Histochemistry (chemistry in the context of biological tissue) is an invaluable set of techniques used to visualize biological structures. This field lies at the interface of organic chemistry, biochemistry, and biology. Integration of these disciplines over the past century has permitted the imaging of cells and tissues using microscopy. Today, by exploiting the unique chemical environments within cells, heterologous expression techniques, and enzymatic activity, histochemical methods can be used to visualize structures in living matter. This review focuses on the labeling techniques and organic fluorophores used in live cells.

Small molecules that modulate protein-protein interactions are of great interest for chemical biology and therapeutics. Here I present a structure-based approach to predict ’bi-functional’ sites able to bind both small molecule ligands and proteins, in proteins of unknown structure. First, I develop a homology-based annotation method that transfers binding sites of known three-dimensional structure onto protein sequences, predicting residues in ligand and protein binding sites with estimated true positive rates of 98% and 88%, respectively, at 1% false positive rates. Applying this method to the human proteome predicts 8463 proteins with bi-functional residues and correctly recovers the targets of known interaction modulators. Proteins with significantly (p < 0.01) more bi-functional residues than expected were found to be enriched in regulatory and depleted in metabolism functions. Finally, I demonstrate the utility of the method by describing examples of predicted overlap and evidence of their biological and therapeutic relevance. The results suggest that combining the structures of known binding sites with established fold detection algorithms can predict regions of protein-protein interfaces that are amenable to small molecule modulation. Open-source software and the results for several complete proteomes are available at http://pibase.janelia.org/homolobind.

An important open question in biophysics is to understand how mechanical forces shape membrane-bounded cells and their organelles. A general solution to this problem is to calculate the bending energy of an arbitrarily shaped membrane surface, which can include both lipids and cytoskeletal proteins, and minimize the energy subject to all mechanical constraints. However, the calculations are difficult to perform, especially for shapes that do not possess axial symmetry. We show that the spherical harmonics parameterization (SHP) provides an analytic description of shape that can be used to quickly and reliably calculate minimum energy shapes of both symmetric and asymmetric surfaces. Using this method, we probe the entire set of shapes predicted by the bilayer couple model, unifying work based on different computational approaches, and providing additional details of the transitions between different shape classes. In addition, we present new minimum-energy morphologies based on non-linear models of membrane skeletal elasticity that closely mimic extreme shapes of red blood cells. The SHP thus provides a versatile shape description that can be used to investigate forces that shape cells.

Rodents move their whiskers to locate and identify objects. Cortical areas involved in vibrissal somatosensation and sensorimotor integration include the vibrissal area of the primary motor cortex (vM1), primary somatosensory cortex (vS1; barrel cortex), and secondary somatosensory cortex (S2). We mapped local excitatory pathways in each area across all cortical layers using glutamate uncaging and laser scanning photostimulation. We analyzed these maps to derive laminar connectivity matrices describing the average strengths of pathways between individual neurons in different layers and between entire cortical layers. In vM1, the strongest projection was L2/3→L5. In vS1, strong projections were L2/3→L5 and L4→L3. L6 input and output were weak in both areas. In S2, L2/3→L5 exceeded the strength of the ascending L4→L3 projection, and local input to L6 was prominent. The most conserved pathways were L2/3→L5, and the most variable were L4→L2/3 and pathways involving L6. Local excitatory circuits in different cortical areas are organized around a prominent descending pathway from L2/3→L5, suggesting that sensory cortices are elaborations on a basic motor cortex-like plan.

Motivation: Digital reconstruction, or tracing, of 3D neuron structures is critical toward reverse engineering the wiring and functions of a brain. However, despite a number of existing studies, this task is still challenging, especially when a 3D microscopic image has low signal-to-noise ratio (SNR) and fragmented neuron segments. Published work can handle these hard situations only by introducing global prior information, such as where a neurite segment starts and terminates. However, manual incorporation of such global information can be very time consuming. Thus, a completely automatic approach for these hard situations is highly desirable.

