Label-free nonlinear optical imaging of mouse retina

Abstract

A nonlinear optical (NLO) microscopy system integrating stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) was developed to image fresh mouse retinas. The morphological and functional details of various retinal layers were revealed by the endogenous NLO signals. Particularly, high resolution label-free imaging of retinal neurons and nerve fibers in the ganglion cell and nerve fiber layers was achieved by capturing endogenous SRS and TPEF signals. In addition, the spectral and temporal analysis of TPEF images allowed visualization of different fluorescent components in the retinal pigment epithelium (RPE). Fluorophores with short TPEF lifetime, such as A2E, can be differentiated from other long-lifetime components in the RPE. The NLO imaging method would provide important information for investigation of retinal ganglion cell degeneration and holds the potential to study the biochemical processes of visual cycle in the RPE. References and links1. C. J. Jeon, E. Strettoi, and R. H. Masland, "The major cell populations of the mouse retina," J. Neurosci. 18(21), 8936-8946 (1998). 2. E. Prokofyeva and E. Zrenner, "Epidemiology of major eye diseases leading to blindness in Europe: a literature review," Ophthalmic Res. 47(4), 171-188 (2012). 3. J. P. Dunn, S. W. Noorily, M. Petri, D. Finkelstein, J. T. Rosenbaum, and D. A. Jabs, "Antiphospholipid antibodies and retinal vascular disease," Lupus 5(4), 313-322 (1996). 4. O. Strauss, "The retinal pigment epithelium in visual function," Physiol. Rev. 85(3), 845-881 (2005). contrast mechanism for label-free imaging of microvasculature in vivo," Opt. Lett. 36(6), 834-836 (2011).

In vivo study of metabolic dynamics and heterogeneity in brown and beige fat by label‐free multiphoton redox and fluorescence lifetime microscopy

“…Over the past decades, the ORR has been widely used as an intrinsic indicator of redox states in cells and tissues [26][27][28]. In addition to redox ratio imaging, fluorescence lifetime imaging microscopy (FLIM) has also been used for mapping of cellular metabolism [29][30][31] and detection of pathogens [32]. We found that the ORR of BAT increases significantly after NE injection in mice, which is correlated with the temperature rise in BAT induced by thermogenesis [14].…”

Multimodal nonlinear optical microscopy reveals critical role of kinesin-1 in cartilage development

“…Details of the integrated multimodal NLO microscopy system used in this work have been described in our previous study [21]. Briefly, in SRS imaging, portion of a femtosecond Ti:sapphire laser of 140 fs pulse duration (Chameleon Ultra II, Coherent, Santa Clara, CA) tuned at 830 nm was used as the pump beam.…”

“…1. Nonlinear optical (NLO) microscopy provides an ideal solution to resolve the structural characteristics in tissues and cells, featured with its inherent 3D resolution and capacity for deep penetration [19][20][21]. Recently, NLO microscopy has been used to study the chondrocytes and ECM of cartilage [22][23][24][25].…”

Epi-direction detected multimodal imaging of an unstained mouse retina with a Yb-fiber laser

“…Fiber lasers require no water-cooling, are less sensitive to air currents or changes in temperature, and their applications to three-photon processes are above the DNA damaging UV regime. While Ti: sapphire lasers have been used for multiphoton microscopy of the retina, 14 their third-harmonic is centered at 266 nm, which matches the absorption maximum of DNA. By establishing the native multiphoton signals that arise from wild type ex vivo retinas, in vivo and clinical use for the diagnosis and treatment of retinal diseases, such as age-related macular degeneration (AMD) can be established.…”