Advances in nonlinear optical microscopy for biophotonics

Rui Li; Xinxin Wang; Yi Zhou; Huan Zong; Maodu Chen; Mengtao Sun

doi:10.1117/1.JNP.12.033007

14 August 2018 Advances in nonlinear optical microscopy for biophotonics

Rui Li, Xinxin Wang, Yi Zhou, Huan Zong, Maodu Chen, Mengtao Sun

Author Affiliations +

Journal of Nanophotonics, Vol. 12, Issue 3, 033007 (August 2018). https://doi.org/10.1117/1.JNP.12.033007

Abstract

This article reviews recent advances in nonlinear optical microscopy for biophotonics, including stimulated Raman scattering (SRS), coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), second harmonic generation (SHG), and sum frequency generation (SFG). We first introduce the principles and applications of SRS, CARS, TPEF, SHG, and SFG and their individual applications for biophotonics. We then introduce the combination of SRS, CARS, TPEF, SHG, and SFG microscopy for biophotonics. Our review not only summarizes the recent advances in nonlinear optical microscopy but also can deepen the understanding of the combination of these types of nonlinear optical microscopy for biophotonics.

1. Introduction

Over the past decade, the nonlinear optical methods have become widely used tools for biomolecular detection, medical diagnosis in cells or tissues at the micrometer and nanometer level.¹ Advancement of these optical methods promotes and enhances basic research in biology, pharmacy, and medicine.²^–⁴ Compared with the optical method, radiation is usually applied for imaging modalities, including x-rays, computed tomography, plain radiography, magnetic resonance imaging, and other nuclear imaging methods.⁵ However, these techniques are costly and emit radiation. For disease detection and diagnoses and cells development process, optical imaging methods can provide molecular information on human tissues with noninvasive, real-time, accurate, sensitive, and economic properties, as discussed in recent reviews.⁶ When human diseases develop, cells and tissues are used to study the genetics, drug screening, and disease control via optical imaging techniques.⁷^–⁹ Among optical imaging technologies, the nonlinear optical methods have great advantages such as noninvasiveness, depth penetration, high sensitivity, and ultrahigh resolution. The nonlinear signal is generated via nonlinear optical microscopy in a small area (measured in nanometers) in the focal plane of the objective lens using a pico- or femtosecond near-infrared pulsed excited laser. This technology noninvasively enables tissue depth penetration, image sensitivity, and high resolution, as discussed in recent reviews.¹⁰^–¹²

Combined various fluorescent dye labels¹³^,¹⁴ and two-photon excited fluorescence (TPEF) microscopy are potentially used in laboratories and clinics to analyze live cells and accurately localize and completely resect live tumors.¹⁵ Live cells and tissues also can be imaged without external labeling by directly tracking different chemical bonds or proteins with nonlinear optical microscopes such as stimulated Raman scatting (SRS),¹⁶^–¹⁹ coherent anti-Stokes Raman scattering (CARS), TPEF, second harmonic generation (SHG), and sum frequency generation (SFG), among other techniques.

In this review, we will discuss several nonlinear methods began to be used in cells and tissues optics, such as SRS,¹⁶^,²⁰ CARS,⁸^,²¹ TPEF,²² SHG,²³ SFG,²⁴ and multiple combinations of techniques. Although it is not comprehensive, an overview of nonlinear optics method applications with representative references is given in Table 1.

Table 1

Representative example applications of nonlinear optical microscopy.

Nonlinear optics method	Tissues or cells
TPEF	Ovarian cancer²⁵
	Brain⁸^,²¹
	Mouse L929 fibroblastic⁷
	Mouse sciatic nerve²⁶
	Mouse living²⁷
	Axonal myelin²⁸
	Neonatal cardiomyocyte²³^,²⁹
	HeLa cells²²
	C. elegans⁹
	Oocyte, embryo, egg⁴
CARS	Brain⁸^,²¹
	Mouse L929 fibroblastic⁷
	C. elegans⁹
	Axonal myelin²⁸
	Oocyte, embryo, egg⁴
	Human stem cells³⁰
	Human mastoid cortex¹¹
	Breast and prostate cancer cells³¹
SRS	Brain¹⁶^,²⁰
	Mouse sciatic nerve²⁶
	Human lung cancer cells³²
	C. elegans³³
	Mouse skin³²
	HeLa cells²²
	Human macrophages³⁴
SHG	Neonatal cardiomyocyte²⁹^,³³
	Human mastoid cortex¹¹
	Cardiac myocytes²³
SFG	C. elegans²⁴

