A Rapid Image Acquisition Method for Focus Stacking in Microscopy
Douglas Clark * and Brian Brown Paedia LLC , 327 Lake Street , San Francisco , CA 94118
*
dclark@paedia.com Introduction
Modern image acquisition in microscopy benefi ts greatly from the use of well-known focus stacking photomontage procedures. T is oſt en involves the taking of a set of images at successive focal distances, followed by mathematical processing and combining of the images to produce a resultant single image with an extended depth of fi eld (DOF) [ 1 ]. T is is useful when viewing subjects whose visible features lie at depths greater than the DOF of the objective lens in use or when objects are tilted or have irregular surfaces. In the past, images to be included in a focus stack (stack) were taken one-at-a-time. A fi rst image was taken, then the position of a subject relative to an objective lens was changed and a second image was taken, and so forth. T is process has worked well, however it takes time. T is article describes an improved system for rapidly obtaining a set of images at video rates, that is, at tens of images per second and at diff erent focal distances. T e subject- to-objective focal distance remains constant so there is no movement of the microscope stage or objective. T e new system greatly increases speed, convenience, and workfl ow in obtaining images with an extended DOF. T e extended DOF brings life to moving and changing subjects and lets them be seen in the full context of their environment. T e new system also applies to cameras, telescopes, binoculars, and monoculars [ 2 , 3 ].
Materials and Methods Apparatus . Figures 1 and 2 show one arrangement of apparatus used for rapid acquisition of multiple images at diff erent focal distances. In Figure 1 , a lens with electrically variable focal length is secured to the trinocular port of a microscope, although it can just as easily be placed on an ocular port. A digital camera is secured to the variable focus lens (VFL) by an adapter such as a C- or F-mount. Electrical wires connect the camera and variable focus lens to control circuitry Figure 2 is a block diagram showing one arrangement of a control system for varying the focus of the VFL as a camera records images in preparation for focus stacking. T e camera receives images from the microscope through the VFL, and the camera provides a synchronizing signal that causes the VFL to change focus for each image that is recorded. An high-defi nition multimedia interface (HDMI) cable connects the camera to a monitor that is used for setting variables such as the focus and VFL parameters. A vertical sync detector connected to the HDMI cable sends a signal to a microprocessor each time the camera sends an image to the monitor. T e microprocessor is programmed to send signals to the VFL in order to vary its focal length in synchronism with the camera’s video rate. In the present case, the camera is free-running, recording and transmitting images at an internally selected rate. Aſt er acquisition, recorded images are delivered to a computer for processing.
18 Figure 1: Components for rapid photomontage including the VLF lens. Figure 3 is a photograph of the VFL that was used in this
work [ 4 , 5 ]. T e lens is plano-convex and has a focal tuning range of +45 to +125 mm. In the present work, we added a −100 mm off set lens to increase this range to −500 to +82 mm in order to enlarge the image on the camera’s sensor and reduce the deviation from the microscope optical design parameters. T e VFL is driven by a current source and its focal power in diopters (dpt) is linear with applied current. From zero to maximum current, the focal power of the combined lenses ranges from −2 to +13 dpt. T e transient response of the lens is suffi ciently fast to permit operation at 60 Hz, the progressive video refresh rate of the camera used in this work. Method . Figure 4 shows the focal power of the combined
VFL and off set lens as a function of current. T e focal power increases linearly with increasing current. In the apparatus described here, the VFL is located at the exit of the microscope’s trinocular port. Placing the VFL here causes changes in magnifi - cation and fi eld of view (FOV) as the focal length of the VFL is varied [ 5 ]. In focus stacking, magnifi cation changes that occur with changing focal length of the VFL are generally removed during processing by the focus stacking soſt ware.
doi: 10.1017/S1551929515000577
www.microscopy-today.com • 2015 July
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84