New applications enabled by small optical elements and their low costs are driving a rising usage of micro-optics in biomedicine. Applications in animals include minimally invasive imaging of cells in solid tissues, such as the brain and muscles, or in hollow organ tracts1-4
. Miniaturized microscopes (~1 g mass) based on micro-optics5,6
permit imaging in freely moving animals6
. Diagnostics are also arising that involve cellular-level imaging in live subjects7-9
. Emerging lab-on-a-chip devices rely on micro-optics in combination with microfluidics10
. Collectively, there are multiple realms in which micro-optics will become increasingly crucial.
A main class of microlenses used for fluorescence imaging is gradient refractive index (GRIN) lenses, which generally have numerical aperture (NA) values ≤ 0.6. These elements provide superior image quality compared to other microlenses and have enabled the first commercial fluorescence microendoscopes for small-animal and clinical applications7,9
. However, owing to their optical aberrations and lower NA values, GRIN lenses do not provide optical resolution comparable to that of conventional microscopy. The best Rayleigh resolution values achieved by two-photon fluorescence imaging with GRIN lenses are ~1.6 μm (lateral) and ~12 μm (axial), yielding highly elongated point spread functions that impede acquisition of high-quality, three-dimensional image stacks4,5
. By comparison, a diffraction-limited 0.8 NA two-photon imaging system provides ~15 times greater voxel density with 920 nm laser excitation. The loss of resolution impairs image quality and hinders examination of micrometer-scale details. Increasing NA values and correcting aberrations are essential to improving the excitation and collection of signal photons while preserving images’ fine features.
Here we report aberration-corrected imaging by combining GRIN lenses with high-NA plano-convex lenses cut from tiny, mass-produced ball lenses (). Joining the two in series yielded micro-objectives with NA ≤ 0.85. A high-index glass, such as LaSFN9 (refractive index = 1.856), for the plano-convex element permitted high NA values and ~200% greater light collection compared to a single GRIN lens. However, plano-convex lenses introduce considerable spherical aberration, which must be compensated to attain high optical resolution. Thus, once we chose a plano-convex element, we made a matched GRIN lens with a refractive index profile tuned so that the primary spherical aberrations of the two elements cancel on the optical axis. We achieved this by calculating how fourth-order (nr4
) variations in the GRIN lens’s radial refractive index profile, n
) = n0
+ nr2 r2
+ nr4 r4
+ ..., affected spherical aberrations (Supplementary Fig. 1
) and then tuning the GRIN fabrication silver-ion exchange process in glass rods11
to yield index profiles optimally matched to our plano-convex lenses (Online Methods).
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 (more ...)
We validated our strategy for a 1-mm-diameter micro-objective of 230 μm working distance and 0.82 NA that we created for two-photon microscopy. By phase-shifting shearing interferometry12
we measured the residual aberrations across 90% of the micro-objective’s back aperture and found that the root mean square wavefront errors were only 5% of a wavelength, under conditions designed to approximate imaging in tissue ( and Supplementary Fig. 2
). This degree of error is consistent with diffraction-limited performance13
. After integrating our micro-objectives into a laser-scanning instrument for two-photon fluorescence imaging, the Rayleigh resolution values determined using 100-nm-diameter fluorescent beads and 920-nm excitation (1.0 ± 0.2 μm, lateral; 4.4 ± 0.2 μm, axial; mean ± s.e.m.; n
= 12 beads) matched those of a water-immersion 0.80 NA microscope objective (Olympus ×40 LUMPlan Fl/IR) (). These values are consistent with diffraction-limited performance given the degree of laser beam expansion on our two-photon microscope (Prairie Technologies), which has a standard configuration.
To test image quality, we compared images of GFP-expressing CA1 hippocampal pyramidal neurons in live mice, as acquired with an aberration-corrected microlens or a conventional GRIN objective (). These comparisons revealed the improvement in lateral resolution attained by the corrected micro-objective as well as its superior optical sectioning ability, apparent via the reduced number of dendrites in individual images. Images obtained with the corrected microlens required reduced illumination power and often revealed neurons’ dendritic spines (). Spines were largely unresolved in images obtained with the conventional GRIN lens () and were not visible in previous neuronal imaging studies with GRIN microlenses2,14
. Given that two-photon imaging of neocortical dendritic spines near the brain surface has propelled in vivo
studies of neuronal structural plasticity15
, the resolution of dendritic spines with micro-optics might similarly enable plasticity studies in deep brain areas such as hippocampus. A second demonstration of microlens performance demanding resolution of fine details involved simultaneous second-harmonic and two-photon imaging of muscle fibers and neuromuscular junctions, yielding dual-color images ().
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 (more ...)
In conclusion, economical and simply made micro-objectives can provide on-axis resolution comparable to that of commercial water-immersion objectives. A range of working distances and magnifications will be attainable by modest variations in the refractive index profile or by using different glasses for the plano-convex element. These microlenses should be an immediate enabling technology for a wide range of applications in research, pharmaceutical and biotechnology settings.