Boosting 2-photon vision with adaptive optics.

The 2-photon effect in vision occurs when two photons of the same wavelength are absorbed by cone photopigment in the retina and create a visual sensation matching the appearance of light close to half their wavelength. This effect is especially salient for infrared light, where humans are mostly insensitive to 1-photon isomerizations and thus any perception is dominated by 2-photon isomerizations. This phenomenon can be made more readily visible using short-pulsed lasers, which increase the likelihood of 2-photon excitation by making photon arrivals at the retina more concentrated in time. Adaptive optics provides another avenue for enhancing the 2-photon effect by focusing light more tightly at the retina, thereby increasing the spatial concentration of incident photons. This article makes three contributions. First, we demonstrate through color-matching experiments that an adaptive optics correction can provide a 25-fold increase in the luminance of the 2-photon effect-a boost equivalent to reducing pulse width by 96%. Second, we provide image-based evidence that the 2-photon effect occurs at the photoreceptor level. Third, we use our results to compute the specifications for a system that could utilize 2-photon vision and adaptive optics to image and stimulate the retina using a single infrared wavelength and reach luminance levels comparable to conventional displays.

Characterizing cone spectral classification by optoretinography.

Light propagation in photoreceptor outer segments is affected by photopigment absorption and the phototransduction amplification cascade. Photopigment absorption has been studied using retinal densitometry, while recently, optoretinography (ORG) has provided an avenue to probe changes in outer segment optical path length due to phototransduction. With adaptive optics (AO), both densitometry and ORG have been used for cone spectral classification based on the differential bleaching signatures of the three cone types. Here, we characterize cone classification by ORG, implemented in an AO line-scan optical coherence tomography (OCT), and compare it against densitometry. The cone mosaics of five color normal subjects were classified using ORG showing high probability (∼0.99), low error (<0.22%), high test-retest reliability (∼97%), and short imaging durations (< 1 hour). Of these, the cone spectral assignments in two subjects were compared against AO-scanning laser opthalmoscope densitometry. High agreement (mean: 91%) was observed between the two modalities in these two subjects, with measurements conducted 6-7 years apart. Overall, ORG benefits from higher sensitivity and dynamic range to probe cone photopigments compared to densitometry, and thus provides greater fidelity for cone spectral classification.

Miniscope3D: optimized single-shot miniature 3D fluorescence microscopy.

Miniature fluorescence microscopes are a standard tool in systems biology. However, widefield miniature microscopes capture only 2D information, and modifications that enable 3D capabilities increase the size and weight and have poor resolution outside a narrow depth range. Here, we achieve the 3D capability by replacing the tube lens of a conventional 2D Miniscope with an optimized multifocal phase mask at the objective's aperture stop. Placing the phase mask at the aperture stop significantly reduces the size of the device, and varying the focal lengths enables a uniform resolution across a wide depth range. The phase mask encodes the 3D fluorescence intensity into a single 2D measurement, and the 3D volume is recovered by solving a sparsity-constrained inverse problem. We provide methods for designing and fabricating the phase mask and an efficient forward model that accounts for the field-varying aberrations in miniature objectives. We demonstrate a prototype that is 17 mm tall and weighs 2.5 grams, achieving 2.76 µm lateral, and 15 µm axial resolution across most of the 900 × 700 × 390 µm3 volume at 40 volumes per second. The performance is validated experimentally on resolution targets, dynamic biological samples, and mouse brain tissue. Compared with existing miniature single-shot volume-capture implementations, our system is smaller and lighter and achieves a more than 2× better lateral and axial resolution throughout a 10× larger usable depth range. Our microscope design provides single-shot 3D imaging for applications where a compact platform matters, such as volumetric neural imaging in freely moving animals and 3D motion studies of dynamic samples in incubators and lab-on-a-chip devices.

On-chip fluorescence microscopy with a random microlens diffuser.

We present an on-chip, widefield fluorescence microscope, which consists of a diffuser placed a few millimeters away from a traditional image sensor. The diffuser replaces the optics of a microscope, resulting in a compact and easy-to-assemble system with a practical working distance of over 1.5 mm. Furthermore, the diffuser encodes volumetric information, enabling refocusability in post-processing and three-dimensional (3D) imaging of sparse samples from a single acquisition. Reconstruction of images from the raw data requires a precise model of the system, so we introduce a practical calibration scheme and a physics-based forward model to efficiently account for the spatially-varying point spread function (PSF). To improve performance in low-light, we propose a random microlens diffuser, which consists of many small lenslets randomly placed on the mask surface and yields PSFs that are robust to noise. We build an experimental prototype and demonstrate our system on both planar and 3D samples.