Two-color, two-photon, and excited-state absorption microscopy

Fu, Dan; Ye, Tong; Matthews, Thomas E.; Yurtsever, Günay; Warren, Warren S.

doi:10.1117/1.2780173

Cited by 141 publications

(108 citation statements)

References 38 publications

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“…Our approach consists of imaging thin unstained biopsy slides with pump-probe microscopy, an approach that has already shown significant promise in differentiating melanomas from cutaneous melanocytic nevi [14,15], as well as conjunctival melanoma from primary acquired melanosis [16]. Pump-probe microscopy provides subcellular resolution of the chemical composition of endogenous pigments by probing their electronic excited state dynamics [14,17]. A very important advantage of this approach is that molecular signatures of melanins do not degrade over time-we have shown that Jurassic-aged eumelanin has the same pump-probe features as its modern counterpart [18]-which enables us to use archived tissue samples in retrospective databases, an approach that is not possible with immunohistochemistry methods [19].…”

Section: Introductionmentioning

confidence: 99%

Pump-probe imaging of pigmented cutaneous melanoma primary lesions gives insight into metastatic potential

Robles

Deb

Wilson

et al. 2015

Biomed. Opt. Express

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Metastatic melanoma is associated with a poor prognosis, but no method reliably predicts which melanomas of a given stage will ultimately metastasize and which will not. While sentinel lymph node biopsy (SLNB) has emerged as the most powerful predictor of metastatic disease, the majority of people dying from metastatic melanoma still have a negative SLNB. Here we analyze pump-probe microscopy images of thin biopsy slides of primary melanomas to assess their metastatic potential. Pumpprobe microscopy reveals detailed chemical information of melanin with subcellular spatial resolution. Quantification of the molecular signatures without reference standards is achieved using a geometrical representation of principal component analysis. Melanin structure is analyzed in unison with the chemical information by applying principles of mathematical morphology. Results show that melanin in metastatic primary lesions has lower chemical diversity than non-metastatic primary lesions, and contains two distinct phenotypes that are indicative of aggressive disease. Further, the mathematical morphology analysis reveals melanin in metastatic primary lesions has a distinct "dusty" quality. Finally, a statistical analysis shows that the combination of the chemical information with spatial structures predicts metastatic potential with much better sensitivity than SLNB and high specificity, suggesting pump-probe microscopy can be an important tool to help predict the metastatic potential of melanomas.

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Section: Introductionmentioning

confidence: 99%

Pump-probe imaging of pigmented cutaneous melanoma primary lesions gives insight into metastatic potential

Robles

Deb

Wilson

et al. 2015

Biomed. Opt. Express

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“…7 We have developed a new excited state absorption ͑ESA͒-based method to image hemoglobin and melanin in tissue without any labeling. 8,9 Previously, we have demonstrated high-resolution in vivo imaging of the blood vessels in mouse ears. 8 In this report, we improved the sensitivity by shifting the pump and probe pulses toward longer wavelengths ͑720 to 760 nm and 810 nm͒, which will also give us deeper penetration in tissue.…”

