Invented by Karl A. Deisseroth, William E. Allen, Brian HSUEH, Li Ye, Leland Stanford Junior University

The market for methods for imaging and analyzing biological specimens using a system has experienced significant growth in recent years. This growth can be attributed to several factors, including advancements in technology, increased funding for research and development, and the growing demand for more accurate and efficient methods in the field of biology. One of the key drivers of this market is the rapid development of imaging and analysis systems. These systems utilize various techniques such as microscopy, spectroscopy, and imaging software to capture detailed images of biological specimens and analyze them at a cellular and molecular level. The integration of these techniques into a single system has revolutionized the field of biology by providing researchers with a comprehensive tool for studying and understanding biological processes. Advancements in technology have played a crucial role in the growth of this market. The development of high-resolution imaging systems, such as confocal microscopy and super-resolution microscopy, has allowed researchers to visualize biological specimens with unprecedented detail. These systems enable the imaging of subcellular structures and molecular interactions, providing valuable insights into the mechanisms of various biological processes. Furthermore, the integration of advanced imaging software and data analysis algorithms has enhanced the efficiency and accuracy of biological specimen analysis. These software tools enable researchers to process large amounts of imaging data, extract relevant information, and generate quantitative measurements. This has significantly reduced the time and effort required for data analysis, allowing researchers to focus more on the interpretation and understanding of their results. The market for methods for imaging and analyzing biological specimens using a system has also been fueled by increased funding for research and development. Governments, academic institutions, and private organizations have recognized the importance of advancing the field of biology and have allocated substantial resources for the development of innovative imaging and analysis systems. This funding has not only accelerated the pace of technological advancements but has also facilitated the commercialization and accessibility of these systems. Moreover, the growing demand for more accurate and efficient methods in the field of biology has created a favorable market environment for imaging and analysis systems. Researchers across various disciplines, including cell biology, genetics, neuroscience, and pathology, require reliable tools for studying biological specimens. The ability to visualize and analyze these specimens in a comprehensive and precise manner is crucial for advancing our understanding of complex biological processes and developing new treatments for diseases. In conclusion, the market for methods for imaging and analyzing biological specimens using a system has experienced significant growth due to advancements in technology, increased funding for research and development, and the growing demand for more accurate and efficient methods in the field of biology. As technology continues to evolve and new applications emerge, this market is expected to expand further, providing researchers with increasingly powerful tools for studying and unraveling the mysteries of the biological world.

The Leland Stanford Junior University invention works as follows

The present disclosure describes methods for preparing biological specimens for imaging analysis. This includes fixing and clearing the specimen, and then analyzing the specimen with microscopy. Methods of quantifying cells are also included, such as the ability to determine active populations of cells that respond to stimulants. These methods can also be practiced using the devices described in the present disclosure. A flow-assisted clearing apparatus allows for rapid clearance of hydrogel-embedded biological specimens, without the use of special equipment like perfusion or electrophoresis.

Background for Method for the imaging and analysis a biological specimen using a system

The mammalian cortex has many layers and regions that contain cells with diverse activity patterns. Indeed, otherwise-indistinguishable populations of principal cells exhibiting profoundly distinct changes in activity in response to the same task or stimulus have been characterized by electrophysiological recording and cellular-resolution fluorescence Ca2+ imaging. Datastreams of molecular and anatomical information on prefrontal cells typology, derived from different methods, have also revealed a rich diversity of principal excitatory neuron types. These findings highlight the diversity in morphology, wiring and electrophysiology of principal neurons, even within specific layers and subregions.

The mapping and correspondences among different domains of diversity (e.g., activity during behavior, long-range wiring, and molecular phenotype) has fundamental implications for elucidating the cellular logic of prefrontal cortex function; moreover, differences in wiring, role in behavior, and molecular signatures among differentially-responsive cells could provide insight into the mechanisms of action of current neuromodulation therapies, and perhaps even lay the foundation for developing new kinds of cell-targeted disease treatment. The present disclosure provides a method to quantify neuronal activities at the single-cell level in intact brains to assess the uniqueness and non-stereotyped character of the mammalian neural system.

The present disclosure includes a method for preparing a bio specimen for imaging analysis. This method comprises: fixing the specimen with hydrogel monomers, resulting in a hydrogel fixed specimen; clearing it using a flow assisted clearing device, resulting in a cleared specimen.

The method can also include a flow-assisted clear device that includes a sample compartment with an inlet and outlet. In this case, the inlet draws the buffer from the sample and the outlet delivers it.

The method can also be used if the buffer that is delivered to the inlet by the inlet is new or re-used.

In other aspects, the method includes where the buffer circulator is a temperature-controlled circulator.

The method can also include a flow-assisted clear device that includes: a container, a sample holder allowing the buffer to pass through it, and the sample holders being able to be removed and placed within the container.

In other aspects of the method, the buffer circulator may include a rotating rod. In some cases, the rotating rod can be a magnetic rotating bar and controlled by an external magnet field.

In other aspects of the method, a plurality flow-assisted clearance devices are arranged parallel to the buffer circulation device. In some cases, the flow-assisted clearance devices are arranged in parallel to the buffer circulator.

The flow-assisted clear device is not an eletrophoresis cell in other aspects.

In other aspects, “the method” includes the following: the flow-assisted cleaning device is further equipped with a buffer filter.

In other aspects of the method, the buffer circulator is used to generate a flow unidirectional through the sample holder.

In other aspects, the method may include contacting the specimen with paraformaldehyde. In some cases, paraformaldehyde is used to perfuse the heart.

In other aspects, the method may include a plurality of monomers hydrogels that contain acrylamide. “For example, 1% of acrylamide.

In other aspects, the method comprises removing substantially from the hydrogel fixed specimen a plurality cellular components. In some cases, the plurality cellular components includes lipids.

In other aspects, the method may include where the buffer is a Tris-Boric buffer and an Ionic Surfactant. In some cases the Tris buffer can be Tris-Boric. In some cases the ionic detergent is sodium dodecylsulfate. The buffer in some cases is around pH 8.5.

In other aspects, the method may include where the refractive-index matching solution has an index that is the same as the specimen that was cleared. In some cases the refractive matching solution has refractive indices of 1.45. In some cases the refractive matching solution is RapidClear, or FocusClear.

In other aspects, the method can be used when the biological specimen is a central nerve system tissue, an entire brain or spinal cord.

The instant disclosure includes a method of quantifying the active neuronal populations of an animal subject exposed to a stimulus, comprising: delivering the stimulant to the animal subject; isolating its brain; preparing it according to any prior method to produce a clear brain; imaging the clear brain; and identifying active neuronal populations.

In other aspects, the method involves incubating the brain before imaging in a mounting media. RapidClear Mounting gel is sometimes used as the mounting medium.

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