The simplest (and wrong) way of measuring the density of a region in autoradiograms is shown in the figure below, which is a film autoradiogram of a rat brain slice incubated with S-35 labeled RNA probe.
The wrong way
The histogram method is a crude way of estimating the density of a region and is often employed by users of Photoshop and similar programs. The result can be improved slightly by smoothing the histogram, or dragging the mouse on the histogram to produce a summation of all the pixel values, which is shown on the graph as ``Area''. However, all of these methods are far less accurate than using spot densitometry.
The only legitimate use for the histogram is to evaluate the relative contributions from the signal and background. In a good image, there may be two distinct peaks separated by a clearly-defined minimum; but, as shown in the histogram shown above, more often there is only a single large peak and an indistinct shoulder.
The image below shows the correct way of measuring areas in autoradiograms.
The correct way
An even better way is to select ``automatic background''.
A disadvantage of film autoradiograms is that it is often necessary to expend considerable effort in setting up 12- or 16-bit CCD cameras and expensive light boxes to acquire good quality images. If you use film, a Northern Light light box, costing about $3000, which uses an electronic servo to maintain precisely-regulated light intensity, is recommended to obtain reproducible results. The images can be converted to colorful pseudo-color pictures that give the impression of smooth gradations of labeling. However, this perception is often misleading because it misrepresents the physical process, which is actually a discrete, binary exposure of individual photographic grains in the film or autoradiographic emulsion.
A second source of inaccuracy in film autoradiograms is that using the most probable value introduces errors if the illumination behind the autoradiogram changes. Also, unlike spot densitometry, this method cannot provide sub-pixel-value accuracy.
A better approach is to use a microscope with a 1
objective,
attached to a microscope camera. Under the microscope, what originally appears
to be smooth gray areas can be seen to be collections of black grains surrounded
by unexposed regions. This sort of image is easily analyzed by the histogram
technique just described, and gives a large, sharp peak corresponding to the
black exposed grains. This obviates the need for acquiring 12- or 16-bit images
and using precisely controlled illumination.
The difference in quality between a photomicrograph and an image acquired from a CCD camera with a zoom lens makes it worth the additional effort. Not only is pixel counting of exposed grains more accurate, it is more direct inasmuch as it is closer to a measurement of the actual physical process of exposing silver grains in the film.
Of course, the ideal method for imaging radiolabeled sections is to forget about film and use a phosphorimager. Nowadays, these are available for $10,000 or less and far surpass film autoradiography in terms of speed and resolution.