Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope

Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Morgado, Miguel; König, Karsten

doi:10.1117/1.jbo.21.3.036002

Cited by 24 publications

(24 citation statements)

References 44 publications

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“…This has been demonstrated in both animal and human corneas ex vivo. 13,21,[28][29][30] In the human corneal epithelium and endothelium, two lifetime components were observed, consistent with the lifetimes reported for free (s 1 ) and protein-bound (s 2 ) NAD(P)H (Table 3). 31 The evaluation of corneal cells metabolism can be an important parameter for assessing the suitability of human corneas for transplantation, particularly for endothelial cells, because they do not regenerate, and a high enough cell density is required for proper function of this layer.…”

Section: Discussionsupporting

confidence: 76%

“…The use of TPI AF intensity to investigate the corneal cell morphology was first demonstrated with rabbit's corneas by Piston et al in 1995. 19 Since then, it has been applied by us and other groups ex vivo to cornea samples of porcine, [20][21][22] mouse, 23 and human, 24,25 and in the case of rabbits also in vivo. 26 Here, we could identify different cell types in the human corneal epithelium (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…In this layer, the collagen is not organized macroscopically in noncentrosymmetric structures, which are required for SHG generation. 36 The lack of a SHG signal in the Descemet's membrane has already been reported for the porcine 21,37 and human corneas. 24 Although the main constituent of the Bowman's layer, stroma, and Descemet's membrane is the same (collagen), the collagen types and concentration vary between them.…”

Section: D Imaging Of Human Corneasmentioning

confidence: 99%

See 2 more Smart Citations

Assessment of Human Corneas Prior to Transplantation Using High-Resolution Two-Photon Imaging

Batista

Breunig

König

et al. 2018

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. The purpose of this study was to evaluate the feasibility of using two-photon imaging (TPI) to assess the condition of human corneas for transplantation.METHODS. Human corneas were imaged after different storage times: short-term (STS), mediumterm (MTS), and long-term (LTS) storage. A high-resolution, custom-built 5-dimensional multiphoton microscope with 12-fs pulsed laser excitation was used for image acquisition. RESULTS.Optical discrimination between different corneal layers and sublayers based on their morphologic characteristics revealed by two-photon autofluorescence (AF) is possible. Furthermore, all layers were characterized based on AF lifetimes to gain information on metabolic activities of cells. The NAD(P)H free to protein-bound ratio (a 1 /a 2 ) of epithelial cells increased significantly in both MTS and LTS corneas compared with STS corneas. In endothelial cells, NAD(P)H a 1 /a 2 was significantly increased in MTS samples. For keratocytes, the NAD(P)H a 1 /a 2 decreased significantly with storage time. This could indicate that the metabolic activity of the epithelial and endothelial cells reduces, whereas the activity of keratocytes increases with storage time. The analysis of the stroma SHG images indicated that the organization of collagen fibers decreases with storage time. The feasibility of measuring the endothelial cell density (ECD) using TPI was demonstrated. An ECD of 1461 6 190 cells/ mm 2 was obtained for MTS samples based on TPI.CONCLUSIONS. TPI can provide information not accessible by current clinical methods, such as the cells' metabolic state and structural organization of the stroma, with subcellular resolution. Thus, it may improve the screening process of corneas prior to transplantation and might help to optimize the storage conditions.

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Section: Discussionsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Section: D Imaging Of Human Corneasmentioning

confidence: 99%

See 1 more Smart Citation

Assessment of Human Corneas Prior to Transplantation Using High-Resolution Two-Photon Imaging

Batista

Breunig

König

et al. 2018

Invest. Ophthalmol. Vis. Sci.

