Based on partially-coherent digital in-line holography, we report a field-portable microscope that can render lensfree colour images over a wide field-of-view of e.g., >20 mm2. This computational holographic microscope weighs less than 145 grams with dimensions smaller than 17×6×5 cm, making it especially suitable for field settings and point-of-care use. In this lensfree imaging design, we merged a colorization algorithm with a source shifting based multi-height pixel super-resolution technique to mitigate ‘rainbow’ like colour artefacts that are typical in holographic imaging. This image processing scheme is based on transforming the colour components of an RGB image into YUV colour space, which separates colour information from brightness component of an image. The resolution of our super-resolution colour microscope was characterized using a USAF test chart to confirm sub-micron spatial resolution, even for reconstructions that employ multi-height phase recovery to handle dense and connected objects. To further demonstrate the performance of this colour microscope Papanicolaou (Pap) smears were also successfully imaged. This field-portable and wide-field computational colour microscope could be useful for tele-medicine applications in resource poor settings.
Behaviors emerge from activity throughout the brain, but non-invasive optical access in adult vertebrate brains is limited. We show that three-photon (3P) imaging through the head of intact adult zebrafish allows structural and functional imaging at cellular resolution throughout the telencephalon and deep into the cerebellum and optic tectum. With 3P imaging, considerable portions of the brain become non-invasively accessible from embryo to sexually mature adult in a vertebrate model.
Lens-free holographic on-chip imaging is an emerging approach that offers both wide field-of-view (FOV) and high spatial resolution in a cost-effective and compact design using source shifting based pixel super-resolution. However, color imaging has remained relatively immature for lens-free on-chip imaging, since a 'rainbow' like color artifact appears in reconstructed holographic images. To provide a solution for pixel super-resolved color imaging on a chip, here we introduce and compare the performances of two computational methods based on (1) YUV color space averaging, and (2) Dijkstra's shortest path, both of which eliminate color artifacts in reconstructed images, without compromising the spatial resolution or the wide FOV of lens-free on-chip microscopes. To demonstrate the potential of this lens-free color microscope we imaged stained Papanicolaou (Pap) smears over a wide FOV of ~14 mm(2) with sub-micron spatial resolution.
Optical microscopy is a valuable tool for in vivo monitoring of biological structures and functions because of its noninvasiveness. However, imaging deep into biological tissues is challenging due to the scattering and absorption of light. Previous research has shown that the two optimal wavelength windows for high-resolution deep mouse brain imaging are around 1300 and 1700 nm. However, one-photon fluorescence imaging in the wavelength region has been highly challenging due to the poor detection efficiency of currently available detectors. To fully utilize this wavelength advantage, we demonstrated here one-photon confocal fluorescence imaging of deep mouse brains with an excitation wavelength of 1310 nm and an emission wavelength within the 1700 nm window. Fluorescence emission at 1700 nm was detected by a custom-built superconducting nanowire single-photon detector (SNSPD) optimized for detection between 1600 nm and 2000 nm with low detection noise and high detection efficiency. With the PEGylated quantum dots and SNSPD both positioned at the optimal imaging window for deep tissue penetration, we demonstrated in vivo one-photon confocal fluorescence imaging at approximately 1.7 mm below the surface of the mouse brain, through the entire cortical column and into the hippocampus region with a low-cost continuous-wave laser source and low excitation power. We further discussed the significance of the staining inhomogeneity in determining the depth limit of one-photon confocal fluorescence imaging. Our work may motivate the further development of long wavelength fluorescent probes, and inspire innovations in high-efficiency, high-gain, and low-noise long wavelength detectors for biological imaging.
Multiphoton fluorescence microscopy enables deep in vivo imaging by using long excitation wavelengths to increase the penetration depth of ballistic photons and nonlinear excitation to suppress the out-of-focus fluorescence. However, the imaging depth of multiphoton microscopy is limited by tissue scattering and absorption. This fundamental depth limit for two-photon microscopy has been studied theoretically and experimentally. Long wavelength three-photon fluorescence microscopy was developed to image beyond the depth limit of two-photon microscopy and has achieved unprecedented in vivo imaging depth. Here we extend the theoretical framework for characterizing the depth limit of two-photon microscopy to three-photon microscopy. We further verify the theoretical predictions with experimental results from tissue phantoms. We demonstrate experimentally that high spatial resolution diffraction-limited imaging at a depth of 10 scattering mean free paths, which is nearly twice the transport mean free path, is possible with multiphoton microscopy. Our results indicate that the depth limit of three-photon microscopy is significantly beyond what has been achieved in biological tissues so far, and further technological development is required to reach the full potential of three-photon microscopy.
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