Results: We have developed an automatic graph algorithm, called the all-path pruning (APP), to trace the 3D structure of a neuron. To avoid potential mis-tracing of some parts of a neuron, an APP first produces an initial over-reconstruction, by tracing the optimal geodesic shortest path from the seed location to every possible destination voxel/pixel location in the image. Since the initial reconstruction contains all the possible paths and thus could contain redundant structural components (SC), we simplify the entire reconstruction without compromising its connectedness by pruning the redundant structural elements, using a new maximal-covering minimal-redundant (MCMR) subgraph algorithm. We show that MCMR has a linear computational complexity and will converge. We examined the performance of our method using challenging 3D neuronal image datasets of model organisms (e.g. fruit fly).

Optogenetics is routinely used to activate and inactivate genetically defined neuronal populations in vivo. A second optogenetic revolution will occur when spatially distributed and sparse neural assemblies can be precisely manipulated in behaving animals.

The challenge of recovering the topology of massive neuronal circuits can potentially be met by high throughput Electron Microscopy (EM) imagery. Segmenting a 3-dimensional stack of EM images into the individual neurons is difficult, due to the low depth-resolution in existing high-throughput EM technology, such as serial section Transmission EM (ssTEM). In this paper we propose methods for detecting the high resolution locations of membranes from low depth-resolution images. We approach this problem using both a method that learns a discriminative, over-complete dictionary and a kernel SVM. We test this approach on tomographic sections produced in simulations from high resolution Focused Ion Beam (FIB) images and on low depth-resolution images acquired with ssTEM and evaluate our results by comparing it to manual labeling of this data.

Genetically encoded calcium indicators (GECIs), which are based on chimeric fluorescent proteins, can be used to monitor calcium transients in living cells and organisms. Because they are encoded by DNA, GECIs can be delivered to the intact brain noninvasively and targeted to defined populations of neurons and specific subcellular compartments for long-term, repeated measurements in vivo. GECIs have improved iteratively and are becoming useful for imaging neural activity in vivo. Here we summarize extrinsic and intrinsic factors that influence a GECI's performance and provides guidelines for selecting the appropriate GECI for a given application. We also review recent progress in GECI design, optimization, and standardized testing protocols.

The basal ganglia play a critical role in the regulation of voluntary action in vertebrates. Our understanding of the function of the basal ganglia relies heavily upon anatomical information, but continued progress will require an understanding of the specific functional roles played by diverse cell types and their connectivity. An increasing number of mouse lines allow extensive identification, characterization, and manipulation of specified cell types in the basal ganglia. Despite the promise of genetically modified mice for elucidating the functional roles of diverse cell types, there is relatively little anatomical data obtained directly in the mouse. Here we have characterized the retrograde labeling obtained from a series of tracer injections throughout the dorsal striatum of adult mice. We found systematic variations in input along both the medial-lateral and anterior-posterior neuraxes in close agreement with canonical features of basal ganglia anatomy in the rat. In addition to the canonical features we have provided experimental support for the importance of non-canonical inputs to the striatum from the raphe nuclei and the amygdala. To look for organization at a finer scale we have analyzed the correlation structure of labeling intensity across our entire dataset. Using this analysis we found substantial local heterogeneity within the large-scale order. From this analysis we conclude that individual striatal sites receive varied combinations of cortical and thalamic input from multiple functional areas, consistent with some earlier studies in the rat that have suggested the presence of a combinatorial map.

An agglomerative clustering algorithm merges the most similar pair of clusters at every iteration. The function that evaluates similarity is traditionally handdesigned, but there has been recent interest in supervised or semisupervised settings in which ground-truth clustered data is available for training. Here we show how to train a similarity function by regarding it as the action-value function of a reinforcement learning problem. We apply this general method to segment images by clustering superpixels, an application that we call Learning to Agglomerate Superpixel Hierarchies (LASH). When applied to a challenging dataset of brain images from serial electron microscopy, LASH dramatically improved segmentation accuracy when clustering supervoxels generated by state of the boundary detection algorithms. The naive strategy of directly training only supervoxel similarities and applying single linkage clustering produced less improvement.