Rapid Image Acquisition
or other morphological changes of substances can be done. Inspection of tilted objects is easily done. Machine vision and inspection can benefi t both in speed and image quality from the technology described here. Limited 3D capability is also available in focus stacking soſt ware [ 6 , 7 ]. Additional images are available for viewing on our website [ 13 ].
Although we have improved the rate of image capture, the speed of image processing by focus stacking has room for improvement. T e stack of 15 images in Figure 12 was acquired in 0.25 seconds but required about 6 seconds to process in a reasonably fast desktop personal computer. For this reason, ongoing work includes assembling a fast video processor for image capture, focus control, and focus stacking [ 2 , 3 ].
Conclusion T is article describes a new method that can rapidly acquire focus stacks in less than a second. Processing the focus stack on a personal computer, to produce an image with apparent large DOF, currently takes about 20 times longer than the acquisition time.
Figure 12 : Mammalian tissue specimen. Image 1 is focused at the lowest level where feature A is in focus. Image 7 is focused near the center of the specimen. Image 15 is focused at the top where feature B is in focus. Image All is a processed image showing all features in focus. 50× objective, stack of 15 images, fi nal image diameter ≅0.3 mm, acquisition time = 0.25 sec.
Other methods for changing focal distance are used in microscopy. In one, a microscope objective lens is moved up and down. In another, a focusing stage moves the specimen up and down [ 12 ]. T ese mechanical methods off er tradeoff s to the VFL approach: the magnifi cation at the focus remains constant with changing distances between an objective lens and a specimen. But this is not a problem with focus stacking because stacking soſt ware identifi es and aligns features in each image of a stack by digitally scaling the images. An advantage of a VFL over a moving objective lens is that the VFL is easily inserted between a microscope’s trinocular port and a camera. T is allows use of a microscope’s lens turret to quickly select among diff erent objectives. Another advantage of a VFL over mechanical systems is speed. T e step response settling time of a VFL is generally shorter than found in a mechanical system. T is means that multiple image acquisition can be faster with a VFL than with a mechanical system.
Acquiring images at the maximum possible speed improves work throughput and permits the all-in-focus imaging of moving or changing specimens. Microscopic species can be viewed in a three-dimensional environment, instead of being squeezed between a microscope slide and a cover slip to keep them in focus. Time-lapse imaging of crystal growth
24
References [1] A Agarwala et al ., “Interactive Digital Photomontage,” http://
grail.cs.washington.edu/projects/ photomontage/photomontage.pdf .
[2] LD Clark et al ., US Patent application No. 2010/0283868, “Apparatus and Method for Application of Selective Digital Photomontage to Motion Pictures,” fi led March 27, 2010.
[3] LD Clark et al ., US Patent 8,212,915, “Externally Actuable Photo-Eyepiece Relay Lens System for Focus and Photomontage in a Wide-Field Imaging System,” filed August 10, 2010.
[4] Optotune,
www.optotune.com . [5] Optotune, “Application Note: Optical focusing in microscopy with Optotune’s focus tunable lens EL-10-30,”
www.optotune.com/images/products/Optotune%20 application%20note%20for%20microscopy.pdf .
[6] HeliconSoſt , www.heliconsoſt .com . [7] Zerene Systems,
www.zerenesystems.com . [8] Edmund Optics Worldwide,
www.edmundoptics.com . [9] Holochip,
www.holochip.com . [10] LensVector,
http://lensvector.com . [11] Parrot, Varioptic,
www.varioptic.com . [12] Physik Instrumente (PI),
www.physikinstrumente.com . [13] Paedia LLC, “A Rapid Image Acquisition System and Method for Focus Stacking Photomontage in Microscopy,”
www.paedia.com/Photomontage_microscopy.html .
www.microscopy-today.com • 2015 July
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