As Janusz remarks, your question is ambiguously phrased. I am guessing that you are contemplating two possible measures of length: (a) the length L_a of a straight line joining the two end points of the fiber, or (b) the length L_b of the arc traced by the centroid of the cross-section of the fiber.
The aspect ratio of a fiber with width W can be calculated based on either one of those lengths, depending on the application. For instance, if you use the investigate hydrodynamic resistance of the fibers in Stokes flow, then L_a/W will mainly govern the physics. However, if you e.g. investigate the performance of the fiber in a paper sheet (mainly governed by bonded area), then L_b/W is more appropriate.
My advise is to consult relevant literature of your intended application to find out what was used in the past. Then make sure to define the aspect ratio with mathematical precision whenever you use it.
The microscopic length should be equal with the physical length.
If you do not want to consider the magnification, I think you can use either microscopic or physical measurement for both Length & diameter, then in principle you should get the same aspect ratio.
The ISO definition of aspect ratio (ISO9276-6) is breadth/length so that all shape parameters have values between 0 and 1 for mathematical convenience and manipulation.
A fiber's aspect ratio is defined as its length divided by its diameter.
Long, thin fibers often provide superior properties, but are also often more expensive to produce and may be more difficult to disperse uniformly in the composite.
Small aspect ratio fibers typically provide better compressive properties but these composites are typically less resistant to damage propogation.
Aspect ratio is the ratio of length to diameter or thickness. If you want to practically measure it, you can do TEM images for the fibers and plot histogram for 25 individual fibers using Image J software. You can then have a rough idea of the aspect ratio of the fiber under study.
Vf typically ranges from 0.1 to 3%. Aspect ratio (l/d) is calculated by dividing fiber length (l) by its diameter (d). Fibers with a non-circular cross-section use an equivalent diameter for the calculation of aspect ratio.
The aspect ratio is the value of length divided by the diameter or thickness (both in the same units) of the fibre. Higher this ratio, more flexible the fibre is.