Innovations in living-system spectroscopic imaging

The concept of our technology lies in a kid book “Where is Waldo”, where the kids identify Waldo by its special signatures (a hat, glasses and sweet smile). Similarly, we use spectroscopic signal as molecule fingerprints for label-free chemical imaging of a complex living system. It is important to note that living system spectroscopic imaging is not a simple addition of a spectrometer to a microscope. Innovations are needed to push the physical limits in terms of detection sensitivity, imaging depth, speed and spatial resolution of label-free microscopy. Below are a few examples.

1.1 Multiplex SRS microscopy: spectral acquisition at microsecond scale

Our group invented a 32-channel tuned amplifier array, which enabled acquisition of an SRS spectrum within 5 microseconds to cover a window of 200 wavenumbers [1]. This method enabled high throughput single cell analysis through multichannel SRS flow cytometry [2]. Through multiplex modulation, we demonstrated spectroscopic SRS imaging in a spectrometer free manner [3]. We further developed a resonant optical delay tuner for microsecond-scale hyperspectral SRS imaging with a pair of chirped fs pulses [4].


[1] Chien-Sheng Liao, Mikhail N. Slipchenko, Ping Wang, Junjie Li, Seung-Young Lee, Robert A. Oglesbee, Ji-Xin Cheng*, Microsecond scale vibrational spectroscopic imaging by multiplex stimulated Raman scattering microscopy, Light: Science & Applications, 2015, 4: e265.

 [2] Chi Zhang, Kai-Chih Huang, Bartek Rajwa, Junjie Li, Shiqi Yang, Haonan Lin, Chien-sheng Liao, Gregory Eakins, Shihuan Kuang, Valery Patsekin, J. Paul Robinson, Ji-Xin Cheng*, “Stimulated Raman scattering flow cytometry for label-free single-particle analysis”, Optica, Jan 2017, 4: 103.

[3] Chien-Sheng Liao, Pu Wang, Ping Wang, Junjie Li, Hyeon Jeong Lee, Gregory Eakins, Ji-Xin Cheng*, “Spectrometer-free Vibrational Imaging by Retrieving Stimulated Raman Signal from Highly Scattered Photons”, Science Advances, 2015, 1:e1500738.

[4] Chien-Sheng Liao, Kai-Chih Huang, Weili Hong, Andy J. Chen, Caroline Karanja, Pu Wang, Gregory Eakins, Ji-Xin Cheng*, “Stimulated Raman spectroscopic imaging by microsecond delay-line tuning”, Optica, 2016, 3: 1377.

1.2 Mid-infrared photothermal (MIP) microscopy: Breaking the diffraction limit by sensing the thermal effect

Infrared spectroscopy allows chemical histology but the spatial resolution is limited by the long infrared wavelength. By using a visible beam to sense the thermal lensing effect induced by infrared absorption in fingerprint region, we demonstrated MIP imaging of living cells with a submicron resolution [5]. We are now pushing the speed limit of this method via wide-field illumination.


[5] Delong Zhang, Chen Li, Chi Zhang, Mikhail N. Slipchenko, Gregory Eakins, Ji-Xin Cheng*, “Depth-resolved mid-infrared photothermal imaging of living cells and organism with sub-micron spatial resolution”, Science Advances, 2016, 2: e1600521

1.3 Vibration-based photoacoustic tomography: listening to chemical bond vibration

To enable deep-tissue vibrational imaging, we invented a new modality based on optical excitation of harmonic vibration, subsequent relaxation of vibrational energy into heat, and acoustic detection of the generated ultrasonic waves [6]. This method extended the vibrational imaging depth to centimeter scale, allowing us to see inside the body noninvasively. We further demonstrated two significant biomedical applications of this approach, one for in vivo imaging of lipid-laden plaque [7] and the other for in situ detection of cancer margin.


[6] Han-Wei Wang Ning Chai, Pu Wang, Song Hu, Wei Dou, David Umulis, Lihong V. Wang, Michael Sturek, Robert Lucht, Ji-Xin Cheng*, “Label-free bond-selective imaging by listening to vibrationally excited molecules”, Phys. Rev. Lett., 2011, 106: 238106. Highlighted by Science and “NIH Research Matters.”

[7] Jie Hui, Yingchun Cao, Yi Zhang, Ayeeshik Kole, Pu Wang, Guangli Yu, Gregory Eakins, Michael Sturek, Weibiao Chen, Ji-Xin Cheng*, “Real-time intravascular photoacoustic-ultrasound imaging of lipid-laden plaque in human coronary artery at 16 frames per second”, Scientific Reports, 2017, 7:1417.

1.4 Transient absorption microscopy: spectroscopic imaging in the time domain

We developed high-speed, high-sensitivity transient absorption spectroscopic imaging for characterization of single-walled carbon nanotubes [8] and single-layer graphene [9]. We were the first to use phase-sensitive transient absorption microscopy to separate semiconducting and metallic nanotubes [10]. We further demonstrated saturated transient absorption microscopy [11] for super-resolution imaging of non-fluorescent species using absorption as a contrast mechanism.


[8] Ling Tong, Yuxiang Liu, Bridget D. Dolash, Yookyung Jung, Mikhail N. Slipchenko, Donald E. Bergstrom, Ji-Xin Cheng*, “Label-free Imaging of Semiconducting and Metallic Carbon Nanotubes in Cells and Mice Using Transient Absorption Microscopy,” Nature Nanotechnology, 2012, 7: 56-61.

[9] Junjie Li, Weixia Zhang, Ting-Fung Chung, Mikhail N. Slipchenko, Yong P. Chen, Ji-Xin Cheng*, Chen Yang*, Highly sensitive transient absorption imaging of graphene and graphene oxide in living cells and circulating blood, Scientific Reports, 2015, 5: 12394.

[10] Yookyung Jung, Mikhail N. Slipchenko, Chang Hua Liu, Alexander E. Ribbe, Zhaohui Zhong, Chen Yang and Ji-Xin Cheng*, “Fast detection of the metallic state of individual single-walled carbon nanotubes using a transient-absorption optical microscope”, Phys. Rev. Lett., 2010, 105: 217401.

[11] Pu Wang, Mikhail N. Slipchenko, James Mitchell, Chen Yang, Eric O. Potma, Xianfan Xu, Ji-Xin Cheng*, “Far-field imaging of non-fluorescent species with sub-diffraction resolution,” Nature Photonics, 2013, 7(6): 449-453.

1.5 Volumetric chemical microscopy

Volumetric imaging allows global understanding of three-dimensional (3D) complex systems. Light-sheet fluorescence microscopy and optical projection tomography have been reported to image 3D volumes with high resolutions and at high speeds. Such methods, however, usually rely on fluorescent labels for chemical targeting, which could perturb the biological functionality in living systems. We demonstrated Bessel-beam-based stimulated Raman projection (SRP) microscopy and tomography for label-free volumetric chemical imaging [12].


[12] Xueli Chen, Chi Zhang, Peng Lin, Kai-Chih Huang, Jimin Liang, Jie Tian & Ji-Xin Cheng*, “Volumetric chemical imaging by stimulated Raman microscopy and tomography”, Nature Communication, 2017, 8: 15117.