The effects of spherical aberration on multiphoton fluorescence excitation microscopy

Abstract

Summary Multiphoton fluorescence excitation microscopy is almost invariably conducted with samples whose refractive index differ from that of the objective immersion medium, conditions that cause spherical aberration. Due to the quadratic nature of multiphoton fluorescence excitation, spherical aberration is expected to profoundly affect the depth dependence of fluorescence excitation. In order to determine the effect of refractive index mismatch in multiphoton fluorescence excitation microscopy, we measured signal attenuation, photobleaching rates and resolution degradation with depth in homogeneous samples with minimal light scattering and absorption over a range of refractive indices. These studies demonstrate that signal levels and resolution both rapidly decline with depth into refractive index mismatched samples. Analyses of photobleaching rates indicate that the preponderance of signal attenuation with depth results from decreased rates of fluorescence excitation, even in a system with a descannd emission collection pathway. Similar results were obtained in analyses of fluorescence microspheres embedded in rat kidney tissue, demonstrating that spherical aberration is an important limiting factor in multiphoton fluorescence excitation microscopy of biological samples.

In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging

“…Images were taken at a zoom of 2 or 4 in the x-y dimension (pixel size = 0.207 μm and 0.103 μm, respectively) and at 0.5-μm steps in the z -direction, with a 12-bit dynamic range. Because the axial resolution of the imaging system has been measured previously to be ≥ 1 μm under the imaging conditions employed (Rubart, 2004; Young et al, 2011), a z -step size of 0.5 μm was in accordance with the Nyquist criteria. A Kalman filter of 2 was used.…”

Intravital microscopy

“…As for the “optical transparency” of a given organ, it strongly depends on its homogeneity. Indeed, light undergoes scattering when crossing the interface between component of the tissues with different refractive indexes 38 , 39 . It is also generally true that highly vascularized tissues are less optically transparent due to the light absorption and scattering of the red blood cells.…”

“…Although a rigorous side by side comparison of the various organs has never been performed, it has been empirically determined that tissues such as the brain are optically more suitable for IVM than skeletal muscle, skin, liver or kidney. Notably, in the last couple of years several approaches have been used to “clear” tissues by using special solutions that either reduce the difference between the refractive index in the various tissue layers 38 , 39 or make them optically transparent 40 . This approach has enabled imaging neurons labeled with genetically encoded fluorescent proteins up to 8 mm below the surface with an unprecedented resolution (Fig.…”

Identification of potential mediators of oxidative damage and targets for novel treatments for chronic kidney disease

“…A further advantage of using near-infrared wavelengths, instead of ultraviolet light, for fluorescence excitation is the reduction of scattering and increase in imaging penetration depth in the sample from approximately 20 µm to 500-600 µm. However, in the kidney, the maximal penetration depth is approximately 150 µm owing to substantial scattering of emission photons from the high refractory index and heterogeneity of kidney tissue (Young et al, 2011a;Young et al, 2011b). A useful application of FLIM in conjunction with MPM in the kidney is measuring the fluorescence lifetime of NAD(P)H. NAD(P)H lifetime measurements are widely used for metabolic and redox imaging in vitro and in vivo (Bird et al, 2005;Skala et al, 2007a;Skala et al, 2007b;Leite-Silva et al, 2013;Thorling et al, 2013).…”

“…A further advantage of using near-infrared wavelengths, instead of ultraviolet light, for fluorescence excitation is the reduction of scattering and increase in imaging penetration depth in the sample from approximately 20 µm to 500-600 µm. However, in the kidney, the maximal penetration depth is approximately 150 µm owing to substantial scattering of emission photons from the high refractory index and heterogeneity of kidney tissue (Young et al, 2011a;Young et al, 2011b). …”