Methods for imaging of excised tissue specimens that obviate manual and time-consuming histology processing steps including wax-embedding, sectioning, and separate staining hold considerable promise for improving our ability to render time- and cost-efficient diagnoses. They also could potentiate the clinical adoption of machine learning tools dependent on routine production of digital histology data. A known limitation of point scanning-based ex vivo imaging technologies that precludes ready adoption for clinical use is imaging speed. We have recently described the development of an approach for multiphoton microscopy capable of imaging un-sectioned and un-embedded human tissue samples at millimeter depths with sufficient quality for primary diagnostic interpretation and at speeds more than 30-times faster than a traditional galvanometer-based system. Incorporation of a polygonal mirror combined with stage scanning in an optically efficient geometry yielded significant speed gains, beyond those possible with resonant galvanometers, and without sacrificing image quality. We hypothesized that further gains in speed, with maintenance of image quality, would be attainable by incorporating pulsed laser excitation with a repetition rate beyond the typical 80 MHz speed of standard Ti-Sapphire lasers. In this analysis we describe the use of a newly produced 250 MHz ultra-fast laser in a polygon-based microscope with stage scanning and demonstrate multiphoton human tissue imaging at speeds on par with the fastest whole slide imaging systems and with resolution, contrast, and coloration that matches physical slides but without any of the principal artifacts associated with slide scanning.
Micro-optical probes, including gradient index (GRIN) lenses and microprisms, have expanded the range of in vivo multiphoton microscopy to reach previously inaccessible deep brain structures such as deep cortical layers and the underlying hippocampus in mice. Yet imaging with GRIN lenses has been fundamentally limited by large amounts of spherical aberration and the need to construct compound lenses that limit the field-of-view. Here, we demonstrate the use of 0.5-mm-diameter, 1.7-mm-long GRIN lens singlets with 0.6 numerical aperture in conjunction with a cover glass and a conventional microscope objective correction collar to balance spherical aberrations. The resulting system achieves a lateral resolution of 618 nm and an axial resolution of 5.5 μm, compared to lateral and axial resolutions of ∼ 1 μm and ∼ 15 μm, respectively, for compound GRIN lenses of similar diameter. Furthermore, the GRIN lens singlets display fields-of-view in excess of 150 μm, compared with a few tens of microns for compound GRIN lenses. The GRIN lens/cover glass combination presented here is easy to assemble and inexpensive enough for use as a disposable device, enabling ready adoption by the neuroscience community.
Multiphoton microscopy of cleared tissue has previously been demonstrated to generate large three-dimensional (3D) volumetric image data on entire intact mouse organs using intrinsic tissue fluorescence. This technique holds great promise for performing 3D virtual biopsies, providing unique information on tissue morphology, and guidance for subsequent traditional slicing and staining. Here, we demonstrate the use of fluorescence lifetime imaging in cleared organs for achieving molecular contrast that can reveal morphologically distinct structures, even in the absence of knowledge of the underlying molecular source. In addition, we demonstrate the power of multimodal imaging, combining multiphoton fluorescence, second harmonic generation, and lifetime imaging to reveal exceptional morphological detail in an optically cleared mouse knee.
Typical imaging depths with multiphoton microscopy (MPM) are limited to less than 300 µm in many tissues due to light scattering. Optical clearing significantly reduces light scattering by replacing water in the organ tissue with a fluid having a similar index of refraction to that of proteins. We demonstrate MPM of intact, fixed, cleared mouse organs with penetration depths and fields of view in excess of 2 mm. MPM enables the creation of large 3-D data sets with flexibility in pixel format and ready access to intrinsic fluorescence and second-harmonic generation. We present high-resolution images and 3-D image stacks of the brain, small intestine, large intestine, kidney, lung, and testicle with image sizes as large as 4096×4096 pixels.
A powerful advantage of multiphoton microscopy is its ability to image endogenous
fluorophores such as the ubiquitous coenzyme NADH in discrete cellular populations. NADH is
integral in both oxidative and non-oxidative cellular metabolism. NADH loses fluorescence
upon oxidation to NAD+; thus changes in NADH fluorescence can be used to monitor
metabolism. Recent studies have suggested that hypo metabolic astrocytes play an important
role in cases of temporal lobe epilepsy (TLE). Current theories suggest this may be due to
defective and/or a reduced number of mitochondria or dysfunction of the neuronal-astrocytic
metabolic coupling. Measuring NADH fluorescence changes following chemical stimulation
enables the quantification of the cellular distribution of metabolic anomalies in epileptic brain
tissue compared to healthy tissue. We present what we believe to be the first multiphoton
microscopy images of NADH from the human brain. We also present images of NADH
fluorescence from the hippocampus of the kainate-treated rat TLE model. In some experiments,
human and rat astrocytes were selectively labeled with the fluorescent dye sulforhodamine 101
(SR101). Our results demonstrate that multiphoton microscopy is a powerful tool for assaying
the metabolic pathologies associated with temporal lobe epilepsy in humans and in rodent
models.
Gradient index lenses enable multiphoton microscopy of deep tissues in the intact animal. In order to assess their applicability to clinical research, we present in vivo multiphoton microscopy with gradient index lenses in brain regions associated with Alzheimer's disease and Parkinson's disease in both transgenic and wild-type mice. We also demonstrate microscopy of ovary in wild type mouse using only intrinsic fluorescence and second harmonic generation, signal sources which may prove useful for both the study and diagnosis of cancer.
Optical holographic correlators can perform many correlations simultaneously. Because the output plane must be divided among the individual templates in the system, for many systems shift-invariance limits the number of correlation templates than can be stored in one correlator. When the system is completely shift-invariant, the correlation peak from one correlator can shift into an area that has been reserved for a different template; in this case, a shifted version of one object might be mistaken for a well-centered version of a different object. This paper describes a technique to control the shift-invariance of a correlator system by moving the holographic material away from the Fourier plane.
Holographic memories can be read-out either with the reference or the signal beam. Reference beam read-out reconstructs the stored data whereas signal beam read-out performs a search of the stored data base. This dual mode of holographic memories is explored for the various methods that have been developed for multiplexing holograms.
Holographic techniques and materials have matured sufficiently to allow high capacity in practical systems. We demonstrate a holographic memory with storage density of 10 bits/micrometers 2. Novel techniques, such as shift multiplexing, can be used to attain even higher capacity with simpler implementation.
The trade-off between the number of neurons that can be implemented with a single correlator and the shift invariance that each neuron has is investigated. A new type of correlator implemented with a planar hologram is described whose shift invariance can be controlled by setting the position of the hologram properly. The shift invariance and the capacity of correlators implemented with volume holograms is also investigated.
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