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We present, as well, measurements of the spherical aberration coefficient Cs=(108.7±9.6) mm and minimum achievable focal step δf= 87.6±1.4) nm for the specially designed long-focal-length objective lens of this microscope.
Z-projection images represent stacks that contain 0.5 µm focal step images using 3-times Kalman averaging.
The focal step between sections was typically 1 μm.
A ×60/1.25 water-immersion objective (Olympus) was used to collect 3-D stacks of 17 focal sections (an entire nucleus of 6 8 μm) with 0.5 μm focal step, once a second for 200 seconds.
Similar(56)
This is suitable so long as the focal steps are finely enough sampled that no drastic change in the optical transfer function has occurred in the step.
In each iteration of our algorithm, we find the most similar pair of subsets from the focal partitioning scheme (step 4), and then merge these subsets to create a new subset and a new partitioning scheme (step 5).
Images were collected as either an individual lambda scan with the collection of 32 channels combined to create one image or as a stack of images (32 channels each image) with different focal planes (~1μm step size) where each individual image was globally analyzed.
Collectively, by analyzing the trajectories of 50,000 photons at each focal depth, these steps simulate the detected directionality (F/B) and attenuation as a function of depth into the tissue.
The specimen was 15° normal to the incoming X-ray beam and scanned through the focal plane in steps of 800 nm (horizontal axis, x) and 800 nm (vertical axis, y) orthogonal to the beam axis using an integration of 1 1.5 s per scan point to ensure adequate counting statistics.
Hence, in a second step, discrete focal thickenings were included in our reconstructions.
For element distribution maps, the sample was rastered through the focal spot with a step of 1 2 μm and dwell time of 4 sec.
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