Abstract

Label-free optical imaging is valuable in biology and medicine because of its non-destructive nature. Quantitative phase imaging (QPI) and molecular vibrational imaging (MVI) are the two most successful label-free methods, providing morphological and biochemical information, respectively. These techniques have enabled numerous applications as they have matured over the past few decades; however, their label-free contrasts are inherently complementary and difficult to integrate due to their reliance on different light–matter interactions. Here we present a unified imaging scheme with simultaneous and in situ acquisition of quantitative phase and molecular vibrational contrasts of single cells in the QPI framework using the mid-infrared photothermal effect. The robust integration of subcellular morphological and biochemical label-free measurements may enable new analyses, especially for studying complex and fragile biological phenomena such as drug delivery, cellular disease, and stem cell development, where long-time observation of unperturbed cells is needed under low phototoxicity.

© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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    [Crossref]

2019 (13)

J. M. Lim, C. Park, J. S. Park, C. Kim, B. Chon, and M. Cho, “Cytoplasmic protein imaging with mid-infrared photothermal microscopy: cellular dynamics of live neurons and oligodendrocytes,” J. Phys. Chem. Lett. 10, 2857–2861 (2019).
[Crossref]

P. D. Samolis and M. Y. Sander, “Phase-sensitive lock-in detection for high-contrast mid-infrared photothermal imaging with sub-diffraction limited resolution,” Opt. Express 27, 2643–2655 (2019).
[Crossref]

Y. Bai, D. Zhang, L. Lan, Y. Huang, K. Maize, A. Shakouri, and J. X. Cheng, “Ultrafast chemical imaging by widefield photothermal sensing of infrared absorption,” Sci. Adv. 5, eaav7127 (2019).
[Crossref]

K. Toda, M. Tamamitsu, Y. Nagashima, R. Horisaki, and T. Ideguchi, “Molecular contrast on phase-contrast microscope,” Sci. Rep. 9, 9957 (2019).
[Crossref]

F. Hu, L. Shi, and W. Min, “Biological imaging of chemical bonds by stimulated Raman scattering microscopy,” Nat. Methods 16, 830–842 (2019).
[Crossref]

J. Shi, T. T. W. Wong, Y. He, L. Li, R. Zhang, C. S. Yung, J. Hwang, K. Maslov, and L. V. Wang, “High-resolution, high-contrast mid-infrared imaging of fresh biological samples with ultraviolet-localized photoacoustic microscopy,” Nat. Photonics 13, 609–615 (2019).
[Crossref]

M. Tamamitsu, K. Toda, R. Horisaki, and T. Ideguchi, “Quantitative phase imaging with molecular vibrational sensitivity,” Opt. Lett. 44, 3729–3732 (2019).
[Crossref]

D. Zhang, L. Lan, Y. Bai, H. Majeed, M. E. Kandel, G. Popescu, and J. X. Cheng, “Bond-selective transient phase imaging via sensing of the infrared photothermal effect,” Light Sci. Appl. 8, 116 (2019).
[Crossref]

V. Micó, J. Zheng, J. Garcia, Z. Zalevsky, and P. Gao, “Resolution enhancement in quantitative phase microscopy,” Adv. Opt. Photon. 11, 135–214 (2019).
[Crossref]

S. Chowdhury, M. Chen, R. Eckert, D. Ren, F. Wu, N. Repina, and L. Waller, “High-resolution 3D refractive index microscopy of multiple-scattering samples from intensity images,” Optica 6, 1211–1219 (2019).
[Crossref]

R. Horisaki, K. Fujii, and J. Tanida, “Diffusion-based single-shot diffraction tomography,” Opt. Lett. 44, 1964–1967 (2019).
[Crossref]

L. Shi, F. Hu, and W. Min, “Optical mapping of biological water in single live cells by stimulated Raman excited fluorescence microscopy,” Nat. Commun. 10, 4764 (2019).
[Crossref]

M. Nuriya, H. Yoneyama, K. Takahashi, P. Leproux, V. Couderc, M. Yasui, and H. Kano, “Characterization of intra/extracellular water states probed by ultrabroadband multiplex coherent anti-stokes Raman scattering (CARS) spectroscopic imaging,” J. Phys. Chem. A 123, 3928–3934 (2019).
[Crossref]

2018 (1)

Y. K. Park, C. Depeursinge, and G. Popescu, “Quantitative phase imaging in biomedicine,” Nat. Photonics 12, 578–589 (2018).
[Crossref]

2017 (6)

D. W. Shipp, F. Sinjab, and I. Notingher, “Raman spectroscopy: techniques and applications in the life sciences,” Adv. Opt. Photon. 9, 315–428 (2017).
[Crossref]

R. Kasprowicz, R. Suman, and P. O’Toole, “Characterising live cell behaviour: traditional label-free and quantitative phase imaging approaches,” Int. J. Biochem. Cell Biol. 84, 89–95 (2017).
[Crossref]

Z. Li, K. Aleshire, M. Kuno, and G. V. Hartland, “Super-resolution far-field infrared imaging by photothermal heterodyne imaging,” J. Phys. Chem. B 121, 8838–8846 (2017).
[Crossref]

K. Lee, K. Kim, G. Kim, S. Shin, and Y. Park, “Time-multiplexed structured illumination using a DMD for optical diffraction tomography,” Opt. Lett. 42, 999–1002 (2017).
[Crossref]

J. A. Rodrigo, J. M. Soto, and T. Alieva, “Fast label-free microscopy technique for 3D dynamic quantitative imaging of living cells,” Biomed. Opt. Express 8, 5507–5517 (2017).
[Crossref]

