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Analysing Microscopy Images? What You Should Know About Dynamic Range: Part 2

In the first part of this article (you can read it here), we looked at clipping and saturation in terms of microscope images, followed by a definition of Dynamic Range and an introduction to Bit Depth.

Intrascene Dynamic Range

The dynamic range which can be detected at the same time in the same field of view (between maximum and minimum intensities) is known as the ‘Intrascene Dynamic Range’. Interscene dynamic range is the range of intensities that can be detected when the detector gain, integration time (photon collection time) and other variables are adjusted for different fields. There is another parameter to consider- that is the amount of noise present along with the signal.

Effective Dynamic Range

The effective dynamic range is often calculated to be the maximum signal that can be accumulated, divided by the noise associated with reading the signal. Signal-to-noise ratio refers to actual signal and noise levels for a given image, so its highest value cannot be more than that of the dynamic range. We mentioned in the first part that we can lose detail if below a certain threshold of brightness, all features look equally dark. We can now explain that these features would be at the most as intense as background noise.

Don’t forget the noise

When the contribution of noise is considered, what we have is essentially a raise of the signal level ‘floor’, below which there is insufficient contrast between specimen features that would enable us to distinguish them as separate. Inevitably, this influences the effective resolution of the system. When plotting contrast-distance functions to demonstrate resolution limits, we often forget that the distance required to provide sufficient contrast to resolve two points in an image is increased when noise is taken into account. We speak about resolution as if noise is zero and the dynamic range is infinite, but in real life a constant noise level limits the useable contrast and point separation range.

A range we can’t see

The human visual system is capable of about 5 to 7 bit discrimination. Compressed digital TV signals evidently are capable of less, as one can clearly see discrete grades in the colors of a sunset or similar scenes. So, why is it that, while we are limited to 7 bit, and most computer screens to 8 bit (256 gray levels), there are high dynamic range image recording systems around?

Larger number of gray levels=accurate data

There are indeed valid reasons. One is related to quantitation. Regardless of whether we can see the difference or not, larger numbers of gray levels allow light intensities to be more accurately determined. Additionally, when multiple image-processing operations are being performed, image data sets which are more precisely resolved into many gray level steps can be subjected to a greater degree of manipulation without the appearance of artifacts.

Isolate and expand

We should not forget that the dynamic range of the image is not necessarily the dynamic range of our region of interest. We may decide to select for display only a part of the recorded image. That part may be a ‘gray’ part, with little visible contrast. When we isolate it, we can expand its contrast levels to occupy all 256 levels of an 8-bit monitor or print. If the image had been taken with low bit depth to begin with, then this expansion would result in artefacts- in visible discernible ‘grades’, such as those seen in digital TV, instead of smooth tonal gradations. Having many gray levels may seem redundant in the original image- but may be just what we need when we focus on a specific region of interest.

High Dynamic Range

And a final note, to avoid any misunderstandings, High Dynamic Range (HDR) in photography does not involve 16-bit detectors or anything that fancy! It involves multiple exposures which are combined to produce a single image that contains the information obtained from all of them. It can be very useful sometimes- but its generalized use and abuse can result in some aesthetically rather unfortunate photographs, which, none the less, are proudly displayed in the social media by their owners!!! HDR can have a value in microphotography, too, if the range of the dynamic range that one wishes to record in a single field exceeds the dynamic range of the detector.

Many of the issues discussed here are related to both the topic of dynamic range and image acquisition/digitization- but we will look at this latter subject in future articles.

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