The role of near-infrared vision in the social behavior (cell sorting, tissue formation) of cells.

(The larger their light scattering, the larger the distance from which aggregating cells came together.)

[See ref 21]

What could be the natural sources of the fluctuating, near infrared light which correspond to the artificial light sources to which the cells responded (see Chapter 3.2 , and Chapter 3.3)? The results of the experiments with cells on both sides of a glass film suggested that the cells themselves are the sources.

Consequently, there are 2 logical possibilities to explain how cells could become such light sources: Either they radiate fluctuating, near-infrared signals, or they scatter the near-infrared portion of the ambient black-body radiation in a fluctuating way. Modern single photon detectors and sophisticated data-processing methods, including Fourier and wavelet analysis, demonstrated that the cells did not radiate any near-infrared signals, but merely scattered the ambient black-body radiation (unpublished results).

Experimental manipulation of cellular light scattering.

(a)Darkfield micrographs of normal 3T3 cells to demonstrate the predominance of the peri-nuclear granules as light scattering centers. (b)Substantial increase of the light scattering of cells that had ingested small latex particles('png' points to the peri-nuclear granules of a normal cell '1').
In order to see how cells scatter the ambient light, one needs to employ no more than a darkfield light microscope (see panel a). Obviously, the major cause of cellular light scattering is the corona of the peri-nuclear granules such as lysosomes, mitochondria, phagosomes etc. as shown in the case of a 3T3 cell. Thus, there was an easy way to increase the light scattering of 3T3 cells by offering them small, strongly light scattering particles that are similar in size and shape to the natural peri-nuclear granules. Inevitably, the cells will phagocytose them and add them to the other phagosomes of the corona of peri-nuclear granules. We used inert latex and synthetic diamond particles, as they have no known chemical effect on the genome, membrane and cytoplasm of the cells. Panel b shows an example of a cell that had ingested 1 μm large latex particles. Such cells, called 'hyperscattering cells', scatter much more light than normal cells (compare the light scattering by the peri-nuclear granules ('png') of cell '1' in the upper right corner with the light scattering of the latex-loaded cell). Using synthetic diamond particles and a range of particle concentrations, it was possible to increase the light scattering of the 3T3 cells, including their near-infrared light scattering up to 140-fold. The scattered light was strongly fluctuating because the incessant movements of all peri-nuclear granules generate stochastic groupings of different sizes, that scatter the light in a correspondingly stochastic way.

(a) Fluorescence image of a Hoechst stained aggregate of 'hyperscattering' cells. (b) Brightfield image of the same aggregate, showing that it consists predominantly of particle-labeled cells.
In order to see how the increased light scattering of cells might alter their social behavior, we turned to the biological phenomenon of cell aggregation (cell sorting). Unfortunately, normal 3T3 cells do not aggregate when growing on solid substrates. Therefore, we isolated a spontaneous variant (called 3T3x cells) that did.

The very surprising result is shown in the above figure: Wherever 3T3x cells had formed aggregates (a) the cells that had ingested the most particles, thus scattering the most light (and consequently appearing the blackest in brightfield microscopy), were located at the center of the aggregates (b). Time-lapse observations showed that the most-light scattering cells approached each other from large distances, long before there was any physical contact between them, and eventually formed the aggregates.

How would aggregating cells behave, if they were attracted to each other over large distances?

Thought experiment to argue that aggregating cells would aggregate a distance Ra away from the ends of an adhesive strip on a non-adhesive substrate (see text). (f,g) show a computer simulation of this phenomenon involving an array of parallel strips.
In order to test quantitatively whether there was such a long-range attraction between the cells that was mediated by light scattering, we turned to a thought experiment. Assume an infinitely long, narrow adhesive strip on a non-adhesive substrate ('a'). If cells were plated randomly on such a strip they would attach and spread on it ('b'). Subsequently, they will migrate up and down the strip while avoiding the non-adhesive surface on either side. If the cells are capable of aggregation then they will eventually form aggregates at random locations with an average distance of 2Ra between them ('c'). The quantity Ra will be called the 'range of aggregation'.The midpoint w at distances Ra between any 2 adjacent aggregates may be considered a watershed of cell migration as the cells ahead of or behind this point effectively migrated to opposite sides as they formed the aggregates ('d'). Assume that the strip was cut off at such a location w ('e'). It would not affect the aggregate behind it because the cells ahead of the cut would have migrated in the opposite direction, anyway. Therefore, one may conclude that the first aggregate of every strip will be located at a distance of Ra from the end of the strip. Instead of using a single adhesive strip, it is better to use a parallel array of such strips because the cellular aggregates would be located in register and become much more visible. Computer simulations of the behavior of cells on such arrays (program available upon request) confirmed the above conclusions. No aggregates formed when Ra = 0 ('f'). In contrast, tight aggregates formed and lined up as a band parallel to the ends of the strips at a distance of Ra, when Ra > > cell diameter ('g').

