Seeing is believing…

November 30, 2012

…and understanding!

Most cellular processes are ingeniously and non-intuitively orchestrated. In addition they contain a large number of components. Understanding all this can pose a challenge. Not surprisingly research experience over the last decades has shown that it actually is a challenge! When ignoring crystal structures, the bulk molecular knowledge about cellular processes comes from indirect evidence such as blots, gels and other traditional techniques. Would it not be interesting to observe processes (1) in vivo, (2) at high resolution and magnification, and (3) in real-time? Probably not many biologists will disagree with this. A major challenge towards this aim, however, is the nature and chemical structure of the cell surface/membrane. From an optical perspective this lipid bilayer structure can be viewed as a so-called “opaque surface”. First of all this means that it is non-transparent and scatters light (Fig. 1). Looking through a sample therefore becomes impossible because the incoming light is so distributed all over the place that it is hard to attach any meaningful information to it.

Image Fig. 1: Plane waves that hit a rough surface are scattered instead of being directly reflected. The resulting random speckles lead to a distorted image of the object in the human eye (Source:

In the past techniques have been developed to extract information from opaque layers which at least let through a small amount of direct beams.  Here, however, I want to present a new approach which has recently been published by a team of Dutch and Italian researchers who managed to retrieve images through a completely opaque surface.

Two layers of material were used for this study. The first layer is the opaque layer which scatters the light and does not allow a direct observation of a 50 μm sized object on the second layer (Fig. 2 (A)). In this case the object to become imaged was the Greek letter pi made out of fluorescent polymers. Both layers are 6 mm apart, which is a large distance considering the size of the object. As depicted in Fig. 2 (B) a laser with 532 nm wavelength is consequently directed at the first opaque layer and serves as the light source. Due to scattering of the light that passes the first layer and due to scattering of the light which fluoresce back through the first layer, it becomes impossible to identify which object is hiding behind the layer (Fig. 2 (C)). It is, however, possible to measure the overall amount of light that originates from the fluorescent object. The researchers now assumed that all the information that one would need in order to reconstruct the image is already contained in this recorded light. Due to scattering and speckling this information is disorganized and cannot be readout by conventional means (such as our eyes and brain). Of course the key element of the study was to develop an algorithm and a technical procedure that were able to extract meaningful information from the chaotic light mix (Fig. 4 (D)). In the following I will explain this procedure in a bit more detail. Since I am NOT a physicist I omitted some of the details, but I am sure that I am mentioning enough facts to understand the technique.


Fig. 2: Experimental design involving an opaque first layer and a second layer with a fluorescent object that could be retrieved by an algorithm involving scanning over different angles and autocorrelating the resulting information. For details see text (Source: Press release “Looking through an opaque material” by the University of Twente, the Netherlands).

Four variables are essential for being able to recalculate the nature of the object behind the opaque layer. First the object’s fluorescent response O which roughly translates to the fluorescent intensity at a given point in space. Secondly the speckle intensity S is important. It describes the amount of light speckle formation also at a given point in space. The third important factor is the angle θ at which the laser shines through the first layer. Finally, the measured overall light intensity I was essential.

During the course of the study, the angle of incident light was slightly varied. By iteratively changing the laser angle and consequently measuring the overall intensity I it became possible to calculate correlations between all four factors. The interesting part is that these correlations are the founding block for organizing the information which is hidden in the scattered fluorescent light and that was thought to be of totally random nature before.

The first step in order to achieve this was the discovery of the relation between incident laser angle and the measured fluorescent intensity I. Interestingly, speckle intensity S and the objects response O remained largely unchanged under one set laser angle. This enabled the researchers to use nine different angle scans, which yielded enough information to autocorrelate S and O. Spatial information from the previously randomly distributed intensity, could know be extracted because the relationship how S, O, and θ influence I was known.

In the same paper the authors also demonstrate the use of this technique for imaging a more complex biological sample hidden behind an opaque layer. However, the amount of detail of such a sample is much greater than the amount of detail of a relatively simple pi letter and reconstruction becomes much more calculation intensive. In order to solve this issue the resolution had to be decreased by increasing the size of the speckle spots, thereby lowering the amount of incoming information. Despite these practical limitations the researchers have clearly demonstrated that it is possible to noninvasively image through an opaque layer, such as a cell membrane. I am sure that this discovery has great potential for molecular and cell biological in vivo studies. This potential is enhanced even more by the possibility of increasing resolution by decreasing speckle spot sizes and by introducing 3D imaging by measuring speckle patterns in an additional direction.

Article: Jacopo Bertolotti, Elbert G. van Putten, Christian Blum, Ad Lagendijk, Willem L. Vos, Allard P. Mosk, Non-invasive imaging through opaque scattering layers, Nature 491(7423), 232–234, 2012.


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