Translating two dimensions into three dimensions

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The translation of two dimensions into three dimensions has always been a crux in biology. Life exists in three dimensions, and order to fully grasp the totality of biology it must be studied in three-dimensional space. Throughout most of the history of the microscope only two dimensions were perceptible to biologists.  Compound microscopes used today are designed to focus on sequential focal planes. However, blurred out of focus planes are still visible and can be a hindrance to visualization.

In the early 1980s immunofluorescence techniques were gaining popularity. These fluorescent signals provided insights into the three-dimensional distribution of cellular structures at the light microscope's resolution. However, only samples prepared in the thinnest possible manner provided clear images. Thicker samples were hindered by the unfocused light of fluorescent labels. This unfocused light blurred the samples and virtually masked the signal. Many researchers were frustrated since most of the observed fluorescence was unfocused light. Fluorescence provided superior contrast for visualization but was lacking in resolution due to the competing light from axial focal planes.

In 1957 Marvin Minsky invented the first confocal microscope. However, the first commercial confocal microscope was not available until the 1980s, the Lasersharp SOM from Oxford Optoelectronics. This technology provides the opportunity for the visualization of optical sections. Via the confocal microscope's use of a pinhole the signal from out of focus planes are rejected. This breakthrough was two-fold. It provides the necessary clarity to perceive a single focal plane and it offers the opportunity through image alignment to develop stacks of two-dimensional images that can be reconstructed into a three-dimensional image with focused light throughout. Now samples could be viewed in a multitude of angles, rotated within space, and examined via ortho-slices.

For biological samples that are too large for confocal analysis other techniques such as serial sectioning are available. Tissue can be embedded in a media such as resin or paraffin and section through the region of interest. These sections are then stained with either a histological stain or a fluorescent marker. The sections are photographed, aligned and built into three dimensional models. These techniques have lead to the publication of projects such as the "BigBrain." Using commercial programs such as Bitplane's Imaris or BioVis3D or one of the multitude of freeware programs like ImageJ or Fiji. Many of these programs involve labor intensive "tracing" of structures of interest on each consecutive photograph. This then creates a model of the "traced" structure that can be viewed in three-dimensional space.

Other less labor-intensive three-dimensional methods have been developed in the laboratory of David Livingston. This technique involves the use of the commercial software, Adobe After Effects, whereby serial sections stained with common histological stains such as hematoxylin and eosin or trichrome are then aligned into a three-dimensional stack. All images in the series can be selectively color cleared in order to study isolated and specific structures. This allows three-dimensional modeling of internal structures of large samples without the labor-intensive step of "tracing" on all of the serial sections.

Finally in recent years large unsectioned samples are being studied in three-dimensions via tissue clearing techniques which makes them permeable to fluorescent antibodies. These techniques been documented through the CLARITY technique in the lab of Karl Deisseroth, and similar work by Ali Ertürk at the Max Planck Institute of Neurobiology. However, these newer techniques may be worthy of a write up on their own.

Picture captions: Upper: The confocal image is of a GFP zebrafish embryo with tubulin labeled in green, GFP is shown in blue. Lower: A reconstruction I did using Imaris of the of vasculature of the human eye. This is an ortho-slice view with the capillaries modeled in purple. 

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