2. Single Experimental Methods

In both nonlinear microscopy and confocal laser scanning microscopy, a vibrating mirror scans the sample with focused laser beams.³⁵ In confocal laser scanning microscopy, a high-resolution optical sectioning image is obtained with pinhole apertures.

In biology and medicine, optical microscopy is restricted by phototoxicity. Each measurement often necessitates the minimum average energy of an excited laser to avoid phototoxicity. Compared to linear processes, nonlinear processes can provide momentary extremely high pulsed excited light with low average energy on a live sample. Near-infrared excitation light is typically used in nonlinear excitation microscopy and can also decrease the phototoxicity. Using infrared light can minimize scattering in live cells and tissues. All of these effects increase the penetration depth of nonlinear microscopes.

The technology and theories of nonlinear optical microscopy have made significant contributions to biological research and medical diagnoses.³⁶ The potential applications of these nonlinear optical microscopes are in the fields of biology and medicine. An energy-level diagram of a variety of nonlinear optical processes, including TPEF, SHG, SFG, CARS, and SRS, is shown in Fig. 1.

Fig. 1

Energy-level diagram for nonlinear optical processes of TPEF, SHG, SFG, CARS, and SRS process.

To accurately study nonlinear optical microscopy, the relationship between polarization $P (t)$ and electric field strength is used to precisely describe nonlinear optical microscopy using the following equation:³⁷

Eq. (1)

P (t) {= ϵ}_{0} [χ^{(1)} E (t) {+ χ}^{(2)} E^{2} (t) {+ χ}^{(3)} E^{3} (t) + \dots],

where

χ^{(1)}

,

χ^{(2)}

, and

χ^{(2)}

are the linear, second-order, and third-order susceptibility, respectively.

ϵ_{0}

is the permittivity of free space. The second-order nonlinear optics are described using the following equation:³⁷

Eq. (2)

P (ω_{1} + ω_{2}) = 2 ϵ_{0} χ^{(2)} E_{1} E_{2} .

SHG and SFG are labeled as $ω_{1} = ω_{2}$ and $ω_{1} \neq ω_{2}$ .

The third-order nonlinear optics (CARS and SRS) are described using the following equations:³⁷

Eq. (3)

P (ω_{1} + ω_{2} - ω_{3}) = 6 ϵ_{0} χ^{(3)} E_{1} E_{2} E_{3}^{*},

Eq. (4)

P (2 ω_{1} - ω_{2}) = 3 ϵ_{0} χ^{(3)} E_{1}^{2} E_{2}^{*} .

2.1.

Two-Photon Excited Fluorescence

TPEF microscopy is one of the traditional fluorescence imaging techniques. Due to its nonlinear optical effects, the penetration depth can reach 1 mm in live tissue with high resolution and high sensitivity. Combining TPEF microscopy with fluorescence materials can provide rapid techniques to diagnose and monitor a variety of diseases using encoded fluorescent proteins, exogenous dyes, and nanomaterials.²⁵ A picosecond or femtosecond beam is focused on the sample using a scanning microscope to generate a fluorescence signal (Fig. 2). TPEF microscopy uses exogenous markers to detect and diagnose live cancer cells’ phenotypic changes, metabolic activity, and protein expression.³⁸ Live ovarian cancer cells have been studied using TPEF microscopy to detect $β$ -galactosidase with lysosome-targetable and two-photon fluorescent probe FC- $β gal$ , which are important for the diagnosis of primary ovarian cancer (Fig. 3).²⁵

Fig. 2

Diagram of a TPEF or SHG microscope with epi and forward channel.

