Introduction to Multispectral Imaging 9
2.3 Light Absorbance and Reflection
We perceive objects based on the way they interact with incident light (excepting light emitting
objects such as light sources, and fluorescing or phosphorescing objects). Objects transmit,
absorb, and/or reflect light; in most cases they exhibit a combination of transmittance,
absorbance, and, reflection. The perceived color of a non-transmissive object is a direct result of
both absorbance and reflection of light. An opaque object we would perceive as red is one that
absorbs most wavelengths of light, except for red wavelengths that are reflected back to the eyes of
the observer. An object we would perceive as red in transmission is one that transmits primarily red
wavelengths, while absorbing or reflecting other wavelengths.
Generally speaking, absorbance and reflection of light are very similar phenomena. One can say that
the light transmitted through a semitransparent object is the light that is not absorbed by the object.
Note how similar this is to the definition of reflectance. Only the geometry is different. Reflectance is
more complex than simple transmission due to variations in surface texture, transparency of the
reflecting layers, and the characteristics of the opaque surface beneath the reflecting layers.
In brightfield light microscopy we have a controlled geometry, and are observing light that is
transmitted through a specimen. Excepting a few pigments and inclusions, biological specimens are
essentially invisible, unless we employ some absorbing dye, or specific optical arrangement to
impart contrast. It is this need for contrast that led to the initial development of biological stains and
stain protocols and subsequently to phase contrast and other optical contrast enhancing
techniques.
The amount of light absorbed by a dye or stain in a specimen can provide a measurement of the
amount of the absorbing material present. This is the basis of spectrophotometry. A basic law of
spectrophotometry is the Beer-Lambert law, which states that the amount of material present
(absorbance) is defined by the relationship:
Absorbance = -log(transmission) = (molar absorbtivity) x (path length) x (concentration)
For a given material, the molar absorbtivity, also called the molar extinction, is a constant, and
therefore one only needs to determine the percent transmission of light through the specimen and
the path length in order to calculate the concentration. The transmission is the amount of light
without the specimen versus the amount of light with the specimen, and this is easily measured.
Since transmission is based on a ratio, it is not sensitive to actual light level, assuming that the light
does not change between the measurement of the “blank” or 100% level; and the “specimen” or
sample measurement. It is this basic law that is used in solution spectrophotometry to determine
the concentration of absorbing materials in solution.
The Beer-Lambert law has two specific requirements:
The absorbing material must be homogeneous
The absorbing material must be a true absorber of light, not a light scatterer
This first requirement poses problems for spectrophotometry through a microscope. The very reason
one uses a dye on a microscope preparation is to see structure, and by definition, if one can see
structure, then the dyed material is not homogeneous. While this may seem like an insurmountable
obstacle to performing spectrophotometry through a microscope, the solution is simple. Microscope
optics are characterized by their ability to resolve two points as separate. This is the resolution of a
particular lens system. If the light detector element sees a smaller portion of the specimen than the
lens system can resolve as separate points, then by definition, the spot being measured is
homogeneous.
Using modern CCD or CMOS cameras for image collection, accurate spectrophotometry requires
