the NLO signals. Due to the coherent addition, majority of the CARS signal goes forward and is highly directional. As shown by Cheng et al . [ 94 ] and Volkmer et al . [ 95 ], the forward CARS (F-CARS) signal is highly directional and can be efficiently collected by a NA=0.55 air condenser. Because the fluorescence signal is spectrally broad and incoherent, it is negligible after the narrow bandpass CARS filters and the air condenser in the forward channel. Instead, the backward fluorescence can be effectively collected by the laser-focusing objective with high numerical aperture (NA). Therefore, one is able to record the forward CARS signal and backward TPEF signal simultaneously using the two external photomultiplier tubes (PMTs), as shown in Fig. 2 . Due to the Gouy phase shift, the forward SHG signal is deflected from the optical axis [ 96 ] and is fairly weak if a low-NA air condenser is used. Meanwhile a significant amount of SHG photons from either collagen fibrils or protein crystals are back scattered [ 37 , 97 ]. Thus, the forward CARS and backward SHG signals can be simultaneously detected by using bandpass filters and the external PMTs. Because the TPEF signal is red-shifted from the SHG signal, the SHG and TPEF signals can be spectrally separated. Huff et al . [ 98 ] demonstrated that with two backward detectors, dichroic mirror and appropriate bandpass filters SHG (SFG) and TPEF signals can be detected simultaneously with forward CARS signal.

Implementation of THG modality

The optical properties of tissues, sensitivity of detectors, and transmission of optics together restrict the THG signal wavelength to be longer than 350 nm. Fortunately, CARS requires no electronic resonance, and thus NIR excitation wavelengths above 1.0 μm can be used, such as the 1064 nm beam provided by high-Q laser. Such configuration opens the opportunity to couple CARS with THG imaging. Because THG signal depends on the cube of excitation beam intensity, fs laser system is usually required to produce high enough peak power to generate strong THG signal. Using fs pulses, Chen et al. [ 78 ] demonstrated the use of the second harmonic of the OPO idler beam at 1018 nm as the Stokes beam for CARS, and the OPO signal beam at 1290 nm for THG imaging on the same platform. It is also important to note that THG signal from interfaces is highly directional and is generated in the forward direction, as shown by Cheng et al. [ 40 ].

Implementation of two-photon luminescence

(TPL) and three-photon luminescence (3PL) Implementation of TPL modality is similar to that of TPEF, because TPL has the same excitation and emission characteristics as TPEF. Using a femtosecond Ti:sapphire laser, Wang et al . reported TPL imaging of single gold NRs in vitro and in vivo [ 49 ]. Emission spectrum of 3PL is similar to that of TPL, but 3PL needs longer excitation wavelength. Tong et al. have recently used a femtosecond OPO to implement 3PL imaging of gold-silver nanocages [ 99 ].