F. Merola, P. Memmolo, L. Miccio, R. Savoia, M. Mugnano, A. Fontana, G. D’Ippolito, A. Sardo, A. Iolascon, A. Gambale, and P. Ferraro, “Tomographic flow cytometry by digital holography,” Light Sci. Appl. 6, e16241 (2017).
[Crossref]

2016 (4)

R. Horisaki, R. Egami, and J. Tanida, “Single-shot phase imaging with randomized light (SPIRaL),” Opt. Express 24, 3765–3773 (2016).
[Crossref]

D. Zhang, C. Li, C. Zhang, M. N. Slipchenko, G. Eakins, and J. X. Cheng, “Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution,” Sci. Adv. 2, e1600521 (2016).
[Crossref]

Y. Wakisaka, Y. Suzuki, O. Iwata, A. Nakashima, T. Ito, M. Hirose, R. Domon, M. Sugawara, N. Tsumura, H. Watarai, T. Shimobaba, K. Suzuki, K. Goda, and Y. Ozeki, “Probing the metabolic heterogeneity of live Euglena gracilis with stimulated Raman scattering microscopy,” Nat. Microbiol. 1, 16124 (2016).
[Crossref]

P. Hosseini, R. Zhou, Y.-H. Kim, C. Peres, A. Diaspro, C. Kuang, Z. Yaqoob, and P. T. C. So, “Pushing phase and amplitude sensitivity limits in interferometric microscopy,” Opt. Lett. 41, 1656–1659 (2016).
[Crossref]

2015 (2)

C. H. Camp and M. T. Cicerone, “Chemically sensitive bioimaging with coherent Raman scattering,” Nat. Photonics 9, 295–305 (2015).
[Crossref]

J. X. Cheng and X. S. Xie, “Vibrational spectroscopic imaging of living systems: an emerging platform for biology and medicine,” Science 350, aaa8870 (2015).
[Crossref]

2014 (1)

M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, M. J. Walsh, B. R. Wood, P. Gardner, and F. L. Martin, “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771–1791 (2014).
[Crossref]

2013 (2)

N. Pavillon, A. J. Hobro, and N. I. Smith, “Cell optical density and molecular composition revealed by simultaneous multimodal label-free imaging,” Biophys. J. 105, 1123–1132 (2013).
[Crossref]

Y. Cotte, F. Toy, P. Jourdain, N. Pavillon, D. Boss, P. Magistretti, P. Marquet, and C. Depeursinge, “Marker-free phase nanoscopy,” Nat. Photonics 7, 113–117 (2013).
[Crossref]

2012 (2)

K. Okabe, N. Inada, C. Gota, Y. Harada, T. Funatsu, and S. Uchiyama, “Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy,” Nat. Commun. 3, 705 (2012).
[Crossref]

B. Bahduri, H. Pham, M. Mir, and G. Popescu, “Diffraction phase microscopy with white light,” Opt. Lett. 37, 1094–1096 (2012).
[Crossref]

2009 (1)

2008 (1)

G. Popescu, Y. K. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. 295, C538–C544 (2008).
[Crossref]

2006 (1)

2005 (2)

2003 (1)

A. Salazar, “On thermal diffusivity,” Eur. J. Phys. 24, 351–358 (2003).
[Crossref]

1995 (1)

J. Wang and M. Fiebig, “Measurement of the thermal diffusivity of aqueous solutions of alcohols by a laser-induced thermal grating technique,” Int. J. Thermophys. 16, 1353–1361 (1995).
[Crossref]

1990 (1)

P. Schiebener, J. Straub, J. M. H. Levelt Sengers, and J. S. Gallagher, “Refractive index of water and steam as function of wavelength, temperature and density,” J. Phys. Chem. Ref. Data 19, 677–717 (1990).
[Crossref]

1969 (1)

E. Wolf, “Three-dimensional structure determination of semi-transparent objects from holographic data,” Opt. Commun. 1, 153–156 (1969).
[Crossref]

Aleshire, K.

Z. Li, K. Aleshire, M. Kuno, and G. V. Hartland, “Super-resolution far-field infrared imaging by photothermal heterodyne imaging,” J. Phys. Chem. B 121, 8838–8846 (2017).
[Crossref]

Alieva, T.

Badizadegan, K.

Y. Sung, W. Choi, C. Fang-Yen, K. Badizadegan, R. R. Dasari, M. S. Feld, “Optical diffraction tomography for high resolution live cell imaging,” Opt. Express 17, 266–277 (2009).
[Crossref]

G. Popescu, Y. K. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. 295, C538–C544 (2008).
[Crossref]

Bahduri, B.

Bai, Y.

Y. Bai, D. Zhang, L. Lan, Y. Huang, K. Maize, A. Shakouri, and J. X. Cheng, “Ultrafast chemical imaging by widefield photothermal sensing of infrared absorption,” Sci. Adv. 5, eaav7127 (2019).
[Crossref]

D. Zhang, L. Lan, Y. Bai, H. Majeed, M. E. Kandel, G. Popescu, and J. X. Cheng, “Bond-selective transient phase imaging via sensing of the infrared photothermal effect,” Light Sci. Appl. 8, 116 (2019).
[Crossref]

Baker, M. J.

M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, M. J. Walsh, B. R. Wood, P. Gardner, and F. L. Martin, “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771–1791 (2014).
[Crossref]

Bassan, P.

M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, M. J. Walsh, B. R. Wood, P. Gardner, and F. L. Martin, “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771–1791 (2014).
[Crossref]

Best-Popescu, C.

G. Popescu, Y. K. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. 295, C538–C544 (2008).
[Crossref]

Bhargava, R.

M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, M. J. Walsh, B. R. Wood, P. Gardner, and F. L. Martin, “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771–1791 (2014).
[Crossref]

Boss, D.

