• Laser & Optoelectronics Progress
  • Vol. 59, Issue 6, 0600012 (2022)
Yitao Cao1、2, Xue Wang1、2, Xinchao Lu1、*, and Chengjun Huang1、2、**
Author Affiliations
  • 1Health Electronics Center, Institute of Microelectronics of Chinese Academy of Sciences, Beijing 100029, China
  • 2School of Microelectronics, University of Chinese Academy of Sciences, Beijing 100049, China
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    DOI: 10.3788/LOP202259.0600012 Cite this Article Set citation alerts
    Yitao Cao, Xue Wang, Xinchao Lu, Chengjun Huang. Label-Free Optical Microscopy Technique and Its Biomedical Applications[J]. Laser & Optoelectronics Progress, 2022, 59(6): 0600012 Copy Citation Text show less
    CARS. (a) Principle of CARS[10] (Reproduced under the terms of the CC-BY license, Copyright 2014, Potcoava M C et al.); (b) schematic of CARS microscopy[11](Reproduced with permission, Copyright 2018, Society of Photo‑Optical Instrumentation Engineers)
    Fig. 1. CARS. (a) Principle of CARS[10] (Reproduced under the terms of the CC-BY license, Copyright 2014, Potcoava M C et al.); (b) schematic of CARS microscopy[11](Reproduced with permission, Copyright 2018, Society of Photo‑Optical Instrumentation Engineers)
    Principle of SRS. (a) Energy diagram of SRS; (b) input and output spectra of SRS; (c) detection scheme of SRL; (d) setup of SRS microscopy[12] (Reproduced with permission, Copyright 2008, American Association for the Advancement of Science)
    Fig. 2. Principle of SRS. (a) Energy diagram of SRS; (b) input and output spectra of SRS; (c) detection scheme of SRL; (d) setup of SRS microscopy[12] (Reproduced with permission, Copyright 2008, American Association for the Advancement of Science)
    CARS images of 3T3-L1 cell culture at different time after adding induction media[15]. (a) 0 h; (b) 24 h; (c) 48 h; (d) 60 h; (e) 96 h; (f) 192 h (Reproduced under the terms of the CC-BY license, Copyright 2003, American Society for Biochemistry and Molecular Biology )
    Fig. 3. CARS images of 3T3-L1 cell culture at different time after adding induction media[15]. (a) 0 h; (b) 24 h; (c) 48 h; (d) 60 h; (e) 96 h; (f) 192 h (Reproduced under the terms of the CC-BY license, Copyright 2003, American Society for Biochemistry and Molecular Biology )
    Comparison between SRS images and HES images of pancreatic tissue[28]. (a) SRS image of healthy pancreatic tissue; (b) HES image of healthy pancreatic tissue; (c) SRS image of cancerous pancreatic tissue; (d) HES image of cancerous pancreatic tissue; (e) SRS image of the tumor area of pancreatic tissue; (f) HES image of the tumor area of pancreatic tissue (Reproduced under the terms of the CC-BY license, Copyright 2019, Sarri B et al.)
    Fig. 4. Comparison between SRS images and HES images of pancreatic tissue[28]. (a) SRS image of healthy pancreatic tissue; (b) HES image of healthy pancreatic tissue; (c) SRS image of cancerous pancreatic tissue; (d) HES image of cancerous pancreatic tissue; (e) SRS image of the tumor area of pancreatic tissue; (f) HES image of the tumor area of pancreatic tissue (Reproduced under the terms of the CC-BY license, Copyright 2019, Sarri B et al.)
    Schematic diagram of photothermal microscopy[31](Reproduced with permission, Copyright 2004, American Physical Society)
    Fig. 5. Schematic diagram of photothermal microscopy[31](Reproduced with permission, Copyright 2004, American Physical Society)