Using this rationale we designed a suitable substrate and tested the behavior of 3T3x cells on it in order to determine whether their value of Ra was much larger than a cell diameter (i.e. whether there was, indeed, a long-range attraction between them) and whether Ra depended on near-infrared light scattering (i.e. whether the long-range attraction was mediated by near-infrared light).

Aggregation arcs. Evidence of long-range attraction between cells.

(a,b)The predicted locations of the aggregates of hyperscattering cells on circular (a) and rectangular (b) parallel arrays of strips. (c) Actual experiment showing the aggregation arc that forms on circular arrays. (d) Higher magnification of a segment of an aggregation arc to show that the aggregates of the hyperscattering cells have sorted out from other, less particle-loaded cells that cover the strips all the way to the end.
The test substrates consisted of a Sylgard 184 surface (non-adhesive surface) onto which a thin layer of Ni-Cr (adhesive surface) was evaporated through the openings of a suitable slotted mask. We used electron microscope grids with a bar pattern as masks because of their low cost and easy availability. As a consequence, the ends of the parallel strips did not lie on a straight line (b), but along a circle (a). Indeed, as shown on panel c , when particle loaded 3T3x cells were plated on such substrates, their aggregates formed a circle parallel to the circular arrangement of the ends of the strips at a certain distance Ra ('aggregation arc').The aggregation arcs were not the result of the cells retracting from the ends of the strips but, as shown on panel d, the heavily particle-loaded cells formed predominantly the aggregates behind the ends of the strips. The aggregates even spilled over the edges and connected with each other thus improving the visibility of the aggregation arcs. In contrast, the other, relatively unlabeled cells covered the strips all the way to their ends. In other words, the particle-labeled cells sorted out from the lesser labeled cells a certain distance Ra behind the ends of the strips. It was obvious that the distance Ra was much larger than a cell diameter, which demonstrated the existence of a long-range attraction between the heavily labeled (thus strongly light-scattering) cells.

Light scattering mediates the long-range attraction between aggregating cells.

Relationship between the range of aggregation Ra and the near-infrared light scattering of hyperscattering cells.
The above method offered an easy and accurate way to measure Ra. Likewise, the scattering of near-infrared light by particle-loaded 3T3x cells could be measured accurately. Therefore, one could test whether there was a correlation between the 2 measures. Using many different particles sizes, materials and concentrations we found that the range of aggregation grew monotonically with the near-infrared light scattering of hyperscattering cells up to a maximum (see graph above). At the highes level of light scattering the range of aggregation decreased again because the aggregates formed very fast. As a result they generated smaller, but more numerous aggregates that were placed closer together by virtue of their larger number.

All the above experiments were carried out in the 'darkness' of the culture incubators that contain no visible light. Consequently, the only candidate for the light scattered by the cells was the black body radiation corresponding to the temperature of 37 oC. Additional experiments that irradiated the cells with exogenous light sources at intensities of 12-15 μW/cm2 showed that red light of 600 nm slightly reduced the value of Ra, whereas near-infrared light of 800 nm increased it by 23%.

Significance for cell intelligence:

If cells can recognize each other's 'images' at a distance they must have a 'brain'.
The above experiments suggest that the distances at which aggregating 3T3x cells were able to detect each other were the larger the more these cells scattered near-infrared light. As a result of this kind of recognition millions of cells formed aggregation arcs that were located at a certain distance from the ends of the adhesive strips, dependent on their level of light scattering. In other words, near-infrared light scattering by the peri-nuclear granules seems to be one of the means by which aggregating cells communicate over large (cellular) distances. The end result was a sorting out of the most light scattering cells away form the other cells. Since the work by Holtfreter and Moscona more than half a century ago, cell sorting has been recognized as a vital mechanism for differentiation and tissue formation.Therefore, near-infrared light scattering appears to be a vital signal carrier for these two basic phenomena of development.

One should keep in mind that the intensity of the scattered light of the black body radiation is extremely small. Therefore, it may turn out that cells are not able to detect the scattered light of any single cell, but need the combined scattering of small groups. Either way, it seems inescapable that cells employ to this end a highly sophisticated detection and data-processing system that ultimately coordinates all the actions required for aggregation. Such actions would include adhesion, polarization, cytoskeletal architecture, shape changes, migration, interactions with the extracellular matrix, and possibly many more. Since several of these actions are also known to feed back on the genome, the postulated detection and data-processing systems appear capable of essentially influencing actions of the entire cell. In short, the cell must be intelligent to act as described above.