Y. Cotte, F. Toy, P. Jourdain, N. Pavillon, D. Boss, P. Magistretti, P. Marquet, and C. Depeursinge, “Marker-free phase nanoscopy,” Nat. Photonics 7, 113–117 (2013).
[Crossref]

Butler, H. J.

M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, M. J. Walsh, B. R. Wood, P. Gardner, and F. L. Martin, “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771–1791 (2014).
[Crossref]

Camp, C. H.

C. H. Camp and M. T. Cicerone, “Chemically sensitive bioimaging with coherent Raman scattering,” Nat. Photonics 9, 295–305 (2015).
[Crossref]

Chen, M.

Cheng, J. X.

D. Zhang, L. Lan, Y. Bai, H. Majeed, M. E. Kandel, G. Popescu, and J. X. Cheng, “Bond-selective transient phase imaging via sensing of the infrared photothermal effect,” Light Sci. Appl. 8, 116 (2019).
[Crossref]

Y. Bai, D. Zhang, L. Lan, Y. Huang, K. Maize, A. Shakouri, and J. X. Cheng, “Ultrafast chemical imaging by widefield photothermal sensing of infrared absorption,” Sci. Adv. 5, eaav7127 (2019).
[Crossref]

D. Zhang, C. Li, C. Zhang, M. N. Slipchenko, G. Eakins, and J. X. Cheng, “Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution,” Sci. Adv. 2, e1600521 (2016).
[Crossref]

J. X. Cheng and X. S. Xie, “Vibrational spectroscopic imaging of living systems: an emerging platform for biology and medicine,” Science 350, aaa8870 (2015).
[Crossref]

Y. Fu, H. Wang, R. Shi, and J. X. Cheng, “Characterization of photodamage in coherent anti-Stokes Raman scattering microscopy,” Opt. Express 14, 3942–3951 (2006).
[Crossref]

Cho, M.

J. M. Lim, C. Park, J. S. Park, C. Kim, B. Chon, and M. Cho, “Cytoplasmic protein imaging with mid-infrared photothermal microscopy: cellular dynamics of live neurons and oligodendrocytes,” J. Phys. Chem. Lett. 10, 2857–2861 (2019).
[Crossref]

Choi, W.

Chon, B.

J. M. Lim, C. Park, J. S. Park, C. Kim, B. Chon, and M. Cho, “Cytoplasmic protein imaging with mid-infrared photothermal microscopy: cellular dynamics of live neurons and oligodendrocytes,” J. Phys. Chem. Lett. 10, 2857–2861 (2019).
[Crossref]

Chowdhury, S.

Cicerone, M. T.

C. H. Camp and M. T. Cicerone, “Chemically sensitive bioimaging with coherent Raman scattering,” Nat. Photonics 9, 295–305 (2015).
[Crossref]

Colomb, T.

Cotte, Y.

Y. Cotte, F. Toy, P. Jourdain, N. Pavillon, D. Boss, P. Magistretti, P. Marquet, and C. Depeursinge, “Marker-free phase nanoscopy,” Nat. Photonics 7, 113–117 (2013).
[Crossref]

Couderc, V.

M. Nuriya, H. Yoneyama, K. Takahashi, P. Leproux, V. Couderc, M. Yasui, and H. Kano, “Characterization of intra/extracellular water states probed by ultrabroadband multiplex coherent anti-stokes Raman scattering (CARS) spectroscopic imaging,” J. Phys. Chem. A 123, 3928–3934 (2019).
[Crossref]

Cuche, E.

D’Ippolito, G.

F. Merola, P. Memmolo, L. Miccio, R. Savoia, M. Mugnano, A. Fontana, G. D’Ippolito, A. Sardo, A. Iolascon, A. Gambale, and P. Ferraro, “Tomographic flow cytometry by digital holography,” Light Sci. Appl. 6, e16241 (2017).
[Crossref]

Dasari, R. R.

Y. Sung, W. Choi, C. Fang-Yen, K. Badizadegan, R. R. Dasari, M. S. Feld, “Optical diffraction tomography for high resolution live cell imaging,” Opt. Express 17, 266–277 (2009).
[Crossref]

G. Popescu, Y. K. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. 295, C538–C544 (2008).
[Crossref]

Deflores, L.

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Pham, H.

Popescu, G.

D. Zhang, L. Lan, Y. Bai, H. Majeed, M. E. Kandel, G. Popescu, and J. X. Cheng, “Bond-selective transient phase imaging via sensing of the infrared photothermal effect,” Light Sci. Appl. 8, 116 (2019).
[Crossref]

Y. K. Park, C. Depeursinge, and G. Popescu, “Quantitative phase imaging in biomedicine,” Nat. Photonics 12, 578–589 (2018).
[Crossref]

B. Bahduri, H. Pham, M. Mir, and G. Popescu, “Diffraction phase microscopy with white light,” Opt. Lett. 37, 1094–1096 (2012).
[Crossref]

G. Popescu, Y. K. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. 295, C538–C544 (2008).
[Crossref]

Rappaz, B.

Ren, D.

Repina, N.

Rodrigo, J. A.

Salazar, A.

A. Salazar, “On thermal diffusivity,” Eur. J. Phys. 24, 351–358 (2003).
[Crossref]

Samolis, P. D.

Sander, M. Y.

Sardo, A.

F. Merola, P. Memmolo, L. Miccio, R. Savoia, M. Mugnano, A. Fontana, G. D’Ippolito, A. Sardo, A. Iolascon, A. Gambale, and P. Ferraro, “Tomographic flow cytometry by digital holography,” Light Sci. Appl. 6, e16241 (2017).
[Crossref]

Savoia, R.