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“…Moreover, in these layers, 2 lifetime components, related to the lifetimes of the free and protein‐bound states of NAD(P)H were detected (Table ). Similar AF lifetime values for these layers were reported in the literature . Considering their NAD(P)H a 1 /a 2 ratio, a lower value was observed for endothelial cells (1.07 ±0.19) than for epithelial cells (1.60 ±0.15).…”

Section: Resultssupporting

confidence: 88%

Assessment of the metabolism and morphology of the porcine cornea, lens and retina by 2‐photon imaging

Batista

Breunig

König

et al. 2018

Journal of Biophotonics

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Two-photon imaging is a noninvasive imaging technique with increasing importance in the biological and medical fields since it allows intratissue cell imaging with high resolution. We demonstrate the feasibility of using a single 2-photon instrument to evaluate the cornea, the crystalline lens and the retina based on their autofluorescence (AF). Image acquisition was performed using a custom-built 2-photon microscope for 5-dimensional microscopy with a near infrared broadband sub-15 femtosecond laser centered at 800 nanometers. Signals were detected using a spectral photomultiplier tube. The spectral ranges for the analysis of each tissue/layer AF were determined based on the spectra of each tissue as well as of pure endogenous fluorophores. The cornea, lens and retina are characterized at multiple depths with subcellular resolution based on their morphology and AF lifetime. Additionally, the AF lifetime of NAD(P)H was used to assess the metabolic activity of the cornea epithelium, endothelium and keratocytes. The feasibility to evaluate the metabolic activity of lens epithelial cells was also demonstrated, which may be used to further investigate the pathogenesis of cataracts. The results illustrate the potential of multimodal multiphoton imaging as a novel ophthalmologic technique as well as its potential as a diagnostic tool.

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“…Both the peak value and the peak location of the detected signal (time-intensity cytometric pulse) of each detector channel are influenced by the spectral overlap due to the different fluorochrome lifetimes. That is, the time delays between the observed fluorescence signals ( fl a , fl b ) and fs are influenced by the spectral overlap [12,13,14,15]. …”

Section: Theorymentioning

confidence: 99%

Representation Method for Spectrally Overlapping Signals in Flow Cytometry Based on Fluorescence Pulse Time-Delay Estimation

Zhang

Lou

Meng

et al. 2016

Sensors

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Flow cytometry is being applied more extensively because of the outstanding advantages of multicolor fluorescence analysis. However, the intensity measurement is susceptible to the nonlinearity of the detection method. Moreover, in multicolor analysis, it is impossible to discriminate between fluorophores that spectrally overlap; this influences the accuracy of the fluorescence pulse signal representation. Here, we focus on spectral overlap in two-color analysis, and assume that the fluorescence follows the single exponential decay model. We overcome these problems by analyzing the influence of the spectral overlap quantitatively, which enables us to propose a method of fluorescence pulse signal representation based on time-delay estimation (between fluorescence and scattered pulse signals). First, the time delays are estimated using a modified chirp Z-transform (MCZT) algorithm and a fine interpolation of the correlation peak (FICP) algorithm. Second, the influence of hardware is removed via calibration, in order to acquire the original fluorescence lifetimes. Finally, modulated signals containing phase shifts associated with these lifetimes are created artificially, using a digital signal processing method, and reference signals are introduced in order to eliminate the influence of spectral overlap. Time-delay estimation simulation and fluorescence signal representation experiments are conducted on fluorescently labeled cells. With taking the potentially overlap of autofluorescence as part of the observed fluorescence spectrum, rather than distinguishing the individual influence, the results show that the calculated lifetimes with spectral overlap can be rectified from 8.28 and 4.86 ns to 8.51 and 4.63 ns, respectively, using the comprehensive approach presented in this work. These values agree well with the lifetimes (8.48 and 4.67 ns) acquired for cells stained with single-color fluorochrome. Further, these results indicate that the influence of spectral overlap can be eliminated effectively. Moreover, modulation, mixing with reference signals, and low-pass filtering are performed with a digital signal processing method, thereby obviating the need for a high-speed analog device and complex circuit system. Finally, the flexibility of the comprehensive method presented in this work is significantly higher than that of existing methods.

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Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope

Cited by 24 publications

References 44 publications

Assessment of Human Corneas Prior to Transplantation Using High-Resolution Two-Photon Imaging

Assessment of Human Corneas Prior to Transplantation Using High-Resolution Two-Photon Imaging

Assessment of the metabolism and morphology of the porcine cornea, lens and retina by 2‐photon imaging

Representation Method for Spectrally Overlapping Signals in Flow Cytometry Based on Fluorescence Pulse Time-Delay Estimation

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