Rapid Image Acquisition
Figure 7: A plot of VFL current vs. time in video mode.
Figure 8 : A plot of VFL current vs. time. Regions of interest are selected. Results Figure 6 : A fl ow diagram showing the steps in processing a stack of images.
stream. The VFL current I VFL cycles through all values once per frame. In this case, instead of returning to zero at the end of a frame as shown in Figure 5 , I VFL reverses direction with each frame. This is because smaller current steps have a shorter transient response from one value to the next, thus enabling a higher video rate. T e stack of images captured in each frame is processed into a single resultant image per frame. T e processed images are displayed one at a time on a monitor, or they can be combined to form a movie. T e speed of image capture is limited only by image acquisition time and refocus time. Regions of interest . In some applications only a limited number of regions of interest (ROI) are required. Individual images in each ROI can be selected from a larger stack, such as would be obtained using the timing in Figure 5 , while images that are not selected are ignored. However, images that are ignored represent time wasted in the gathering of image data. Instead, image acquisition time is shortened by acquiring only images of interest. Figure 8 shows the timing of lens focusing currents for 2 ROIs. Images in ROI #1 are captured at times between t 0 and t 2 , while images in ROI #2 are captured at times between t 2 and t 4 . No image is acquired at currents between I1 and I2, thus no time is wasted by gathering an image that will be ignored. Individual ROIs are processed separately in focus stacking soſt ware, as discussed below.
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Circuit board . Figure 9 shows images of a component on a printed circuit board. T e height of the component is 1,118 microns, and the DOF of the 5× objective used here is 44 microns. T e FOV is 3.0 mm. T e calculated number of images required for focus stacking, N, is 25. A total of 27 images were acquired to allow some DOF overlap. Leſt to right: in the fi rst image, the printed circuit board and solder connections are in focus. T e next image is number 14 in the stack, and the focus is halfway to the top of the component. T e third image is number 27, and the focus is at the top of the component. T e fourth image is the resultant processed, all-in-focus image. All images were captured in 0.45 sec. Ronchi ruling . Figure 10 shows the setup used in photographing a tilted subject. A 250 lp/in, chrome-on-glass Ronchi ruling, a lined resolution test target [ 8 ], was tilted at about 10 degrees with respect to the microscope stage and illuminated diascopically. A10× objective lens with a DOF of 8 microns was used, and its FOV was 1.5 mm. T e height diff erence across the FOV was 300 microns, thus a minimum of 37 images were required for a stack. A total of 38 were used. All images were captured in 0.63 sec. Figure 11 shows successive images of the Ronchi ruling. Leſt to right: the fi rst image shows image number 1 taken with focus at the leſt -hand end of the FOV. T e next image is number 19, and the focus is at the center of the FOV. T e third image is number 38, with focus at the right-hand end of the FOV. T e fourth image is the resultant processed, all-in-focus image.
www.microscopy-today.com • 2015 July
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