F. Merola, P. Memmolo, L. Miccio, R. Savoia, M. Mugnano, A. Fontana, G. D’Ippolito, A. Sardo, A. Iolascon, A. Gambale, and P. Ferraro, “Tomographic flow cytometry by digital holography,” Light Sci. Appl. 6, e16241 (2017).
[Crossref]

Schiebener, P.

P. Schiebener, J. Straub, J. M. H. Levelt Sengers, and J. S. Gallagher, “Refractive index of water and steam as function of wavelength, temperature and density,” J. Phys. Chem. Ref. Data 19, 677–717 (1990).
[Crossref]

Shakouri, A.

Y. Bai, D. Zhang, L. Lan, Y. Huang, K. Maize, A. Shakouri, and J. X. Cheng, “Ultrafast chemical imaging by widefield photothermal sensing of infrared absorption,” Sci. Adv. 5, eaav7127 (2019).
[Crossref]

Shi, J.

J. Shi, T. T. W. Wong, Y. He, L. Li, R. Zhang, C. S. Yung, J. Hwang, K. Maslov, and L. V. Wang, “High-resolution, high-contrast mid-infrared imaging of fresh biological samples with ultraviolet-localized photoacoustic microscopy,” Nat. Photonics 13, 609–615 (2019).
[Crossref]

Shi, L.

F. Hu, L. Shi, and W. Min, “Biological imaging of chemical bonds by stimulated Raman scattering microscopy,” Nat. Methods 16, 830–842 (2019).
[Crossref]

L. Shi, F. Hu, and W. Min, “Optical mapping of biological water in single live cells by stimulated Raman excited fluorescence microscopy,” Nat. Commun. 10, 4764 (2019).
[Crossref]

Shi, R.

Shimobaba, T.

Y. Wakisaka, Y. Suzuki, O. Iwata, A. Nakashima, T. Ito, M. Hirose, R. Domon, M. Sugawara, N. Tsumura, H. Watarai, T. Shimobaba, K. Suzuki, K. Goda, and Y. Ozeki, “Probing the metabolic heterogeneity of live Euglena gracilis with stimulated Raman scattering microscopy,” Nat. Microbiol. 1, 16124 (2016).
[Crossref]

Shin, S.

Shipp, D. W.

Sinjab, F.

Slipchenko, M. N.

D. Zhang, C. Li, C. Zhang, M. N. Slipchenko, G. Eakins, and J. X. Cheng, “Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution,” Sci. Adv. 2, e1600521 (2016).
[Crossref]

Smith, N. I.

N. Pavillon, A. J. Hobro, and N. I. Smith, “Cell optical density and molecular composition revealed by simultaneous multimodal label-free imaging,” Biophys. J. 105, 1123–1132 (2013).
[Crossref]

So, P. T. C.

Sockalingum, G. D.

M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, M. J. Walsh, B. R. Wood, P. Gardner, and F. L. Martin, “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771–1791 (2014).
[Crossref]

Soto, J. M.

Straub, J.

P. Schiebener, J. Straub, J. M. H. Levelt Sengers, and J. S. Gallagher, “Refractive index of water and steam as function of wavelength, temperature and density,” J. Phys. Chem. Ref. Data 19, 677–717 (1990).
[Crossref]

Strong, R. J.

M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, M. J. Walsh, B. R. Wood, P. Gardner, and F. L. Martin, “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771–1791 (2014).
[Crossref]

Sugawara, M.

Y. Wakisaka, Y. Suzuki, O. Iwata, A. Nakashima, T. Ito, M. Hirose, R. Domon, M. Sugawara, N. Tsumura, H. Watarai, T. Shimobaba, K. Suzuki, K. Goda, and Y. Ozeki, “Probing the metabolic heterogeneity of live Euglena gracilis with stimulated Raman scattering microscopy,” Nat. Microbiol. 1, 16124 (2016).
[Crossref]

Sulé-Suso, J.

M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, M. J. Walsh, B. R. Wood, P. Gardner, and F. L. Martin, “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771–1791 (2014).
[Crossref]

Suman, R.

R. Kasprowicz, R. Suman, and P. O’Toole, “Characterising live cell behaviour: traditional label-free and quantitative phase imaging approaches,” Int. J. Biochem. Cell Biol. 84, 89–95 (2017).
[Crossref]

Sung, Y.

Suzuki, K.

Y. Wakisaka, Y. Suzuki, O. Iwata, A. Nakashima, T. Ito, M. Hirose, R. Domon, M. Sugawara, N. Tsumura, H. Watarai, T. Shimobaba, K. Suzuki, K. Goda, and Y. Ozeki, “Probing the metabolic heterogeneity of live Euglena gracilis with stimulated Raman scattering microscopy,” Nat. Microbiol. 1, 16124 (2016).
[Crossref]

Suzuki, Y.

Y. Wakisaka, Y. Suzuki, O. Iwata, A. Nakashima, T. Ito, M. Hirose, R. Domon, M. Sugawara, N. Tsumura, H. Watarai, T. Shimobaba, K. Suzuki, K. Goda, and Y. Ozeki, “Probing the metabolic heterogeneity of live Euglena gracilis with stimulated Raman scattering microscopy,” Nat. Microbiol. 1, 16124 (2016).
[Crossref]

Takahashi, K.

