In vivo two-photon imaging of the mouse retina

Abstract

Though in vivo two-photon imaging has been demonstrated in non-human primates, improvements in the signal-to-noise ratio (SNR) would greatly improve its scientific utility. In this study, extrinsic fluorophores, expressed in otherwise transparent retinal ganglion cells, were imaged in the living mouse eye using a two-photon fluorescence adaptive optics scanning laser ophthalmoscope. We recorded two orders of magnitude greater signal levels from extrinsically labeled cells relative to previous work done in two-photon autofluorescence imaging of primates. Features as small as single dendrites in various layers of the retina could be resolved and predictions are made about the feasibility of measuring functional response from cells. In the future, two-photon imaging in the intact eye may allow us to monitor the function of retinal cell classes with infrared light that minimally excites the visual response.

Multimodal and multiplex spectral imaging of rat cornea ex vivo using a white‐light laser source

“…As shown in the upper axis in Figure 2, almost all fundamental Raman bands are simultaneously observed by using the white-light Stokes pulses. Figure 3 shows the spectral profiles of the imaginary part of the CARS signal (Im [χ (3) ]), each of which is retrieved from the spectra shown in Figure 2. These Im [χ (3) ] spectra were calculated by maximum entropy method (MEM) [25].…”

“…Figure 3 shows the spectral profiles of the imaginary part of the CARS signal (Im [χ (3) ]), each of which is retrieved from the spectra shown in Figure 2. These Im [χ (3) ] spectra were calculated by maximum entropy method (MEM) [25]. The MEM does not require any a priori knowledge of the vibrational bands but still is able to retrieve the phase information on the third-order nonlinear susceptibility χ (3) , whose imaginary part corresponds to ordinary (spontaneous) Raman spectra [26].…”

“…These Im [χ (3) ] spectra were calculated by maximum entropy method (MEM) [25]. The MEM does not require any a priori knowledge of the vibrational bands but still is able to retrieve the phase information on the third-order nonlinear susceptibility χ (3) , whose imaginary part corresponds to ordinary (spontaneous) Raman spectra [26]. Therefore, the intensity of the retrieved band, Im [χ (3) ], is proportional to the molecular concentration.…”

“…The capability of quantitative spectral analysis attained by the combination of multiplex CARS and MEM is one of the most important advantages of our system. It is noted that we have performed noise filtering of the Im [χ (3) ] spectra by using the singular value decomposition (SVD) analysis [27]. The Im [χ (3) ] spectra show rich information on the molecular composition of the cornea tissue.…”

“…The signal is generated from a tightly-focused confined volume, resulting in three-dimensional sectioning capability. So far, various kinds of nonlinear optical processes such as two-or three-photon excited fluorescence [3,4], second-or third-harmonic generation (SHG or THG) [5][6][7][8][9][10][11], sum frequency generation (SFG) [12], third-order sum frequency generation (TSFG) [13], coherent anti-Stokes Raman scattering (CARS) [14][15][16] and stimulated Raman scattering (SRS) [17][18][19] have been applied to microscopy. Since these nonlinear optical processes simultaneously take place, we are able to combine various multiphoton microscopic techniques [13,20].…”