Implementation of electronic-resonance-enhanced FWM modality

The electronic-resonance-enhanced FWM is a two-beam modality with emission similar to CARS, however unlike CARS it is originated from broad electronic and/or plasmonic resonances, which usually have dephasing time on the fs time scale [ 100 ]. Therefore, fs lasers are preferred over ps ones for optimizing the efficiency of the electronic-resonance-enhanced FWM process, as has been demonstrated by Jung et al. [ 51 ] and discussed in the recent review by Wang et al. [ 11 ]. A single-beam near-degenerate FWM microscopy has been demonstrated by Min et al. [ 101 ] and applied to ultrasensitive detection of chromophores and hemoglobin. Implementation of SRS modality Because SRS and CARS occur simultaneously, it is natural to implement SRS imaging on a CARS microscope. The SRS signal originates from an intensity gain of the Stokes beam (SRG setup) or an intensity loss of the pump beam (SRL setup). The small change in intensity of the detected beam due to SRS process is buried in the noise, and so phase-sensitive detection via a lock-in amplifier is required to recover the signal. For high-speed detection of SRG signal, the intensity of pump beam is modulated, usually at MHz frequency, and the intensity of Stokes beam is detected with large area photodiode (see Fig. 2 ). As in any heterodyne detected processes, the noise in the detected beam limits the ultimate sensitivity. Freudiger et al. [ 14 ] and Nandakumar et al. [ 13 , 15 ] have achieved high-speed SRS microscopy by using high-frequency modulation (>1 MHz) that avoids the low-frequency laser noise. Ozeki et al. compared the signal-to-noise ratio in CARS and SRS [ 12 ]. It is important to understand that the detection strategy is different for these modalities: (i) While CARS signal appears at a new wavelength, the SRS signal is detected at the exact wavelength of pump or Stokes beams; (ii) The CARS beam is highly directional, and can be effectively collected with low numerical aperture (NA) collection optics. In contrast, the SRS beam exactly follows pump and Stokes beams, and therefore, the focusing and collection optics with similar NA are required for optimal collection of SRS signal; (iii) The epi-detected CARS signal arises from small objects or from reflection of the forward signal, whereas the epi-detected SRS solely relies on the back-scattered photons. Because the SRS signal has the same wavelengths as the excitation beams, polarization separation is used for epi-SRS detection [ 14 , 59 ]; (iv) In contrast to CARS, the phase matching conditions of SRS are always satisfied, which eliminates the need for tight focusing. Moreover, in the case of Gaussian pump and Stokes beams focused collinearly through a sample, Owyoung and Jones [ 57 ] derived in 1977 an analytical expression for the SRS signal intensity, which is independent of focusing conditions and linear to both pump and Stokes powers. In microscopy, this independence of focusing conditions has been demonstrated by Slipchenko et al. [ 59 ], who imaged large particles in pharmaceutical solid dosage forms using low NA objective. Although the SRS signal is free from the non-resonant background, there are a number of optical processes, including stimulated absorption or emission [ 18 , 69 ], the optical Kerr effect, cross phase modulation, and the thermal lensing effect [ 102 ], which could potentially produce the background. In practice, stimulated absorption or emission [ 18 , 69 ] can be minimized by using longer excitation wavelengths. The optical Kerr effect and cross phase modulation can be minimized using frequency modulation instead of amplitude modulation [ 103 ]. Thermal lensing effect [ 102 ] can be minimized by using a high NA objective for signal collection [ 14 , 104 ] or by high-frequency modulation (> 1 MHz) [ 105 , 106 ]. Most SRS experiments have been done with crystal-based OPOs, but a recent paper showed the promise of using a fiber laser for SRS imaging [ 107 ].

Implementation of pump-probe and photothermal modalities

The presence of two excitation beams in CARS microscope also opens opportunities for developing pump-probe microscopy. Since the pump-probe technique measures the modulation of the probe beam intensity induced by the interaction of the modulated pump beam with a sample, the implementation of pump-probe modality is similar to SRS and can be realized by phase-sensitive detection [ 18 , 69 ]. In previous work, photothermal microscopy was done with cw lasers [ 20 ]. The pulsed laser on a CARS microscope enabled photothermal imaging based on two-photon absorption [ 108 ].

Coupling CARS and SRS with spontaneous Raman

A drawback of single-frequency CARS and SRS microscopy is that each image only contains information from a single Raman shift. The lack of spectral information can be partially compensated by acquiring Raman spectra at the points of interest in a CARS or SRS image. To realize Raman microspectroscopy on the CARS microscope, the laser source should have sufficient spectral resolution, and therefore ps laser source is optimal. In case of fs laser system, the spectral bandwidth can be reduced by narrowband filter. Slipchenko et al. [ 104 ] first realized Raman microspectroscopy on a ps laser based CARS microscope by adding a spectrometer to one of the optical ports of the microscope and using appropriate filters to reject the scattering of the excitation laser. To reject background signal and achieve 3-D spatial resolution, the spectrometer slit assembly has been replaced with a pinhole. Due to NIR nature of the Raman signal, the spectrum obtained by charge-coupled device (CCD) might show strong etalon effect. Such effect can be avoided by using front-illuminated or deep-depleted CCD detectors. Coupling of CARS and Raman has been used for spectral analysis of lipids in C. elegans [ 109 ], drug tablets [ 59 ], and cellulose fibers [ 110 ].

4.1.

