In vivo fluorescence imaging with high-resolution microlenses

Abstract

Micro-optics are increasingly used for minimally invasive in vivo imaging, in miniaturized microscopes and in lab-on-a-chip devices. Owing to optical aberrations and lower numerical apertures, a main class of microlens, gradient refractive index lenses, has not achieved resolution comparable to conventional microscopy. Here we describe high-resolution microlenses, and illustrate two-photon imaging of dendritic spines on hippocampal neurons and dual-color nonlinear optical imaging of neuromuscular junctions in live mice.

Figure 1

Aberration-corrected micro-objectives enable high-resolution imaging. (a) Optical ray diagram of micro-objectives combining a plano-convex high-index lens and a 1.0-mm-diameter GRIN lens (0.45 NA) with an adapted index profile, yielding compound microlenses with NA ≤ 0.85. Scale bar, 1 mm. (b) Wavefront errors in units of λ measured by shearing interferometry using light of 633 nm, as a function of normalized radial position (0 = on axis, 1.0 = the lens perimeter) across two orthogonal axes of the back pupil of a 0.82 NA micro-objective of 230 μm working distance in water. (c) Lateral and axial resolution measurements, using 920-nm excitation to image a 100-nm-diameter fluorescent bead, for a 0.82 NA micro-objective (microlens) and a water-immersion Olympus LUMPlan Fl/IR 0.8 NA ×40 objective ( ×40). The bead’s cross-sectional profiles in the lateral and axial dimensions are shown with Gaussian curve fits (fit), which are nearly indistinguishable from fits to the square of an Airy disc. Mean ± s.e.m. Rayleigh resolution values (1.0 ± 0.2 μm, lateral; 4.4 ± 0.2 μm, axial) were determined using n = 12 beads and measurements of this type.

Figure 2

In vivo two-photon imaging with high-resolution micro-objectives permits superior resolution than with uncorrected GRIN lenses and enables visualization of neuronal dendritic spines. (a–e) Images of GFP-expressing hippocampal pyramidal neurons in live mice. Images in a,c,e were acquired using a 0.82 NA micro-objective; b and d are corresponding images of the cells shown in a and c, acquired with a 0.48 NA singlet silver-doped GRIN objective. The image in f was obtained by dual-color imaging of a neuromuscular junction with the 0.82 NA micro-objective. Two-photon fluorescence from a YFP-expressing motor neuron (green) and second-harmonic generation from sarcomeres (blue) of the post-synaptic muscle. Images in a,b,e,f are 512 × 512 pixels. Images in c,d were cropped to 256 × 128 pixels. Scale bars, 5 μm (a–e) and 10 μm (f). All images but that in e are axial projections of three-dimensional image stacks (see Online Methods for parameters).