M. Nuriya, H. Yoneyama, K. Takahashi, P. Leproux, V. Couderc, M. Yasui, and H. Kano, “Characterization of intra/extracellular water states probed by ultrabroadband multiplex coherent anti-stokes Raman scattering (CARS) spectroscopic imaging,” J. Phys. Chem. A 123, 3928–3934 (2019).
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K. Toda, M. Tamamitsu, Y. Nagashima, R. Horisaki, and T. Ideguchi, “Molecular contrast on phase-contrast microscope,” Sci. Rep. 9, 9957 (2019).
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M. Tamamitsu, K. Toda, R. Horisaki, and T. Ideguchi, “Quantitative phase imaging with molecular vibrational sensitivity,” Opt. Lett. 44, 3729–3732 (2019).
[Crossref]

Tanida, J.

Toda, K.

K. Toda, M. Tamamitsu, Y. Nagashima, R. Horisaki, and T. Ideguchi, “Molecular contrast on phase-contrast microscope,” Sci. Rep. 9, 9957 (2019).
[Crossref]

M. Tamamitsu, K. Toda, R. Horisaki, and T. Ideguchi, “Quantitative phase imaging with molecular vibrational sensitivity,” Opt. Lett. 44, 3729–3732 (2019).
[Crossref]

Toy, F.

Y. Cotte, F. Toy, P. Jourdain, N. Pavillon, D. Boss, P. Magistretti, P. Marquet, and C. Depeursinge, “Marker-free phase nanoscopy,” Nat. Photonics 7, 113–117 (2013).
[Crossref]

Trevisan, J.

M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, M. J. Walsh, B. R. Wood, P. Gardner, and F. L. Martin, “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771–1791 (2014).
[Crossref]

Tsumura, N.

Y. Wakisaka, Y. Suzuki, O. Iwata, A. Nakashima, T. Ito, M. Hirose, R. Domon, M. Sugawara, N. Tsumura, H. Watarai, T. Shimobaba, K. Suzuki, K. Goda, and Y. Ozeki, “Probing the metabolic heterogeneity of live Euglena gracilis with stimulated Raman scattering microscopy,” Nat. Microbiol. 1, 16124 (2016).
[Crossref]

Uchiyama, S.

K. Okabe, N. Inada, C. Gota, Y. Harada, T. Funatsu, and S. Uchiyama, “Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy,” Nat. Commun. 3, 705 (2012).
[Crossref]

Wakisaka, Y.

Y. Wakisaka, Y. Suzuki, O. Iwata, A. Nakashima, T. Ito, M. Hirose, R. Domon, M. Sugawara, N. Tsumura, H. Watarai, T. Shimobaba, K. Suzuki, K. Goda, and Y. Ozeki, “Probing the metabolic heterogeneity of live Euglena gracilis with stimulated Raman scattering microscopy,” Nat. Microbiol. 1, 16124 (2016).
[Crossref]

Waller, L.

Walsh, M. J.

M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, M. J. Walsh, B. R. Wood, P. Gardner, and F. L. Martin, “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771–1791 (2014).
[Crossref]

Wang, H.

Wang, J.

J. Wang and M. Fiebig, “Measurement of the thermal diffusivity of aqueous solutions of alcohols by a laser-induced thermal grating technique,” Int. J. Thermophys. 16, 1353–1361 (1995).
[Crossref]

Wang, L. V.

J. Shi, T. T. W. Wong, Y. He, L. Li, R. Zhang, C. S. Yung, J. Hwang, K. Maslov, and L. V. Wang, “High-resolution, high-contrast mid-infrared imaging of fresh biological samples with ultraviolet-localized photoacoustic microscopy,” Nat. Photonics 13, 609–615 (2019).
[Crossref]

Watarai, H.

Y. Wakisaka, Y. Suzuki, O. Iwata, A. Nakashima, T. Ito, M. Hirose, R. Domon, M. Sugawara, N. Tsumura, H. Watarai, T. Shimobaba, K. Suzuki, K. Goda, and Y. Ozeki, “Probing the metabolic heterogeneity of live Euglena gracilis with stimulated Raman scattering microscopy,” Nat. Microbiol. 1, 16124 (2016).
[Crossref]

Wolf, E.

E. Wolf, “Three-dimensional structure determination of semi-transparent objects from holographic data,” Opt. Commun. 1, 153–156 (1969).
[Crossref]

Wong, T. T. W.

J. Shi, T. T. W. Wong, Y. He, L. Li, R. Zhang, C. S. Yung, J. Hwang, K. Maslov, and L. V. Wang, “High-resolution, high-contrast mid-infrared imaging of fresh biological samples with ultraviolet-localized photoacoustic microscopy,” Nat. Photonics 13, 609–615 (2019).
[Crossref]

Wood, B. R.

M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, M. J. Walsh, B. R. Wood, P. Gardner, and F. L. Martin, “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771–1791 (2014).
[Crossref]

Wu, F.

Xie, X. S.

J. X. Cheng and X. S. Xie, “Vibrational spectroscopic imaging of living systems: an emerging platform for biology and medicine,” Science 350, aaa8870 (2015).
[Crossref]

Yaqoob, Z.

Yasui, M.

M. Nuriya, H. Yoneyama, K. Takahashi, P. Leproux, V. Couderc, M. Yasui, and H. Kano, “Characterization of intra/extracellular water states probed by ultrabroadband multiplex coherent anti-stokes Raman scattering (CARS) spectroscopic imaging,” J. Phys. Chem. A 123, 3928–3934 (2019).
[Crossref]

Yoneyama, H.

M. Nuriya, H. Yoneyama, K. Takahashi, P. Leproux, V. Couderc, M. Yasui, and H. Kano, “Characterization of intra/extracellular water states probed by ultrabroadband multiplex coherent anti-stokes Raman scattering (CARS) spectroscopic imaging,” J. Phys. Chem. A 123, 3928–3934 (2019).
[Crossref]

Yung, C. S.

