Using light to describe the ancient world

“…There is a substance so strange, and so beautiful, that whenever people encountered it, they thought they’d found something magical. And its magic is real, because this material has travelled through time, bringing with it passengers from the distant past, that have wonderful tales to tell…”

–  Sir David Attenborough, The Amber Time Machine (1994)

Amber is crystallised tree sap, and according to Sir David Attenborough, a Time Machine. Amber flowing down a tree trunk can freeze insects in time; Howell Edwards and colleagues showed in 2007 that the proteins of those insects can remain intact for tens of millions of years.

I have, on two previous occasions, described spectroscopy studies of amber. I freely admit I have a fascination with amber, which probably began in 1993 with Jurassic Park. Mostly though, I see amber as a spyglass into the past, and a source for very good paleontology. Another example of good amber spectroscopy is the work by Howell Edwards and colleagues, who showed that insects trapped in amber retain their original, biological proteins. That is, keratin from tens of millions of years ago. The amber was not cut, small pieces of the insect were not chipped away for chemical analysis, and yet fossilised remains of an animal were described while trapped inside the fossilised remains of a plant.

Incredible.

This was made possible by confocal microscopy, which involves a clever arrangement of lenses to capture light from a focal plane. For standard, non-confocal microscopy, light from a point source – like a light bulb – is used to illuminate a sample. Light reflecting off the sample produces an image, which is channelled through a set of lenses into eyepieces, if you are directly looking at the sample, or a detector (like a charge-coupled device from a camera), if you are viewing the sample on a display screen. Confocal microscopy is different, as explained by Olaf Hollricher and Wolfram Ibach (2010). Light from a point source – like a laser – “…is focused with a lens or an objective onto a sample…[t]he image spot is then focused…onto an aperture (pinhole) in front of a detector. The size of the pinhole is chosen so that only the central part of the focus can pass through the pinhole to reach the detector…” This means that only a small amount of light from the most in-focus part of the image can be detected.

A confocal pinhole can limit the light that reaches a detector in Raman spectroscopy. Only light from the focal plane is detected, which allows objects within other, translucent objects, to be analysed. Image from Wikipedia

How is this useful? Imagine a lump of amber with an inclusion. You can see the inclusion, which means the amber is translucent, so you can shine a light into it, meaning you can collect a Raman spectrum from within the amber. If you place that amber on the stage of a Raman microscope then you will be able to focus through the amber matrix and see the inclusion. The laser will also focus on the inclusion, but it must travel through the amber to reach it, which will give you Raman scattering (or fluorescence) from the amber as well as a spectrum from the inclusion. This is diffuse, unfocused light, but it will produce scattering all the same. If we use a confocal pinhole though, we will only collect light from the centre of the focal plane – just giving us a spectrum of the inclusion.

Howell Edwards and colleagues were the first researchers to use confocal Raman microscopy for studying insects in amber. Edwards and colleagues (2007) studied both Baltic and Dominican Amber (195-38 and ~17 million years old, respectively) and were immediately faced with a problem. “The insect remains are expected to be keratotic and of an unknown state of preservation; the protein signatures from these inclusions are predicted to be very close in wavenumber to those of the supporting amber matrix, namely, 1660, 1450 and 1220 cm-1 compared with 1650, 1440 and 1260 cm-1…” The insect spectrum had the potential to be subtly different from the amber spectrum, so high precision data collection was needed. The authors used confocal Raman spectroscopy to collect light from the insect alone, and discovered that proteins of the Dominican insect were present, but degraded, whereas the proteins of the Baltic insect well-preserved. The amber matrix didn’t have a chance to interfere with the spectrum because the confocal pinhole only allowed the in-focus light to reach the detector. “…The potential of Raman spectroscopic techniques for the non-destructive analysis of relict biomaterials in closely similar organic host minerals has been demonstrated and the use of these techniques for the screening of specimens for molecular preservation studies prior to the application of destructive characterisation techniques or for the extraction of amino acids and DNA is advocated… [Edwards et al. 2007]”

Ollricher O, Ibach W. 2010. High-resolution optical and confocal microscopy. In: Confocal Raman Microscopy.Springer-Verlag,Berlin,Germany. p. 3.

Edwards HGM, Farwell DW, Jorge Villar SE. 2007. Raman microspectroscopic studies of amber resins with insect inclusions. Spectrochimica Acta Part A 68: 1089–1095

Confocal image from Wikipedia

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