Rapid Image Acquisition
Figure 9 : The circuit board. Left to right: the circuit board in focus, halfway between the circuit board and the top of the component, the top of the component, and the processed image completely in focus. 5× objective, stack of 27 images, fi nal image diameter ≅3.0 mm, acquisition time = 0.45 sec.
scale and FOV as the VFL current was varied. T us, despite the large height of the circuit board and ruling specimens, it was not necessary to scale the images before processing the stack. In our experience, feature tracking and scaling within the stacking soſt ware works very well except when there are discontinuities in specimen height across the FOV.
Figure 10: Ronchi ruling setup. The ruling is tilted 10 degrees.
Mammalian tissue . Figure 12 shows successive images of a diascopically illuminated stained mammalian tissue specimen. T e FOV is 0.3 mm, the thickness of the specimen is 15 microns, and the DOF of the 50× objective used here is 1.18 microns. A total of 15 images were acquired at 60 Hz for a total acquisition time of 0.25 sec. Two features, A and B, are noted in the images. In image 1, focused on the lowest part of the specimen, feature A is visible, and feature B is barely discernible. In image 7, focused halfway between the lowest and highest parts of the specimen, feature A is less visible, and feature B is partially visible. In image 15, focused at the highest part of the specimen, feature A is nearly invisible, and feature B is clearly visible. Both features are easily seen in the fi nal stacked image. With a fi xed focus, a viewer or an automated image analyzer could easily miss one or the other of these features, but with focus stacking, both are easily seen.
Discussion
Note that the focus stacking soſt ware was able to properly scale the individual images in spite of the apparent change in
When separate ROIs are imaged as described in connection with Figure 8 , a discontinuity in specimen height occurs. T e magnifi cations in the two ROIs are diff erent, and the focus stacking soſt ware has no image features to follow from one image to the next in order to provide proper scaling of the images with respect to one another. T e resultant image shows the two ROIs at two diff erent magnifi cations. T is discrepancy is easily removed by doing a one-time calibration of the magnifi cation versus lens current. If ROI #1 is used as a standard, images acquired in ROI #2 are simply scaled by the new magnifi cation factor prior to focus stacking. Focusing without changing magnifi cation and FOV can be done, however this is generally not possible with most commercial microscopes. It requires placing the VFL in a part of the optical path that is not usually reachable [ 5 ]. We have demonstrated a system for rapid acquisition of images for focus stacking. T e DOF of a lens system and the height of a specimen determine the minimum number of images required to obtain an all-in-focus image. Synchronizing lens focus with image capture and selecting only the minimum required number of overlapping DOF images minimizes the time required to acquire images for processing by focus stacking. T e speed of image capture is limited only by the camera and the VFL. Instead of synchro- nizing focusing with the camera, a controller can trigger the camera in sync with changing focal lengths of the VFL. A number of VFL technologies are available, and some may be suited to particular applications [ 9 – 11 ].
Figure 11 : Ronchi ruling. Left to right: image focused at the left, image focused at the center, image focused at the right, processed image completely in focus. 10× objective, stack of 38 images, fi nal image diameter ≅1.5 mm, acquisition time = 0.63 sec.
2015 July •
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