    Photothermal microscopy images. (a) COS-7 cells before digitonin addition[32]; (b)(c) COS-7 cells after digitonin addition[32](Reproduced under the terms of OSA Open Access Publishing Agreement, Copyright 2007, Optical Society of America); (d) single mitochondria[33] (Reproduced with permission, Copyright 2010, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim); (e) HeLa cells[34] (Reproduced under the terms of OSA Open Access Publishing Agreement, Copyright 2019, Optical Society of America); (f) melanoma cells[35] (Reproduced under the terms of OSA Open Access Publishing Agreement, Copyright 2015, Optical Society of America); (g) oligodendrocytes[36] (Reproduced with permission, Copyright 2019, American Chemical Society); (h) vesicular stomatitis virus[37]; (i) poxvirus[37] (Reproduced with permission, Copyright 2021, American Chemical Society)
    Fig. 6. Photothermal microscopy images. (a) COS-7 cells before digitonin addition[32]; (b)(c) COS-7 cells after digitonin addition[32](Reproduced under the terms of OSA Open Access Publishing Agreement, Copyright 2007, Optical Society of America); (d) single mitochondria[33] (Reproduced with permission, Copyright 2010, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim); (e) HeLa cells[34] (Reproduced under the terms of OSA Open Access Publishing Agreement, Copyright 2019, Optical Society of America); (f) melanoma cells[35] (Reproduced under the terms of OSA Open Access Publishing Agreement, Copyright 2015, Optical Society of America); (g) oligodendrocytes[36] (Reproduced with permission, Copyright 2019, American Chemical Society); (h) vesicular stomatitis virus[37]; (i) poxvirus[37] (Reproduced with permission, Copyright 2021, American Chemical Society)
    3D images of photothermal microscopy. (a) Zebrafish gill larvae blood vessels[38] (Reproduced with permission, Copyright 2010,American Institute of Physics); (b) melanoma[39] (Reproduced under the terms of the CC-BY license, Copyright 2016,He J P et al.); (c) lipid droplets stored in PC-3 prostate cancer cells[40] (Reproduced under the terms of the CC-BY license, Copyright 2016,Zhang Delong et al.)
    Fig. 7. 3D images of photothermal microscopy. (a) Zebrafish gill larvae blood vessels[38] (Reproduced with permission, Copyright 2010,American Institute of Physics); (b) melanoma[39] (Reproduced under the terms of the CC-BY license, Copyright 2016,He J P et al.); (c) lipid droplets stored in PC-3 prostate cancer cells[40] (Reproduced under the terms of the CC-BY license, Copyright 2016,Zhang Delong et al.)
    Schematic diagrams of SPRM. (a) Schematic diagram of wide-field SPRM; (b) schematic diagram of scanning SPRM[43](Reproduced under the terms of OSA Open Access Publishing Agreement, Copyright 2012, Optical Society of America)
    Fig. 8. Schematic diagrams of SPRM. (a) Schematic diagram of wide-field SPRM; (b) schematic diagram of scanning SPRM[43](Reproduced under the terms of OSA Open Access Publishing Agreement, Copyright 2012, Optical Society of America)
    SPRM imaging. (a) Single H1N1 influenza virus[46]; (b) single T4 bacteriophage[47] (Reproduced with permission, Copyright 2017, Society of Photo‑Optical Instrumentation Engineers); (c) single DNA molecular chain[48] (Reproduced with permission, Copyright 2014, American Chemical Society)
    Fig. 9. SPRM imaging. (a) Single H1N1 influenza virus[46]; (b) single T4 bacteriophage[47] (Reproduced with permission, Copyright 2017, Society of Photo‑Optical Instrumentation Engineers); (c) single DNA molecular chain[48] (Reproduced with permission, Copyright 2014, American Chemical Society)
    Study on molecular dynamic using SPRM. (a) 3D structure of microtubule[51] (Reproduced with permission, Copyright WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim); (b) z-axis motility profile of single bacteria[52] (Reproduced with permission, Copyright 2016, American Chemical Society); (c) 2D tracking of single exosome[53]