Study of white matter

White matter of the central nervous system

(CNS) is enriched in myelin sheath, which is a multi-lamellar oligodendroglial cell membranes wrapping around axons [ 111 ]. The lamellar structure of myelin sheath has been characterized by traditional methods including electron microscopy, histology, and immunofluorescence [ 112 ]. However, these methods can only examine fixed tissues, and so prevent dynamic studies of cellular activities during the disease process. Clinical imaging tools, such as magnetic resonance imaging (MRI), have been used for in vivo imaging of white matter, but they lack single-cell resolution [ 75 ]. By taking advantage of high lipid content in myelin sheath (~70% lipid by weight) [ 113 ], Wang et al . in 2005, for the first time, reported the application of CARS microscopy to label-free visualization of myelin sheaths under physiological conditions [ 114 ]. Owing to submicron resolution of CARS microscopy, detailed structures of the node of Ranvier and Schmidt-Lanterman incisure were resolved [ 114 ]. In the past several years, the integration of multiple NLO modalities on the same CARS microscopic platform has enabled CARS imaging of myelin sheath, SFG or SHG imaging of astrocyte processes and microtubules, and TPEF imaging of other important biological components (e.g. calcium activity) with the aid of fluorescence probes, simultaneously [ 98 , 115 – 118 ]. The interactions between myelinated axons and other essential components (e.g. astrocyte processes, calcium activity) in the white matter were directly visualized. These capabilities of multimodal NLO microscopy open up new opportunities to investigate the pathological pathways of demyelinating diseases and explore better therapeutic strategies for the diseases. Demyelination, or the loss of myelin sheath around the axons, impairs action potential conduction along the axons, which in turn accounts for numerous neurological disorders including multiple sclerosis, and spinal cord injury [ 112 , 113 ]. The molecular mechanisms underlying myelin damage, however, is not well understood. By using multimodal NLO microscopy, Fu et al . have carried out mechanistic studies of myelin damage in different demyelination models [ 115 , 116 ]. By incorporating TPEF imaging of the calcium activity indicator with CARS imaging of myelin sheath, Lysophosphatidyl choline (Lyso-PtdCho) was shown to not only induce myelin swelling but also cause Ca 2+ influx into the axons [ 116 ]. In another study, Fu et al . first used CARS imaging to show that glutamate induced paranodal myelin splitting and retraction [ 115 ]. TPEF imaging and immunohistochemistry were then used to reveal that glutamate inhibits axonal conduction via breaking axo-glial junctions and exposing juxtaparanodal K + channels [ 115 ]. As one of the neurological disorders, spinal cord injury (SCI) causes immediate disruption to neuronal membranes and Ca 2+ influx into axons, followed by subsequent secondary damages [ 119 ]. A key therapeutic strategy for SCI is sealing the damaged membranes at an early stage [ 120 , 121 ]. Recently, Shi et al . used multimodal NLO microscopy to evaluate the effectiveness of copolymer micelles in repairing SCI in vivo [ 122 ]. In this study, CARS imaging was used to monitor the morphological changes of myelin sheath after injury and treatments with different polymers. TPEF imaging was used to map the calcium distribution in myelinated axons [ 122 ]. As shown in Fig. 3 , intraaxonal free Ca 2+ level was much higher in compression-injured spinal cord ( Fig. 3b ) than that in healthy one ( Fig. 3a ). With low-concentration micelle treatment, injured spinal cord showed significantly reduced Ca 2+ influx into axons ( Fig. 3c ). To summarize, the capability of simultaneous observation of various components and their interactions in the white matter makes multimodal NLO microscopy an attractive tool for the studies of neurological diseases. Future studies focusing on in vivo multimodal NLO imaging of the white matter would significantly contribute to the understanding of the dynamics of disease progression.

4.2.

Study of lipid metabolism 4.2.1 Lipid droplet biology Lipid droplets

(LDs) are complex proteolipid organelles that often play dynamic roles in various aspects of lipid metabolism [ 123 – 125 ]. The aberrant accumulation of LDs is associated with some of the most widespread human diseases, such as obesity, diabetes type II, and atherosclerosis [ 123 – 125 ]. With the capability of label-free visualization of LDs, CARS microscopy has been used to monitor LDs dynamics in many systems [ 126 – 130 ]. By combining CARS with TPEF imaging, further in-depth studies on LD biology have been conducted. Nan et al. revealed the interaction between LDs and mitochondria, which were labeled with fluorescent probe, in adrenal cortical cells [ 130 ]. By coupling the immunostaining of LDs-associated proteins, Yamaguchi et al. found that Comparative Gene Identification-58 protein facilitated lipolysis of LDs in cooperation with perilipin [ 128 ]. With the help of fluorophore-labeled insulin, Le et al. unraveled that insulin accounted for the phenotypic variability in LDs accumulation among clonal cells [ 126 ]. By utilizing curcumin autofluorescence, Kim et al. found that curcumin could inhibit adipocyte differentiation [ 129 ]. Through live cell fluorescence imaging of viral RNA and simultaneously CARS imaging of LDs, Lyn et al. highlighted the significance of lipid metabolism in viral replication [ 127 ]. Given that fluorescent proteins-tagged LDs-associated proteins have been made available [ 131 ], simultaneous CARS and TPEF imaging would be able to provide new insights into the dynamic association between lipids and proteins, which are critical for LDs formation and function in living cells.