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confidence: 99%

Label-free in vivo optical imaging of microvasculature and oxygenation level

Matthews

Ye³

et al. 2008

J. Biomed. Opt.

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Abstract. The ability to perform high-resolution imaging of microvasculature and its oxygenation is very important in studying early tumor development. Toward this goal, we improved upon our excited state absorption ͑ESA͒-based imaging technique to allow us to not only image hemoglobin directly but also differentiate between oxyand deoxyhemoglobin in tissue. We demonstrate the separation of arterioles from venules in a live nude mouse ear using our imaging technique. Blood vessel structure and oxygenation provides important information about angiogenesis and hypoxia in tumor development.1-3 Microscopic imaging provides a very intuitive way of visualizing the angiogenic vasculature differences between tumors and healthy tissue. Recent developments in confocal and multiphoton fluorescence microscopy ͑MFM͒ based on fluorescent labeling of blood plasma or vessel walls allow submicrometer-resolution imaging of microvasculature and provide sectioning capabilities. [4][5][6] These methods can measure a number of important parameters such as blood flow rate, blood vessel diameter, vascular density, and endothelial permeability. However, the necessary use of a tracer or fluorescence tag is an obvious limitation, especially for human studies. Another important aspect that is missing from confocal or multiphoton imaging of blood vessels is the oxygenation level, which is a key determinant of the physiological state of tissue. Because of the irregular pattern and organization of vasculature in tumors, some cancerous cells are located more than 100 m ͑the diffusion limit for oxygen͒ away from blood vessels and become hypoxic. Tumor hypoxia has been suggested as a cause of malignant transformation and a source of resistance to current cancer therapies. Therefore, measuring blood oxygen delivery and hypoxia at the microvascular level could be of important prognostic value for tumor diagnosis and treatment. 7 We have developed a new excited state absorption ͑ESA͒-based method to image hemoglobin and melanin in tissue without any labeling. 8,9 Previously, we have demonstrated high-resolution in vivo imaging of the blood vessels in mouse ears. 8 In this report, we improved the sensitivity by shifting the pump and probe pulses toward longer wavelengths ͑720 to 760 nm and 810 nm͒, which will also give us deeper penetration in tissue. More importantly, the ratios of two-color ESA signal sizes of oxyhemoglobin and deoxyhemoglobin are very different when we simply switch the pump and probe sequence. Therefore, it is possible to determine their ratio by comparing images taken under these two different conditions. We are able to use this technique to differentiate between arterioles and venules in a live mouse ear. Our two-color ESA imaging technique could potentially be very useful in studying the microvasculature and oxygenation level changes in early tumor development.The experimental setup has been described in detail previously.9,10 Here, we made some modifications to the original setup. Both the 810-nm beam and the 740-nm beam wer...

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“…In addition, Lakowicz et al proposed a method for rejecting background noise by detuning the repetition rate of each light source, resulting in beating of the fluorescence, which can be detected using a lock-in amplifier. Fu et al subsequently demonstrated imaging using this repetition rate detuning scheme [23]. Cambaliza and Saloma [24], Ibáñez-López et al [25], Blanca and Saloma [26], Xiao et al [27], Caballero et al [28], Wang et al [29] and Dang et al [30] have computed increased axial resolution of ND-2PE over D-2PE using various constructed illumination geometries.…”

Section: Introductionmentioning

confidence: 99%

Non-degenerate 2-photon excitation in scattering medium for fluorescence microscopy

Yang¹,

Abashin²,

Saisan³

et al. 2016

Opt. Express

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Non-degenerate 2-photon excitation (ND-2PE) of a fluorophore with two laser beams of different photon energies offers an independent degree of freedom in tuning of the photon flux for each beam. This feature takes advantage of the infrared wavelengths used in degenerate 3-photon excitation (D-3PE) microscopy to achieve increased penetration depths, while preserving a relatively high 2-photon excitation cross section in comparison to that of D-3PE. Here, using spatially and temporally aligned Ti:Sapphire laser and optical parametric oscillator beams operating at near infrared (NIR) and short-wavelength infrared (SWIR) optical frequencies, we employ ND-2PE and provide a practical demonstration that a constant fluorophore emission intensity is achievable deeper into a scattering medium using ND-2PE as compared to the commonly used degenerate 2-photon excitation (D-2PE). References and links1. K. Svoboda and R. Yasuda, "Principles of two-photon excitation microscopy and its applications to neuroscience," Neuron 50(6), 823-839 (2006). 2. J. N. Kerr and W. Denk, "Imaging in vivo: watching the brain in action," Nat. Rev. Neurosci. 9(3), 195-205 (2008). 3. W. Denk and K. Svoboda, "Photon upmanship: why multiphoton imaging is more than a gimmick," Neuron 18(3), 351-357 (1997). 4. F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2(12), 932-940 (2005 11. D. Kobat, N. G. Horton, and C. Xu, "In vivo two-photon microscopy to 1.6-mm depth in mouse cortex," J. Biomed. Opt. 16(10), 106014 (2011). 12. R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, "Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser," Sci. Rep. 3, 1014 (2013). 13. P. Theer and W. Denk, "On the fundamental imaging-depth limit in two-photon microscopy," J. Opt. Soc. Am.A adaptive optical recovery of optimal resolution over large volumes," Nat. Methods 11(6), 625-628 (2014).

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Two-color, two-photon, and excited-state absorption microscopy

Cited by 141 publications

References 38 publications

Pump-probe imaging of pigmented cutaneous melanoma primary lesions gives insight into metastatic potential

Pump-probe imaging of pigmented cutaneous melanoma primary lesions gives insight into metastatic potential

Label-free in vivo optical imaging of microvasculature and oxygenation level

Non-degenerate 2-photon excitation in scattering medium for fluorescence microscopy

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