    Fig. 10. Study on molecular dynamic using SPRM. (a) 3D structure of microtubule[51] (Reproduced with permission, Copyright WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim); (b) z-axis motility profile of single bacteria[52] (Reproduced with permission, Copyright 2016, American Chemical Society); (c) 2D tracking of single exosome[53]
    Study on cell-substrate interaction using SPRM. (a) Distribution of adhesion intensity of cell-substrate[55]; (b) distribution of cell-substrate distance[56]; (c) 3D reconstruction process of cell-substrate[57] (Reproduced with permission, Copyright 2012,2014,2018, American Chemical Society)
    Fig. 11. Study on cell-substrate interaction using SPRM. (a) Distribution of adhesion intensity of cell-substrate[55]; (b) distribution of cell-substrate distance[56]; (c) 3D reconstruction process of cell-substrate[57] (Reproduced with permission, Copyright 2012,2014,2018, American Chemical Society)
    Schematic diagram of iSCAT[62]. (a) Wide-field iSCAT; (b) fast beam-scanning iSCAT (Reproduced under the terms of the CC-BY-NC-ND license, Copyright 2021, Priest L et al.)
    Fig. 12. Schematic diagram of iSCAT[62]. (a) Wide-field iSCAT; (b) fast beam-scanning iSCAT (Reproduced under the terms of the CC-BY-NC-ND license, Copyright 2021, Priest L et al.)
    iSCAT imaging of single virus and biomolecule. (a) Single SV40[63]; (b) single streptavidin[66] (Reproduced with permission, Copyright 2007,2017, American Chemical Society)
    Fig. 13. iSCAT imaging of single virus and biomolecule. (a) Single SV40[63]; (b) single streptavidin[66] (Reproduced with permission, Copyright 2007,2017, American Chemical Society)
    Study on biomolecular dynamics using iSCAT. (a) Vesicle changes[67](Reproduced with permission, Copyright 2013, American Chemical Society); (b) nanodomain formation[68]; (c) myosin filament dynamics[70](Reproduced under the terms of the CC-BY license, Copyright 2020, Biophysical Society)
    Fig. 14. Study on biomolecular dynamics using iSCAT. (a) Vesicle changes[67](Reproduced with permission, Copyright 2013, American Chemical Society); (b) nanodomain formation[68]; (c) myosin filament dynamics[70](Reproduced under the terms of the CC-BY license, Copyright 2020, Biophysical Society)
    Study on structure of living cells using iSCAT[71]. (a) Cell focaladhesions and filopodium structure;(b) cell edge structure (Reproduced under the terms of the CC-BY-NC license, Copyright 2018, The Royal Society of Chemistry)
    Fig. 15. Study on structure of living cells using iSCAT[71]. (a) Cell focaladhesions and filopodium structure;(b) cell edge structure (Reproduced under the terms of the CC-BY-NC license, Copyright 2018, The Royal Society of Chemistry)
    TechniqueLateral resolutionAxial resolutionLaser powerSensitivityTemporal resolutionImaging timeRef
    CRSCARS600 nm1.1 µm

    14 mW(pump)

    7 mW(Stokes)

    -10 μs~1.6 s20
    SRS400 nm2 µm

    60 mW(pump)

    100 mW(Stokes)

    -2 μs~1 s27
    PTM290 nm860 nm

    0.9 mW(pump)

    7 mW(probe)

    5 nm5 μs

    several

    seconds

    34
    SPRMWide-field260 nm-5 mW15 nm2 ms2 ms4951
    Scanning186 nm2.3 nm--100 ms73 min57
    iSCATWide-field~190 nm-10 mW65 ku~0.33 ms~0.33 ms64
    Fast beam-scanning1 μm-2.5 kW/cm260 ku-~100 µs65
    Table 1. Performance comparison of four label-free optical microscopic imaging techniques
    Yitao Cao, Xue Wang, Xinchao Lu, Chengjun Huang. Label-Free Optical Microscopy Technique and Its Biomedical Applications[J]. Laser & Optoelectronics Progress, 2022, 59(6): 0600012
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