Lipid metabolism in model organism Although nematode

C. elegans is distant from mammals, the pathways of lipid metabolism in human are highly conserved in C. elegans [ 132 , 133 ]. Highly tractable genetics that are critical in lipid metabolism make C. elegans an attractive model for the integrative studies of lipid metabolism regulation and related metabolic diseases [ 134 , 135 ]. The transparent nature of C. elegans facilitates the studies of lipid storage by microscopy-based methods [ 136 ]. Traditionally, the lipid stores in intestinal and hypodermal cells are visualized by staining with lipophilic dyes including Sudan black and Nile Red. However, the labeling efficiency and accuracy have recently been challenged [ 136 ]. Although fixative staining methods provide reproducible data, their results may be interfered with fluorescence signals from the autofluorescent particles in lysosome-related organelles [ 137 , 138 ]. Feeding worms with vital dyes was thought to monitor dynamics of lipid storage, but the vital dyes have been recently shown to be just sequestrated in autofluorescent particles, rather than label any fat stores in intestine or hypodermis [ 137 , 139 ]. Moreover, the dynamic changes of lipid composition for individual LDs are not possible to be measured with traditional analytical chemistry methods such as gas chromatography. Therefore, a non-invasive and label-free method to quantitatively analyze lipid storage and composition in living C. elegans is critically needed. Several pilot studies showcase the capability of CARS microscopy for label-free imaging of lipid storage in living C. elegans . As the first study, Hellerer et al. demonstrated that CARS microscopy not only overcame the limitations of fluorescent staining methods, but also accurately assessed the impact of genetic deficiencies in lipid metabolism (e.g. insulin signaling pathway) on lipid storage, distribution, and packing density [ 140 ]. In a follow-up study by Morck et al ., CARS imaging did not detect any cholesterol lowering drug-induced reduction of lipid storage as detected by Nile Red staining [ 141 ]. More recently, by combining CARS imaging with TPEF imaging, Le et al. and Yen et al. revealed two distinctive lipid species in C. elegans : a neutral lipid species in both intestine and hypodermis and an autofluorescent particle species only found in intestine [ 109 , 137 ]. Le et al. also found that genetic mutations of lipid signaling pathways including peroxidation and desaturation altered the accumulation of neutral lipids and autofluorescent particles ( Fig. 4a–b ) [ 109 ]. Other than C. elegans , some other model organisms, such as zebrafish and yeast Saccharomyces cerevisiae, are also gaining popularity in lipid research, as shown by and Holtta-Vuori et al. [ 142 ] and Brackmann et al. [ 143 ] (for review see [ 144 ]). By coupling confocal Raman microspectroscopy with CARS and TPEF imaging, the chemical composition of neutral lipids and antofluorescent particles can be characterized. The intensity ratio between Raman bands at 1660 cm −1 and 1445 cm −1 has been used as a reliable measure of lipid chain unsaturation [ 109 ]. As shown in Fig. 4d , neutral lipid droplets in eggs, whose structure is depicted in Fig. 4c , have higher level of lipid chain unsaturation compared with those in adult intestine. Interestingly, the ester bond around 1742 cm −1 is absent in autofluorescent particles, which suggests that these particles do not contain neutral lipids. Moreover, the prominent Raman bands for phenytalanine around 1004 cm −1 , for amide III region 1225–1350 cm −1 , for amide I around 1660 cm −1 , and for CH 3 bonds around 2940 cm −1 imply that the autofluorescent particles are rich in proteins rather than lipids. Taking advantage of recent advances in C. elegans genetics [ 134 ] and microfluidic chamber-based lifelong C. elegans imaging [ 145 ], it is foreseeable that multimodal NLO microscopy presents an unprecedented opportunity to study dynamic lipid regulation in living C. elegans .

Intestinal lipid absorption

Intestinal lipid absorption happens in enterocytes, where free fatty acids (FFAs) and monoacylglycerols are resynthesized into triacylglycerols (TGs) and packaged into the chylomicrons for export into the lymphatic system through exocytosis. [ 146 ]. Traditionally, the process of intestinal lipid absorption was visualized by histology and electron microscopy, which hinder the investigation of the dynamic process. CARS microscopy has been first used by Zhu et al. to reveal a dynamic, cytoplasmic triacylglycerol pool in enterocytes, which challenges the conventional paradigm [ 147 ]. In the follow-up studies by Lee et al. , the role of diacylglycerol acyltransferase 1, a key enzyme in TG biosynthesis, in dietary fat absorption was carefully studied [ 148 ]. Moreover, TPEF imaging of lipid droplet associated proteins was recently combined with CARS imaging to study the association of the proteins with cytoplasmic LDs in mouse enterocytes during dietary fat absorption [ 149 ]. Future applications of multimodal NLO microscopy are expected to contribute new knowledge on the impact of genes and enzymes on intestinal lipid absorption, and the responses of drugs for reduction of energy intake.