J. Shi, T. T. W. Wong, Y. He, L. Li, R. Zhang, C. S. Yung, J. Hwang, K. Maslov, and L. V. Wang, “High-resolution, high-contrast mid-infrared imaging of fresh biological samples with ultraviolet-localized photoacoustic microscopy,” Nat. Photonics 13, 609–615 (2019).
[Crossref]

Zalevsky, Z.

Zhang, C.

D. Zhang, C. Li, C. Zhang, M. N. Slipchenko, G. Eakins, and J. X. Cheng, “Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution,” Sci. Adv. 2, e1600521 (2016).
[Crossref]

Zhang, D.

Y. Bai, D. Zhang, L. Lan, Y. Huang, K. Maize, A. Shakouri, and J. X. Cheng, “Ultrafast chemical imaging by widefield photothermal sensing of infrared absorption,” Sci. Adv. 5, eaav7127 (2019).
[Crossref]

D. Zhang, L. Lan, Y. Bai, H. Majeed, M. E. Kandel, G. Popescu, and J. X. Cheng, “Bond-selective transient phase imaging via sensing of the infrared photothermal effect,” Light Sci. Appl. 8, 116 (2019).
[Crossref]

D. Zhang, C. Li, C. Zhang, M. N. Slipchenko, G. Eakins, and J. X. Cheng, “Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution,” Sci. Adv. 2, e1600521 (2016).
[Crossref]

Zhang, R.

J. Shi, T. T. W. Wong, Y. He, L. Li, R. Zhang, C. S. Yung, J. Hwang, K. Maslov, and L. V. Wang, “High-resolution, high-contrast mid-infrared imaging of fresh biological samples with ultraviolet-localized photoacoustic microscopy,” Nat. Photonics 13, 609–615 (2019).
[Crossref]

Zharov, V. P.

V. P. Zharov and D. O. Lapotko, “Photothermal imaging of nanoparticles and cells,” IEEE J. Sel. Top. Quantum Electron. 11, 733–751 (2005).
[Crossref]

Zheng, J.

Zhou, R.

Adv. Opt. Photon. (2)

Am. J. Physiol. (1)

G. Popescu, Y. K. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. 295, C538–C544 (2008).
[Crossref]

Biomed. Opt. Express (1)

Biophys. J. (1)

N. Pavillon, A. J. Hobro, and N. I. Smith, “Cell optical density and molecular composition revealed by simultaneous multimodal label-free imaging,” Biophys. J. 105, 1123–1132 (2013).
[Crossref]

Eur. J. Phys. (1)

A. Salazar, “On thermal diffusivity,” Eur. J. Phys. 24, 351–358 (2003).
[Crossref]

IEEE J. Sel. Top. Quantum Electron. (1)

V. P. Zharov and D. O. Lapotko, “Photothermal imaging of nanoparticles and cells,” IEEE J. Sel. Top. Quantum Electron. 11, 733–751 (2005).
[Crossref]

Int. J. Biochem. Cell Biol. (1)

R. Kasprowicz, R. Suman, and P. O’Toole, “Characterising live cell behaviour: traditional label-free and quantitative phase imaging approaches,” Int. J. Biochem. Cell Biol. 84, 89–95 (2017).
[Crossref]

Int. J. Thermophys. (1)

J. Wang and M. Fiebig, “Measurement of the thermal diffusivity of aqueous solutions of alcohols by a laser-induced thermal grating technique,” Int. J. Thermophys. 16, 1353–1361 (1995).
[Crossref]

J. Phys. Chem. A (1)

M. Nuriya, H. Yoneyama, K. Takahashi, P. Leproux, V. Couderc, M. Yasui, and H. Kano, “Characterization of intra/extracellular water states probed by ultrabroadband multiplex coherent anti-stokes Raman scattering (CARS) spectroscopic imaging,” J. Phys. Chem. A 123, 3928–3934 (2019).
[Crossref]

J. Phys. Chem. B (1)

Z. Li, K. Aleshire, M. Kuno, and G. V. Hartland, “Super-resolution far-field infrared imaging by photothermal heterodyne imaging,” J. Phys. Chem. B 121, 8838–8846 (2017).
[Crossref]

J. Phys. Chem. Lett. (1)

J. M. Lim, C. Park, J. S. Park, C. Kim, B. Chon, and M. Cho, “Cytoplasmic protein imaging with mid-infrared photothermal microscopy: cellular dynamics of live neurons and oligodendrocytes,” J. Phys. Chem. Lett. 10, 2857–2861 (2019).
[Crossref]

J. Phys. Chem. Ref. Data (1)

P. Schiebener, J. Straub, J. M. H. Levelt Sengers, and J. S. Gallagher, “Refractive index of water and steam as function of wavelength, temperature and density,” J. Phys. Chem. Ref. Data 19, 677–717 (1990).
[Crossref]

Light Sci. Appl. (2)

F. Merola, P. Memmolo, L. Miccio, R. Savoia, M. Mugnano, A. Fontana, G. D’Ippolito, A. Sardo, A. Iolascon, A. Gambale, and P. Ferraro, “Tomographic flow cytometry by digital holography,” Light Sci. Appl. 6, e16241 (2017).
[Crossref]

D. Zhang, L. Lan, Y. Bai, H. Majeed, M. E. Kandel, G. Popescu, and J. X. Cheng, “Bond-selective transient phase imaging via sensing of the infrared photothermal effect,” Light Sci. Appl. 8, 116 (2019).
[Crossref]

Nat. Commun. (2)

K. Okabe, N. Inada, C. Gota, Y. Harada, T. Funatsu, and S. Uchiyama, “Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy,” Nat. Commun. 3, 705 (2012).
[Crossref]

