Main Page/BPHS 4090/In-Vivo Spectrocopy
Required Components
- USB Ocean Optics spectrometer
- Collection fiber for spectrometer
- PC with spectrometer software and MS Excel (or equivalent spreadsheet) for data processing
- White reflectance reference target
- White LED illumination source
- Blood pressure compression cuff
- Excel calculation spreadsheets
- Stopwatch timer (can use windows clock for this)
- A usb stick or some way to take your data with you for analysis
Objective
To measure the in-vivo oxygenation state of haemoglobin, and calculate the change in oxygenation before, during, and after reactive hyperaemia by analyzing the colour content of light diffusely reflected off of the skin.
Introduction
Optical methods of skin analysis are ideal because they can be performed non-invasively, and in real-time. It is quite intriguing that so much information can be discovered from something as simple as launching some photons at an object and analyzing what comes back. In this lab, you will be exploring the use of light as a non-invasive measurement tool to determine the in-vivo oxygenation status of haemoglobin in your blood. These measurements will be made in a non-invasive sense, so as much as you may enjoy slicing up your lab partner to get at their blood, it ain’t gonna happen here! You will, on the other hand, have the opportunity to cut off the blood flow to one of your lab partner(s) limbs, though sadly, this will only be temporary. In this lab you will get familiar with the concept of light propagation in turbid (scattering) media, as well as gain experience with optical spectroscopy methods. High resolution spectral information can be analyzed to allow semi-quantitative and fully quantitative analysis of biological materials, and is a very powerful technique, useful for a variety of biophysical applications.
 Figure 1 - Absorption spectrum typical for melanin.
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Determination of physiologically relevant parameters in a quick, reliable and repeatable fashion is of paramount importance in healthcare and biological research. The optical properties of human skin have been the subject of numerous investigations over the years, and two of the most relevant parameters to measure are the haemoglobin (Hb) oxygenation state and melanin content. Hb and melanin are the two major cutaneous chromophores within human skin, which means that their concentrations are essentially responsible for the colour of your skin. Upon exposure to ultraviolet (UV) light, melatinocytes increase their production of melanin within the skin, we know this process by its more common name, a suntan. The absorption spectrum of melanin is shown in figure 1, and is almost linear over the visible spectrum. It is best measured in the spectral rang above 600 nm, as it is the main source of light absorption in the skin at this wavelength and beyond.
The main target we are after in this lab is the oxygenation state of Hb. Hb is the iron-containing protein attached to red blood cells, and aside from giving blood its red colour, it is responsible for transporting oxygen from the lungs to the rest of the body. The mechanism of oxygen binding in Hb is due to a single iron atom, contained within the protein structure of Hb, and just below a porphyrin ring. A 2D representation of the ring and iron is shown in Figure 2. This structure serves to trap an oxygen molecule and hold it for transport around the body. A typical Hb molecule consists of four of these binding sites surrounded by a protein matrix, and the overall structure of the Hb molecule changes when carrying oxygen. This structural change results in a change in the absorption spectrum of haemoglobin in the 400-600 nm spectral range (Figure 3).
 Figure 2 - Schematic representation of the haeme porphyrin ring in Hb.
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 Figure 3 - Absorption spectra of oxygenated Hb (double peak) and de-oxygenated Hb (single peak).
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There is a significant shift of the absorption peak in the 400-450 nm spectral range. However, we will focus on measuring the changes between 500-600 nm since the measurements are easier to perform and more reliable in this range. The absorption spectrum of oxygenated Hb exhibits two peaks in the 500-600 nm spectral range, while the spectrum of de-oxygenated Hb exhibits only a single peak. It is the change between these two states that you will quantify, and to do this we will focus on measuring diffusely reflected light from your palm and the inside of your forearm. These diffuse reflectance measurements will allow us to calculate the absorption spectra of Hb, correct for the effects of melanin from different skin types, and monitor the oxygen saturation state of Hb as we simulate a state of reactive hyperaemia, which is a brief increase in blood flow following a period of ischemia, or arterial occlusion.
Figure 4 - Specular reflection from a surface.
Figure 5 - Diffuse reflection from a multi-layer structure.
To understand how we can make measurements of absorption by analysing diffusely reflected light, we should first define what is meant by the term reflection. In general light reflection can be defined in two ways; specular reflection, and diffuse reflection. Specular reflection refers to light that has been directly reflected from an interface, and is directional. A highly polished metal surface, such as a mirror, is an example of a specular reflector. This type of reflector will follow the law of reflection first described by Descartes, namely that the angle of incidence equals the angle of reflection (Figure 4). Another property of a specular reflector is that it will retain image information, which is why you can see your reflection in a mirror.
In diffuse reflection, the light can be thought of as penetrating a small distance into the reflector and scattering multiple times before exiting (Figure 5). This type of reflectance is non-directional, and does not produce any image since all image information in the wavefront is lost due to multiple scatterings. An example of a diffuse reflector would be a piece of white marble. No matter how much you polish the marble, most of the light striking its surface is diffusely reflected, which is why marble makes for a very poor mirror. A perfect diffuse reflector will reflect light uniformly into the 2π steradian space above it, while a perfect specular reflector will reflect light at an angle defined by the angle of incidence. In general most objects will reflect light both specularly and diffusely.
Human skin can also be thought of an object which is diffusely reflective, like the marble, only that it also contains absorbers, namely Hb and melanin. Skin is a heterogeneous, multi-layered structure consisting of three basic layers, each containing numerous sub-layers. The basic layers of skin are the epidermis, which is the outermost layer and provides protection, the dermis, which serves as the location for hair follicles, sweat glands, etc, and the hypodermis, which consists of connective tissue to secure the skin to bones and muscle, as well as blood vessels to deliver oxygen and nutrients to the skin (Figures 6 & 7). Note that it is not important for you to memorize the various layers that make up the skin, but it is important to note where the chromophores we will be measuring reside and originate from.