4.5.

Study of skin

As the soft outer covering, skin performs various functions including protection, heat regulation, sensation, storage and synthesis [ 178 ]. Because many components in the skin are rich in lipids, Evans et al. for the first time demonstrated CARS imaging of the lipid-rich structures including corneocytes, sebaceous glands, and subcutaneous adipocytes in the skin of a live mouse [ 77 ]. Label-free and high-speed CARS imaging enabled real-time monitoring of time-dependent diffusion of mineral oil in the skin [ 77 ]. Freudiger et al. furthered demonstrated SRS imaging of mouse ear skin with high sensitivity and no nonresonant background [ 14 ]. More recently, by combining SHG imaging with CARS imaging, Zimmerley et al. found that DMSO gradually caused morphological changes to the collagen type I matrix in the skin [ 179 ]. By combining confocal Raman microspectroscopy with CARS imaging, Slipchenko et al. showed that the lipids accumulated in sebaceous glands had much higher saturation level and lipid packing density compared with those in adipocytes [ 104 ]. These studies demonstrate the ability of multimodal NLO microscopy to evaluate the effects of chemical compounds, such as skin care product, on the skin tissue.

4.8.

Study of biomass conversion

Biomass conversion provides alternative ways to produce energy [ 183 ]. However, the biomass conversion kinetics has not been well understood yet. Very recently, Saar et al. used SRS microscopy to map the distribution of two important chemical species in the biomass conversion process, lignin and cellulose, in corn stover, without labeling [ 60 ]. They further showed real-time SRS imaging of a delignification reaction in corn stover ( Fig. 6 ). This study suggests that SRS imaging holds the promise for more thorough studies of biomass conversion process.

4.10. NLO imaging of nanomaterials in vitro and in vivo Owing to unique optical properties, nanomaterials have been extensively used as probes for live cell and animal imaging [ 49 , 189 – 192 ]. The strong intrinsic nonlinearity of these nanomaterials has recently caught substantial attentions [ 193 – 195 ]. In this section, we discuss the application of electronic-resonance-enhanced FWM and transient absorption microscopy to imaging various types of nanomaterials.

Gold nanostructures

Despite the wide use of gold nanostructures as multiphoton luminescent imaging agents [ 193 ], the broad fluorescent spectral profile makes gold nanostructures difficult to be distinguished from other fluorescent molecules. In contrast, electronic-resonance-enhanced FWM signals from various gold nanostructures including coupled gold nanoparticles [ 196 ], gold NRs [ 51 ], and gold NWs [ 53 ] have shown narrow spectral profiles at well defined frequencies. In the study of gold NR, Jung et al. found that FWM signal was enhanced not only by surface plasmon resonance, but also by aggregation of NRs [ 51 ]. In another study by Kim et al. , gold zigzag NWs exhibited a strong polarization-dependent FWM signal [ 53 ]. Such strong FWM responses from gold nanostructures are intriguing for future applications of these nanostructures in biomedical imaging. Silicon nanowire (SiNW) SiNW is a nanomaterial with precisely controllable size, shape, and surface chemistry. Very recently, Jung et al. found intrinsically intensive and stable FWM and THG emissions from SiNWs as small as 5 nm with the narrow and well defined spectral profiles ( Fig. 8a–b ) [ 52 ]. The strong FWM signals from SiNWs permitted real-time monitoring of the circulation of SiNWs in the peripheral blood of a live mouse. The biodistribution of SiNWs in various tissues is shown in Fig. 8c–e [ 52 ]. SiNWs is anticipated to be a novel agent for deep tissue and in vivo imaging. The interactions between nanomaterials and biological cells or tissues could be addressed by combining electronic-resonance-enhanced FWM microscopy with well-established TPEF, CARS and SFG microscopy.

Carbon nanotube

Owing to intrinsic optical properties, carbon nanotubes have been visualized in biological environment by Raman [ 197 – 199 ] and photoacoustic [ 200 ] microscopy. Recently, Kim et al. utilized FWM microscopy to examine individual carbon nanotubes [ 54 ]. When the excitation was resonant with electronic transitions of the nanotube, SWNTs exhibited large FWM contrast at the anti-Stokes frequency [ 54 ]. Jung et al. have used transient absorption microscopy to distinguish semiconducting and metallic SWNTs [ 68 ]. With the capability of detecting the metallic state in individual SWNTs, transient absorption microscopy holds a great potential for screening of carbon nanotubes in nano-electronics applications.