Label-free imaging of heme proteins with two-photon excited photothermal lens microscopy

Lü, Sijia; Min, Wei; Chong, Shasha; Holtom, Gary R.; Xie, Xi Shan

doi:10.1063/1.3308485

Cited by 79 publications

(68 citation statements)

References 19 publications

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“…In the SRS spectra the signal almost disappears off resonance and therefore allowing chemically specific imaging. The small background in the SRS images may be due to a combination of photo-thermal effects [25,26] and two-photon two-colour absorption [27,28] or cross phase modulation. …”

Section: Comparison Of Cars and Srsmentioning

confidence: 99%

Chemically specific imaging and in-situ chemical analysis of articular cartilage with stimulated Raman scattering

et al. 2013

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Stimulated Raman Scattering has been applied to unstained samples of articular cartilage enabling the investigation of living cells within fresh tissue. Hyperspectral SRS measurements over the CH vibrational region showed variations in protein and lipid content within the cells, pericellular matrix and interteritorial matrix. Changes in the cells and pericellular matrix were investigated as a function of depth into the cartilage. Lipid was detected in the pericellular matrix of superficial zone chondrocytes. The spectral profile of lipid droplets within the chondrocytes indicated that they contained predominantly unsaturated lipids. The mineral content has been imaged by using the PO4 3-vibration at 959cm -1 and the CO3 2-vibration at 1070cm -1 . Both changes in cells and mineralization are known to be important factors in the progression of osteoarthritis. SRS enables these to be visualised in fresh unstained tissue and consequently should benefit osteoarthiritis research

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Section: Comparison Of Cars and Srsmentioning

confidence: 99%

Chemically specific imaging and in-situ chemical analysis of articular cartilage with stimulated Raman scattering

et al. 2013

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“…The chloroplast signal followed a linear power dependence for both the pump and Stokes beams ( figure 4C and D) which follows the expected trend for a two-color two-photon absorption (TPA) process 16 . The discrepancy in phase can be explained by two-photon photothermal lensing (TPPL) 22,23 . Local heating resulting from resonant two-photon absorption of the pump beam causes a refractive index gradient, which results in a deflection of the probe (in this case the Stokes) beam that is detected as a loss in intensity.…”

Section: Resultsmentioning

confidence: 99%

Label-free Chemically Specific Imaging in Planta with Stimulated Raman Scattering Microscopy

Mansfield

Littlejohn²,

Seymour³

et al. 2013

Anal. Chem.

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The growing world population puts ever-increasing demands on the agricultural and agrochemical industries to increase agricultural yields. This can only be achieved by investing in fundamental plant and agrochemical research and in the development of improved analytical tools to support research in these areas.There is currently a lack of analytical tools that provide non-invasive structural and chemical analysis of plant tissues at the cellular scale. Imaging techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy provide labelfree chemically specific image contrast based on vibrational spectroscopy. Over the past decade these techniques have been shown to offer clear advantages for a vast range of biomedical research applications. The intrinsic vibrational contrast provides label-free quantitative functional analysis; it does not suffer from photobleaching; and allows near realtime imaging in 3D with submicron spatial resolution. However, due to the susceptibility of current detection schemes to optical absorption and fluorescence from pigments (such as chlorophyll) the plant science and agrochemical research communities have not been able to benefit from these techniques and their application in plant research has remained virtually unexplored.In this paper we explore the effect of chlorophyll fluorescence and absorption in CARS and SRS microscopy. We show that with the latter it is possible to use phase-sensitive detection to separate the vibrational signal from the (electronic) absorption processes. Finally we demonstrate the potential of SRS for a range of in planta applications by presenting in-situ chemical analysis of plant cell wall components, epicuticular waxes, and the deposition of agrochemical formulations onto the leaf surface.

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“…It has been applied to visualize the distribution of endogenous chromoproteins in biological specimens such as cytochromes in mitochondria [1][2][3][4], hemoglobin in red blood cells in blood vessels [5,6], and melanin pigments in skin cancer [7]. Furthermore, gold nanoparticles have been used to identify biomolecules in antibody labelling techniques applied in cellular imaging [8][9][10][11][12].…”

Section: Introductionmentioning

confidence: 99%

Photothermal Microscopy for High Sensitivity and High Resolution Absorption Contrast Imaging of Biological Tissues

Miyazaki

Kobayahsi

2017

Photonics

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Photothermal microscopy is useful to visualize the distribution of non-fluorescence chromoproteins in biological specimens. Here, we developed a high sensitivity and high resolution photothermal microscopy with low-cost and compact laser diodes as light sources. A new detection scheme for improving signal to noise ratio more than 4-fold is presented. It is demonstrated that spatial resolution in photothermal microscopy is up to nearly twice as high as that in the conventional widefield microscopy. Furthermore, we demonstrated the ability for distinguishing or identifying biological molecules with simultaneous muti-wavelength imaging. Simultaneous photothermal and fluorescence imaging of mouse brain tissue was conducted to visualize both neurons expressing yellow fluorescent protein and endogenous non-fluorescent chromophores.

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Label-free imaging of heme proteins with two-photon excited photothermal lens microscopy

Cited by 79 publications

References 19 publications

Chemically specific imaging and in-situ chemical analysis of articular cartilage with stimulated Raman scattering

Chemically specific imaging and in-situ chemical analysis of articular cartilage with stimulated Raman scattering

Label-free Chemically Specific Imaging in Planta with Stimulated Raman Scattering Microscopy

Photothermal Microscopy for High Sensitivity and High Resolution Absorption Contrast Imaging of Biological Tissues

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