EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization

AbstractDetermining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In SituHybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine novel spatially and molecularly defined subregions. EASI-FISH also facilitates iterative re-analysis of scRNA-Seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.Highlights-EASI-FISH enables robust gene expression profiling in thick brain slices-A turnkey analysis pipeline for facile analysis of large EASI-FISH image datasets-EASI-FISH reveals novel subregions of the lateral hypothalamus-Identification of rare cell types based on morphological and spatial heterogeneity

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Expansion-Assisted Iterative-Fish Defines Lateral Hypothalamus Spatio-Molecular Organization

SSRN Electronic Journal ◽

10.2139/ssrn.3797275 ◽

2021 ◽

Author(s):

Yuhan Wang ◽

Mark Eddison ◽

Greg Fleishman ◽

Martin Weigert ◽

Shengjin Xu ◽

...

Keyword(s):

Lateral Hypothalamus ◽

Molecular Organization

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Bimolecular and Monomolecular Lipid Films: Selected Area Electron Diffraction

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100063603 ◽

1969 ◽

Vol 27 ◽

pp. 338-339

Author(s):

Robert M. Glaeser ◽

David W. Deamer

Keyword(s):

Electron Diffraction ◽

Membrane Structure ◽

Molecular Organization ◽

Membrane Material ◽

X Ray Diffraction ◽

Membrane Systems ◽

X Ray ◽

Selected Area Electron Diffraction ◽

Lipid Films ◽

Ultimate Objective

In the investigation of the molecular organization of cell membranes it is often supposed that lipid molecules are arranged in a bimolecular film. X-ray diffraction data obtained in a direction perpendicular to the plane of suitably layered membrane systems have generally been interpreted in accord with such a model of the membrane structure. The present studies were begun in order to determine whether selected area electron diffraction would provide a tool of sufficient sensitivity to permit investigation of the degree of intermolecular order within lipid films. The ultimate objective would then be to apply the method to single fragments of cell membrane material in order to obtain data complementary to the transverse data obtainable by x-ray diffraction.

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Evaluation of Freezing Methods by Low Temperature X-Ray Diffraction

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079176 ◽

1977 ◽

Vol 35 ◽

pp. 330-333

Author(s):

T. Gulik-Krzywicki ◽

M.J. Costello

Keyword(s):

Biological Materials ◽

Molecular Organization ◽

X Ray Diffraction ◽

Glass Capillary ◽

X Ray ◽

Rate Dependent ◽

Before And After ◽

Freeze Etching ◽

Diffraction Patterns ◽

Set Up

Freeze-etching electron microscopy is currently one of the best methods for studying molecular organization of biological materials. Its application, however, is still limited by our imprecise knowledge about the perturbations of the original organization which may occur during quenching and fracturing of the samples and during the replication of fractured surfaces. Although it is well known that the preservation of the molecular organization of biological materials is critically dependent on the rate of freezing of the samples, little information is presently available concerning the nature and the extent of freezing-rate dependent perturbations of the original organizations. In order to obtain this information, we have developed a method based on the comparison of x-ray diffraction patterns of samples before and after freezing, prior to fracturing and replication.Our experimental set-up is shown in Fig. 1. The sample to be quenched is placed on its holder which is then mounted on a small metal holder (O) fixed on a glass capillary (p), whose position is controlled by a micromanipulator.

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On the structural organization of biological pumps and channels

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100110878 ◽

1984 ◽

Vol 42 ◽

pp. 138-141

Author(s):

G. Zampighi ◽

M. Kreman

Keyword(s):

Active Transport ◽

Transport System ◽

Plasma Membranes ◽

Transport Proteins ◽

Molecular Organization ◽

Passive Transport ◽

Concentration Gradients ◽

Cell Functions ◽

Natural Concentration ◽

Active Transport System

The plasma membranes of most animal cells contain transport proteins which function to provide passageways for the transported species across essentially impermeable lipid bilayers. The channel is a passive transport system which allows the movement of ions and low molecular weight molecules along their concentration gradients. The pump is an active transport system and can translocate cations against their natural concentration gradients. The actions and interplay of these two kinds of transport proteins control crucial cell functions such as active transport, excitability and cell communication. In this paper, we will describe and compare several features of the molecular organization of pumps and channels. As an example of an active transport system, we will discuss the structure of the sodium and potassium ion-activated triphosphatase [(Na+ +K+)-ATPase] and as an example of a passive transport system, the communicating channel of gap junctions and lens junctions.

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High-resolution measurement of birefringent fine structure in living cells using a new polarized light microscope

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136647 ◽

1995 ◽

Vol 53 ◽

pp. 54-55

Author(s):

Rudolf Oldenbourg

Keyword(s):

Light Microscope ◽

Fluorescent Dyes ◽

Polarized Light ◽

Processing System ◽

Video Camera ◽

Molecular Organization ◽

Computer Assisted ◽

Image Recording ◽

Birefringent Crystal ◽

Technological Advances

The recent renaissance of the light microsope is fueled in part by technological advances in components on the periphery of the microscope, such as the laser as illumination source, electronic image recording (video), computer assisted image analysis and the biochemistry of fluorescent dyes for labeling specimens. After great progress in these peripheral parts, it seems timely to examine the optics itself and ask how progress in the periphery facilitates the use of new optical components and of new optical designs inside the microscope. Some results of this fruitful reflection are presented in this symposium.We have considered the polarized light microscope, and developed a design that replaces the traditional compensator, typically a birefringent crystal plate, with a precision universal compensator made of two liquid crystal variable retarders. A video camera and digital image processing system provide fast measurements of specimen anisotropy (retardance magnitude and azimuth) at ALL POINTS of the image forming the field of view. The images document fine structural and molecular organization within a thin optical section of the specimen.

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