Fig. 3

TPEF images of living ovarian cancer SKOV-3cells, (a) probe FC- $β gal$ image of $β$ -gal, (b) probe lysosome image of LysoTracker Red DND-99 by the tracker, (c) merged image of (a) and (b), and (d) intensity profile of regions of interest across SKOV-3 cells, adapted from Ref. 25.

2.2.

Second-Harmonic Generation

In general, for the second-order nonlinear optical techniques of three-wave mixing, the generation of a w3 photo has to emit one w1 and one w2 photon ( $w 1 + w 2 = w 3$ ). When w1 equals w2, it is defined as SHG (Fig. 1); otherwise, it is defined as SFG (Fig. 1).³⁹ SHG occurs when an incident laser beam passes through a noncentrosymmetric and highly ordered medium such as tendons, axons, and striated muscle. Due to the nonabsorptive effects of nonlinear processes, SHG inhibits photobleaching.⁴⁰ SHG was discovered by Franken in 1961.⁴¹ Because it uses the same laser scanning microscope and laser source with an additional proper narrow band filter, SHG can be combined with other nonlinear equipment. SHG is suitable for the collagen, microtubules, and muscles in live tissues and cells⁴² and is widely applied for biological research, medical diagnoses,⁴³ and medicine.¹⁰^,²⁹^,²³^,⁴²^,⁴⁴ SHG microscopy has been used to measure the muscles’ contractile integrity via sarcomeric myosin imaging. Previous studies found that the muscle contractile integrity and neuromuscular health are strongly correlated in mice (Fig. 4).⁴⁴

Fig. 4

SHG images of normal and diseased gastrocnemius muscle dissected, (a) normal muscle and (b) diseased muscle, adapted from Ref. 44.

2.3.

Coherent Anti-Stokes Raman Scattering

CARS is one of the most powerful Raman techniques for imaging molecular vibrations in live cells and tissues as discussed in recent reviews.³^,⁴⁵^,⁴⁶^–⁵⁰ Similar to SRS, CARS is also generated by four-wave mixing with a three-order nonlinear effect, and these work together in the same process. However, unlike SRS, anti-Stokes signal is used for molecular imaging, and a variation in the pump intensity is applied in SRS microscopy.⁵¹ Collinear picosecond or femtosecond beams (pump and Stokes) are focused on the sample using a scanning microscope to generate anti-Stokes signals (Fig. 5). Similar to SRS, CARS microscopy is widely applied for imaging live cancer cells,³¹^,⁵² probing the interactions between live cells and plasmas,⁵³ investigating intracellular lipid storage and dynamics in live tissues,⁵⁴ and assessing lipid uptake in live stem cells during differentiation,³⁰ with the same source, ease of use, real-time, label-free, and ultrahigh resolution. CARS is used to image lipids and measure their number and size to analyze their effects on hormone-treated breast and prostate cancer cells as shown in Fig. 6.

Fig. 5

Diagram of a CARS or SFG microscope with epi and forward channel.

Fig. 6

CARS images of treated versus vehicle control living breast and prostate cancer cells. (a) Medroxyprogesterone acetate treated and (b) untreated breast cells, (c) synthetic androgen R1881treated, and (d) untreated prostate cells, scale bar $30 μ m$ , adapted from Ref. 52.

2.4.

Stimulated Raman Scattering

SRS is a form of Raman scattering. Similar to Raman, it uses femtosecond or picosecond lasers to directly image the vibrational fingerprints of live cells and tissues. SRS is also a special case of four-wave mixing with the three-order nonlinear effect. SRS can provide high-speed, ultrahigh resolution, high-sensitivity, free label, real-time, and three-dimensional⁵⁵ properties to image the distribution of specific molecules⁵⁶ and monitor glucose metabolic activity,² intracellular drug uptake,¹⁹^,³²^,⁵⁷^,⁵⁸ and image the vibration of newly synthesized proteins,¹⁸^,⁵⁹ image multiple proteins in situ,¹⁶^,⁶⁰^,⁶¹ detect brain tumors,²⁰ and probe the interactions between nanoparticles and live cancer cells.⁶² Based on the configuration of CARS microscopy, electro-optical modulators or acousto-optic modulators and lock-in amplifiers are added to the Stokes beam and photomultiplier tube, respectively, to generate an SRS signal (Fig. 7). Macrophages are among the most important white blood cells in the immune systems of animals. They phagocytose cancer cells and cell debris, among other functions. SRS microscopy was first used to characterize the different uptake kinetics of d31-palmitic acid by macrophages between individual cells as shown in Fig. 8.

