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Rapid Image Acquisition


Figure 2: A block diagram showing one arrangement of components required to produce a focus stack.


Figure 5 : A plot of VFL current vs. time.


current. Minimizing the overlap from one current value to the next hastens image capture and reduces the number of images acquired in a stack, which in turn minimizes the amount of image processing required to form a fi nal image. Setup . Prior to recording a series of images, values for I 0 , I 4, and the number of steps, N, between I 0 and I 4 are determined. T e height of the specimen to be examined is determined manually using the microscope’s calibrated focusing knob or other means.


1. With the lens current at I 0 (I 0 = 0 in most cases), the camera’s “live view” mode is used to manually focus the microscope on the lowest point in the specimen.


Figure 3: The VFL and lens mount adapter used in this work.


2. Next, the lens current is manually increased until the highest point in the specimen is in focus. T is current is I 4 .


3. T e height of the specimen is divided by the DOF of the objective lens being used. T e quotient is N, the minimum number of images to be included in the focus stack.


4. The values for I 0 , I 4 , and N are entered into the micropro- cessor’s program.


5. T e camera’s video recording function is started and runs until images for all N steps are recorded. In the present example, 5 images are recorded, each at a diff erent focal depth. T e camera used in this work records 60 images per second, so all 5 images for a stack are recorded in 5/60 s = 83 ms.


Figure 4 : The VFL focal power as a function of current.


Figure 5 is a plot of VFL current versus time during capture of a stack that will form a single image. In the present work, the timing is supplied by a digital single-lens refl ex (DSLR) camera operating in video mode. T e VFL current is set to a diff erent value for each time slot, that is, t 0 to t 1 , t 1 to t 2 , and so forth. T e timing of image capture is marked by asterisks. Capture occurs near the end of each current step to allow settling of the lens at one end and time for image transfer from the camera’s sensor to memory at the other. T e values of current are selected so that the DOF at each current overlaps the DOF at the next


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For routine work I 0 , I 4 , and N are pre-assigned values and are held in the microprocessor’s memory as constants. When an objective is changed, only N is updated so that setup time is minimized. T e above setup steps can easily be automated. In the present apparatus, I 4 and N can be conveniently changed on the fl y. Figure 6 shows the steps in processing of a stack of images. A stack of images is saved either in the camera’s memory or in the computer. A focus stacking program [ 6 , 7 ] extracts the in-focus parts of each image in the stack and combines them into a fi nal, all-in-focus image. Video image capture . The above techniques are easily applied to video image capture. Figure 7 is a plot of VFL current versus time during capture of a multi-frame video


www.microscopy-today.com • 2015 July


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