L. Shi, F. Hu, and W. Min, “Optical mapping of biological water in single live cells by stimulated Raman excited fluorescence microscopy,” Nat. Commun. 10, 4764 (2019).
[Crossref]

Nat. Methods (1)

F. Hu, L. Shi, and W. Min, “Biological imaging of chemical bonds by stimulated Raman scattering microscopy,” Nat. Methods 16, 830–842 (2019).
[Crossref]

Nat. Microbiol. (1)

Y. Wakisaka, Y. Suzuki, O. Iwata, A. Nakashima, T. Ito, M. Hirose, R. Domon, M. Sugawara, N. Tsumura, H. Watarai, T. Shimobaba, K. Suzuki, K. Goda, and Y. Ozeki, “Probing the metabolic heterogeneity of live Euglena gracilis with stimulated Raman scattering microscopy,” Nat. Microbiol. 1, 16124 (2016).
[Crossref]

Nat. Photonics (4)

Y. K. Park, C. Depeursinge, and G. Popescu, “Quantitative phase imaging in biomedicine,” Nat. Photonics 12, 578–589 (2018).
[Crossref]

C. H. Camp and M. T. Cicerone, “Chemically sensitive bioimaging with coherent Raman scattering,” Nat. Photonics 9, 295–305 (2015).
[Crossref]

Y. Cotte, F. Toy, P. Jourdain, N. Pavillon, D. Boss, P. Magistretti, P. Marquet, and C. Depeursinge, “Marker-free phase nanoscopy,” Nat. Photonics 7, 113–117 (2013).
[Crossref]

J. Shi, T. T. W. Wong, Y. He, L. Li, R. Zhang, C. S. Yung, J. Hwang, K. Maslov, and L. V. Wang, “High-resolution, high-contrast mid-infrared imaging of fresh biological samples with ultraviolet-localized photoacoustic microscopy,” Nat. Photonics 13, 609–615 (2019).
[Crossref]

Nat. Protoc. (1)

M. J. Baker, J. Trevisan, P. Bassan, R. Bhargava, H. J. Butler, K. M. Dorling, P. R. Fielden, S. W. Fogarty, N. J. Fullwood, K. A. Heys, C. Hughes, P. Lasch, P. L. Martin-Hirsch, B. Obinaju, G. D. Sockalingum, J. Sulé-Suso, R. J. Strong, M. J. Walsh, B. R. Wood, P. Gardner, and F. L. Martin, “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771–1791 (2014).
[Crossref]

Opt. Commun. (1)

E. Wolf, “Three-dimensional structure determination of semi-transparent objects from holographic data,” Opt. Commun. 1, 153–156 (1969).
[Crossref]

Opt. Express (4)

Opt. Lett. (6)

Optica (1)

Sci. Adv. (2)

D. Zhang, C. Li, C. Zhang, M. N. Slipchenko, G. Eakins, and J. X. Cheng, “Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution,” Sci. Adv. 2, e1600521 (2016).
[Crossref]

Y. Bai, D. Zhang, L. Lan, Y. Huang, K. Maize, A. Shakouri, and J. X. Cheng, “Ultrafast chemical imaging by widefield photothermal sensing of infrared absorption,” Sci. Adv. 5, eaav7127 (2019).
[Crossref]

Sci. Rep. (1)

K. Toda, M. Tamamitsu, Y. Nagashima, R. Horisaki, and T. Ideguchi, “Molecular contrast on phase-contrast microscope,” Sci. Rep. 9, 9957 (2019).
[Crossref]

Science (1)

J. X. Cheng and X. S. Xie, “Vibrational spectroscopic imaging of living systems: an emerging platform for biology and medicine,” Science 350, aaa8870 (2015).
[Crossref]

Supplementary Material (1)

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Figures (6)