Fig. 7

Diagram of an SRS microscope with epi and forward channel.

Fig. 8

SRS image of a fixed living human macrophage cells in d31-palmitic acid environment with the corresponding Raman shift being $2125 {cm}^{- 1}$ . (a) Nonresonant background of CARS signal, (b) SRS image, (c) merge image of SRS (red), and CARS (gray), adapted from Ref. 34.

3. Multiple Experimental Methods

Picosecond or femtosecond pulsed laser and multi- or two-photon scanning microscopes are used in SRS and CARS. Many useful signals can be applied to biological research and medical diagnosis, such as TPEF, SHG, and SFG.

3.1.

Two Methods

3.1.1.

TPEF and SHG

Both TPEF and SHG are second-order nonlinear processes with minimum device, single incident lasers, and the same optical system.¹⁰^,²⁹^,²³ The distribution of alpha-actinin with fluorescence drugs and sarcomeric structures in live embryonic cardiomyocytes is imaged using TPEF and SHG channels, respectively. This second-order nonlinear optical technique can effectively reveal long-term structural changes in the live DiO-stained myofibrillogenesis of a single cardiomyocyte using real-time, high-resolution, and high-speed (4 SPF) features as shown in Fig. 9.

Fig. 9

TPEF and SHG images of living cultured neonatal cardiomyocyte: (a) TPEF image, (b) SHG image of sarcomeric structure, and (c) merged of images (scale bars: $10 μ m$ ), adapted from Ref. 29.

3.1.2.

SRS and TPEF

TPEF and SRS microscopy are combined with new features to image live cells and tissues. They can provide similar quality images with the same organelles of live cells as shown in Fig. 10.²² Deferent organelles can also be imaged using TPEF and SRS, respectively. In a recent study, the distribution of lipid droplets and the endoplasmic reticulum was obtained in cancer cells in situ. The spatial–temporal dynamics of lipid droplets and the endoplasmic reticulum in live cancer cells can be monitored to study organelle dynamics and metabolism as shown in Fig. 10(d).⁶³

Fig. 10

(a) TPEF image, (b) SRS image, and (c) merged image of living Hela cancer cells, adapted from Ref. 22. (d) SRS, TPEF, merged, and trajectories images of living Hela cancer cells, adapted from Ref. 63.

3.1.3.

CARS and TPEF

Due to the spectra overlap of TPEF and CARS signals, they cannot be easily separated. Using the correct wavelength of the pump and probe source solves this problem. As female gametocytes and germ cells are involved in reproduction, oocytes are the earliest stages of mammals. As with early embryos, they are widely used to study genetic diseases, cloned animals, genetic breeding, organ transplantation, and cell differentiation mechanisms. Lipid droplets store fatty acids and play a significant role in the preimplantation development of oocytes. Combined CARS and differential interference contrast (DIC) microscopy have been used to quantitatively image lipids in live mouse oocytes and early embryos at different stages of cell division as shown in Fig. 11.

Fig. 11

(a) DIC, (b) CARS, (c) TPEF, and (d) merged images in living early embryonic stages. The CARS images are shown with the corresponding Raman shift being $2850 {cm}^{- 1}$ , adapted from Ref. 4.

3.1.4.

CARS and SHG

Stem cell-based bone engineering is a treatment for bone regeneration. Combined CARS and SHG microscopy has been used to image the apatite and collagen of live cells, respectively, as shown in Fig. 12.¹¹

Fig. 12

CARS (magenta) and SHG (green) three-dimensionality images of calcium hydroxyapatite deposits embedded in collagen. The SHG image of collagen is shown with 532 nm, adapted from Ref. 11.

3.2.

