Field-portable pixel super-resolution colour microscope.

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 mm(2). 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

THE DEPTH LIMIT OF MULTIPHOTON MICROSCOPY AND APPLICATIONS TO IMAGING MINIATURE ADULT VERTEBRATE BRAINS

125 pagesMultiphoton microscopy has enabled unprecedented access to biological systems in their native environment. Two-photon microscopy was invented at Cornell University in 1990 and has since been adopted all over the world for deep in vivo imaging. While three-photon microscopy was demonstrated not much later (1996), the required technology and incentive for further development of this technique was not available until much later in 2013 once again at Cornell, the original home of multiphoton imaging. Three-photon microscopy has since enabled unprecedented access deep in highly scattering biological tissue.While the advantage of three-photon microscopy for deep imaging has been demonstrated, the depth limit of this technique had not been established. In this dissertation I provide a comprehensive theoretical and experimental investigation of the depth limit of multiphoton microscopy techniques. I 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. Additionally, I demonstrate unique possibilities that multiphoton microscopy enables in neuroscience research by applying three-photon microscopy and third harmonic generation microscopy to a miniature adult vertebrate, Danionella dracula. My work demonstrates that two- and three-photon microscopy can be used to access the entire depth of the adult wild type Danionella dracula brain without any modifications to the animal other than mechanical stabilization. These results show that multiphoton microscopy is ideal for readily penetrating the entire adult brain within the geometry of these miniature animals’ head structures, without the need for pigment removal. With multiphoton microscopy enabling optical access to the adult brain and a repertoire of methods that allow observation of the larval brain, Danionella provides a model system for readily studying the entire brain over the lifetime of a vertebrate for the first time.2023-01-0

Radiofrequency Encoded Angular-Resolved Light Scattering

Label-free classification of microstructures is a valuable approach in a variety of fields including cytometry and atmospheric science. The sensitive classification of microscopic cells and organisms is especially an important outstanding problem in biology. Flow cytometry is a routine method for cell classification. Current flow cytometers use light scattering at two fixed angles to infer information about size and internal complexity of cells at rates of more than 10,000 cells per second. However, this approach limits the precision and information that can be deduced by the cell population from the light scattering patterns. Capturing the full angular scattering spectrum of cells and particles would enable classification of cells with higher resolution and specificity. By capturing the angular dependence of scattering intensity we will be able to extract information about the scattering particle, hence providing a label-free method for particle classification. Current systems that provide angular scattering patterns do not have the throughput required to be implemented in state-of-the-art flow cytometers. Inverse scattering has been one of the more difficult problems to solve in electromagnetic wave interaction problems. Yet there have been many solutions obtained by analytical and computational modeling for various cases. The angular light scattering profile of particles is dependent on their morphological parameters, such as size, shape, and their internal structure. One of the most interesting applications of modeling these scattering profiles is in characterizing cells to identify abnormalities. Methods to take advantage of this angular dependent information have been demonstrated, however, these methods have various limitations such as low speed and precision. Here I present a new high throughput multi-angle resolved light scattering measurement technique that is able to capture the full angular scattering profile of particles in a single shot using a single detector. Termed Radiofrequency Encoded Angular-resolved Light Scattering (REALS), this technique uses one-to-one radiofrequency-to-angle mapping to measure angular dependence of light scattered from particles in a single shot using a photomultiplier tube. Using this technique it is possible to capture the continuous scattering profile over a wide dynamic range without mechanical scanning. This information allows us to characterize particle morphology and size with increased accuracy and high throughput, enabling label-free and high speed flow cytometry. As a proof of concept we distinguish the radius of tapered silica fiber over a range of radii

Radiofrequency Encoded Angular-Resolved Light Scattering

Label-free classification of microstructures is a valuable approach in a variety of fields including cytometry and atmospheric science. The sensitive classification of microscopic cells and organisms is especially an important outstanding problem in biology. Flow cytometry is a routine method for cell classification. Current flow cytometers use light scattering at two fixed angles to infer information about size and internal complexity of cells at rates of more than 10,000 cells per second. However, this approach limits the precision and information that can be deduced by the cell population from the light scattering patterns. Capturing the full angular scattering spectrum of cells and particles would enable classification of cells with higher resolution and specificity. By capturing the angular dependence of scattering intensity we will be able to extract information about the scattering particle, hence providing a label-free method for particle classification. Current systems that provide angular scattering patterns do not have the throughput required to be implemented in state-of-the-art flow cytometers. Inverse scattering has been one of the more difficult problems to solve in electromagnetic wave interaction problems. Yet there have been many solutions obtained by analytical and computational modeling for various cases. The angular light scattering profile of particles is dependent on their morphological parameters, such as size, shape, and their internal structure. One of the most interesting applications of modeling these scattering profiles is in characterizing cells to identify abnormalities. Methods to take advantage of this angular dependent information have been demonstrated, however, these methods have various limitations such as low speed and precision. Here I present a new high throughput multi-angle resolved light scattering measurement technique that is able to capture the full angular scattering profile of particles in a single shot using a single detector. Termed Radiofrequency Encoded Angular-resolved Light Scattering (REALS), this technique uses one-to-one radiofrequency-to-angle mapping to measure angular dependence of light scattered from particles in a single shot using a photomultiplier tube. Using this technique it is possible to capture the continuous scattering profile over a wide dynamic range without mechanical scanning. This information allows us to characterize particle morphology and size with increased accuracy and high throughput, enabling label-free and high speed flow cytometry. As a proof of concept we distinguish the radius of tapered silica fiber over a range of radii

Field-portable pixel super-resolution colour microscope.

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 mm(2). 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

Field-portable pixel super-resolution colour microscope.

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 mm(2). 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

Image processing block diagrams.

<p>(a) For creating a lower resolution colour image of the specimen, three lower resolution holograms are acquired, each with a different illumination wavelength (λ  =  470 nm, 527 nm and 625 nm). A previous approach that simply combines these three holograms into a RGB image results in a ‘rainbow’ like colour artefact. The current approach eliminates the colour noise by averaging the colours in the YUV colour space. (b) The flowchart for acquiring and processing a super-resolved multi-height phase-recovery based grey-scale image. (c) A colour image with sub-micron spatial resolution (shown on the left) is rendered by replacing the lower resolution brightness component from (a) with the super-resolved brightness component from (b) in the YUV colour space.</p

A different view of Moore’s law.

<p>A comparison of transistor counts in central processing units (CPUs) versus the pixel counts on cellular phone cameras. The transistor count has several data points for each year, while the cellular phone pixel count has only one data point, which corresponds to the maximum pixel count introduced in that year.</p

Quantification of the spatial resolution as a function of the number of heights used in our multi-height phase-recovery process.

<p>(a) Amplitude image that was reconstructed using one height. (b) Amplitude image that was reconstructed using two heights. (c) Amplitude image that was reconstructed using three heights. Cross sections of the smallest resolved gratings are provided below each reconstructed image. Pixel size of the monochrome CMOS chip used in our field-portable microscope is 1.67 µm. Note that using a CMOS imager chip that has a smaller pixel size (e.g., ∼1.1 µm) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076475#pone.0076475-McLeod1" target="_blank">[37]</a>, a higher spatial resolution of e.g., ∼300 nm can also be achieved using the same lensfree imaging technique <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076475#pone.0076475-Greenbaum2" target="_blank">[41]</a>.</p