Fig. 1.
Fig. 1. Concept of MV-QPI. (a) Principle of the MV-contrast acquisition in the QPI framework. The MIR light of a certain wavenumber is irradiated to the wide area of the sample, where the resonant biomolecules are selectively excited to their fundamental vibrational states. The vibrational energy is eventually transformed into heat that diffuses into the surrounding medium. The resulting photothermal RI decrease is detected by the QPI system with the spatial resolution of the VIS probe light. (b) Cross-correlative analysis enabled by MV-QPI. The phase or RI image obtained at the MIR OFF state reveals the quantitative and comprehensive morphology of the sample containing rich information about cellular shapes and distributions of intracellular organelles. Scanning the MIR wavenumber visualizes contrasts of various MV resonances at each spatial point in the FOV, which can be decomposed into individual biomolecular constituents through chemometric analysis. (c) Mechanism of the diffraction limit in the standard 2D QPI. The object is illuminated at the normal angle with a plane wave, and only a limited range of spatial-frequency information of the diffracted light is collected with the objective lens. (d) Mechanism of the depth- and super-resolution in the synthetic-aperture QPI. The object is illuminated with the angled plane wave such that higher-frequency contents that used to be outside the NA of the objective lens in (c) can be collected. Scanning the angle of the illumination allows us to computationally synthesize the 3D frequency aperture. The depth- and super-resolved imaging performance can be achieved with the expanded axial and lateral bandwidths of the 3D synthetic aperture, respectively. The black dotted curves in the frequency spectrum indicate the Ewald’s spherical cap, which determines the 3D coverage of the NA of the objective lens under a certain angle of illumination.
Fig. 2.
Fig. 2. Experimental implementations. (a) Synchronization of the pulse trains and image sensor. The VIS and MIR lasers are electrically controlled to synchronize their pulse repetitions (${\sim}{1}\;{\rm kHz}$) and relative time delay. The MIR beam is intensity modulated to be in phase with the half-harmonic of the image sensor’s frame rate (${\sim}{100}\;{\rm Hz}$). (b) MV-DH system. The DH microscope is built based on a commercial microscope housing IX73 (Olympus). The collimated VIS laser beam is used as the plane-wave probe illumination, and the magnified image of the sample is formed at the output port of the microscope. The subsequent ${4}{f}$ system is used to perform the common-path off-axis interferometry. The MIR laser beam is loosely focused to the sample with a ${{\rm CaF}_2}$ lens. QCL, quantum cascade laser. (c) MV-ODT system. The collimated VIS laser beam is split into two paths to create the Mach–Zehnder off-axis interferometer. The deflection angle of the probe beam created by the wedge prism is magnified and relayed into the sample plane by the subsequent tube lens and the illumination objective lens. The resulting angled plane-wave illumination is then collected by another objective lens, and the subsequent tube lens forms the sample’s magnified image on the image sensor. The MIR and VIS beams are combined by the dichroic mirror (DM). To avoid absorption of the MIR light by, e.g.,  ${{\rm SiO}_2}$-based optics, a reflective objective lens is chosen for illumination.
Fig. 3.
Fig. 3. Basic performance of the MV-ODT system. The liquid oil sandwiched between two ${{\rm CaF}_2}$ substrates is used as the sample, which is excited by the MIR beam with the focus diameter of ${\sim}{30}\;\unicode{x00B5}{\rm m}$. (a) Linearity of the photothermal RI change with respect to the MIR excitation pulse energy. (b) Exponential temporal decay of the photothermal RI change with the decay constant of ${\sim}{130}\;\unicode{x00B5} {\rm s}$. (c) MIR spectrum of the liquid oil obtained by the MV-ODT system, showing good agreement with the FTIR reference spectrum. Each measurement point shown in (a)–(c) represents one voxel of the FOV.
Fig. 4.
Fig. 4. Comparison of the depth-resolving capability between MV-DH and MV-ODT. (a), (b) Raw phase and RI images of the fixed HEK293 cells in ${{\rm D}_2}{\rm O}$-based PBS at the MIR OFF state, respectively. The image shown in (b) is a cross-section of one particular height of the reconstructed 3D RI tomogram. (c), (d) Photothermal contrasts of the same FOVs as those shown in (a) and (b), respectively, obtained with the MIR wavenumber tuned to ${1548}\;{{\rm cm}^{ - 1}}$. In (c), the cellular structures are contaminated by the photothermal signals originating from the out-of-focus aqueous layers. In (d), the depth resolution provided by ODT results in the higher contrasts of the cellular structures (indicated by the red arrows) as well as the more uniform and flattened background distribution originating from the MIR absorption of the in-focus water layer (indicated by the blue arrows). The depth-resolved quantification of the RI values also allows for accurate estimation of the photothermal temperature rise inside the cells (${\sim}{0.1}\;{\rm K}$) using the thermo-optic coefficient of water (${\sim}{1.4} \times {{10}^{ - 4}}$ [${1/\!K}$]). In this experiment, the MIR fluence is intentionally made nonuniform within the FOV to make a clearer comparison.
Fig. 5.
Fig. 5. Live-cell, broadband MIR-fingerprint MV-DH microscopy. (a) Raw phase image of the live COS7 cell in ${{\rm H}_2}{\rm O}$-based culture medium at the MIR OFF state. (b) MIR spectrum of the nucleolus (orange), cytoplasm (blue), and empty area (gray) indicated by the arrows of the respective colors in (a). The scanned MIR wavenumbers are in the MIR fingerprint region, where spectroscopic signatures of ${{\rm CH}_2}$ bending and peptide bond’s amide bands can be found, which are abundant in lipids and proteins, respectively. Compared to the cytoplasm, the nucleolus shows the stronger signal of the broad absorption centered at ${\sim}{1550}\;{{\rm cm}^{ - 1}}$, which coincides with the amide II band. Each spectral point represents the spatial average of ${3} \times {3}$ diffraction-limited pixels (${1.3}\;\unicode{x00B5}{\rm m} \times {1.3}\;\unicode{x00B5}{\rm m}$). (c), (d) MV images of the cell resonant to 1472 and ${1548}\;{{\rm cm}^{ - 1}}$, respectively, after the spatial and spectral normalization (see Supplement 1 for more detail). In (c), the small cytoplasmic localizations of the MV contrast at the cellular boundary could represent the existence of lipid droplets. In (d), the MV contrast shows strong selectivity on the nucleoli that could represent rich proteins.
Fig. 6.
Fig. 6. Depth-resolved, broadband MIR-fingerprint MV-ODT microscopy. (a), (b) Cross-sectional images in two different axial planes of the reconstructed RI tomogram of the fixed HEK293 cells in ${{\rm D}_2}{\rm O}$-based PBS at the MIR OFF state. The images section the podia (red arrows) and nucleoli (red square) of the cells, respectively. (c), (d) MV contrasts of the same FOVs as those shown in (a) and (b), respectively, resonant to ${1563}\;{{\rm cm}^{ - 1}}$. The photothermal temperature rise inside the cells can be estimated to be ${\sim}{0.1}\;{\rm K}$ using the thermo-optic coefficient of water. (e) MIR spectrum at one voxel of the FOV in the nucleolus indicated by the white arrow in (d), resolving the characteristic signature of the amide II band. (f) Enlargement of the red-square regions in (a)–(d). At ${z}={0}\;{\unicode{x00B5}{\rm m}}$, the nucleolus indicated by the red arrow is not visible in the RI or the photothermal contrast. At ${z}={3.3}\;{\unicode{x00B5}{\rm m}}$, the nucleolus appears in the RI contrast, which also gives the signal in the photothermal contrast, demonstrating the depth-resolving capability.

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