Three Methods

With the appropriate filters, fluorescent labels, and excited wavelengths, three or more types of nonlinear optic microscopy can be combined in the same scanning microscope. The results indicate that these multiple processes are advantageous for imaging live tissues or animals.⁷^,²⁴ Caenorhabditis elegans, a free-living (nonparasitic) transparent nematode (roundworm) $\sim 1 mm$ in length, is widely used in genetics and developmental biology, behavior and neurobiology, aging and longevity, human genetic diseases, pathogen and biological interactions, drug screening, animal emergency response, and other fields. CARS, SFG, and TPEF have been used to study the muscle of live C. elegans using a scanning microscope with an excited laser as shown in Fig. 13.

Fig. 13

(a) CARS (Red), (b) SFG (Blue), and (c) TPEF (green fuorescent protein, Green) images of muscle of C. elegans for the $2860 {cm}^{- 1}$ vibration mode. Scale bar is 10 and $50 μ m$ , adapted from Ref. 24.

4. Conclusion

We have summarized the principles, applications, and advantages of individual and combination of several types of nonlinear optical microscopy, including SRS, TPEF, SHG, and SFG. The experimental results indicate that these multiple processes are of great advantage for imaging live tissues or animals, C. elegans. The nonlinear optical microscopy can monitor specific molecules and proteins inside cells and tissues in three dimensions with high sensitivity and ultrahigh resolution. Our review can promote further understanding of the advanced application of combination of these types of nonlinear optical microscopy for biophotonics.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant Nos. 91436102, 11374353, 11474141, 51401239, and 11704058), Fundamental Research Funds for the Central Universities in USTB, and National Basic Research Program of China (Grant No. 2016YFA0200802).

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Biography

Rui Li received his PhD from the Department of Physics of Dalian University, China, in 2014. From 2009 to 2012, he worked as an exchanged student at the Department of Electronic Engineering, University of Texas at Arlington. Since 2014, he has worked as a lecturer at the Department of Physics of Dalian University of Technology, China. His current research interests focus on surface enhancement Raman scattering and coherent anti-stokes Raman scattering microscopy.

Xinxin Wang is the PhD candidate supervised by Professor Mengtao Sun, at the School of Mathematics and Physics, University of Science and Technology Beijing, China. Her current research interests focus on one and two photon absorptions, fluorescence, and Raman spectra.

Yi Zhou is the PhD candidate supervised by Professor Rui Li, at Dalian University of Technology. His current research interests focus on one and two photon absorptions, fluorescence, and Raman spectra.

Huan Zong is a PhD candidate supervised by Professor Mengtao Sun, at the School of Mathematics and Physics, University of Science and Technology Beijing, China. Her current research interests focus on one and two photon absorptions, fluorescence, and Raman spectra.

Maodu Chen received his PhD in 2003 from Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS). From 2003 to 2005, he worked as a postdoc at the Department of Chemical Physics, Northwestern University. Since 2005, he has worked as an associate professor at the Department of Physics of Dalian University of Technology. In 2010, he became a full professor at Dalian University of Technology, China.

Mengtao Sun received his PhD in 2003 from the Dalian Institute of Chemical Physics, CAS. From 2003 to 2006, he worked as a postdoc at the Department of Chemical Physics, Lund University. Since 2006, he has worked as an associate professor at the Institute of Physics, CAS. In 2016, he became a full professor at the University of Science and Technology Beijing. His current research interests focus on nonlinear optical microscopy, such as CARS, TPEF, and SHG, applied in biophtonics and two-dimensional materials.

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Rui Li, Xinxin Wang, Yi Zhou, Huan Zong, Maodu Chen, and Mengtao Sun "Advances in nonlinear optical microscopy for biophotonics," Journal of Nanophotonics 12(3), 033007 (14 August 2018). https://doi.org/10.1117/1.JNP.12.033007

Received: 6 June 2018; Accepted: 30 July 2018; Published: 14 August 2018

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KEYWORDS

Microscopy

Second-harmonic generation

Optical microscopy

Biomedical optics

Tissues

Microscopes

Luminescence

1.

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