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OpenStage: a low-cost motorized microscope stage with sub-micron positioning accuracy.

Campbell RA, Eifert RW, Turner GC - PLoS ONE (2014)

Bottom Line: Home-built multiphoton microscopes are easy to build, highly customizable, and cost effective.We obtain positioning repeatability of the order of 1 μm in X/Y and 0.1 μm in Z.Our "OpenStage" controller is sufficiently flexible that it could be used to drive other devices, such as micro-manipulators, with minimal modifications.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.

ABSTRACT
Recent progress in intracellular calcium sensors and other fluorophores has promoted the widespread adoption of functional optical imaging in the life sciences. Home-built multiphoton microscopes are easy to build, highly customizable, and cost effective. For many imaging applications a 3-axis motorized stage is critical, but commercially available motorization hardware (motorized translators, controller boxes, etc) are often very expensive. Furthermore, the firmware on commercial motor controllers cannot easily be altered and is not usually designed with a microscope stage in mind. Here we describe an open-source motorization solution that is simple to construct, yet far cheaper and more customizable than commercial offerings. The cost of the controller and motorization hardware are under $1000. Hardware costs are kept low by replacing linear actuators with high quality stepper motors. Electronics are assembled from commonly available hobby components, which are easy to work with. Here we describe assembly of the system and quantify the positioning accuracy of all three axes. We obtain positioning repeatability of the order of 1 μm in X/Y and 0.1 μm in Z. A hand-held control-pad allows the user to direct stage motion precisely over a wide range of speeds (10(-1) to 10(2) μm·s(-1)), rapidly store and return to different locations, and execute "jumps" of a fixed size. In addition, the system can be controlled from a PC serial port. Our "OpenStage" controller is sufficiently flexible that it could be used to drive other devices, such as micro-manipulators, with minimal modifications.

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Related in: MedlinePlus

Z-Stack behavior.A. Position of the fluorescent bar as the objective is commanded to upwards in ten steps of . Three cycles of motion are shown. Each point represents data from a single frame. Grey points indicate frames when the Z-stage is moving. Colored points indicate frames when the Z-stage is stationary. Points at the same depth share the same color. Upward motion is indicated by more negative numbers. Positioning accuracy is unidirectional, since the objective always approaches each depth from the same direction. B. The location of the objective over 100 Z-stack cycles. Each point represents objective position from one cycle of one depth. There are 100 points for each depth. Motions are highly repeatable over time. Colored lines are linear regression fits. C. Correspondence between target and achieved position. D. Achieved position for the  Z-depth. Note the data are bimodally distributed. E. Same data as D, but plotted as a function of stimulus repeat. Different symbols distinguish between data obtained in the first and second blocks (10 minute gap between blocks). F. Same as D, but for the  depth.
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pone-0088977-g008: Z-Stack behavior.A. Position of the fluorescent bar as the objective is commanded to upwards in ten steps of . Three cycles of motion are shown. Each point represents data from a single frame. Grey points indicate frames when the Z-stage is moving. Colored points indicate frames when the Z-stage is stationary. Points at the same depth share the same color. Upward motion is indicated by more negative numbers. Positioning accuracy is unidirectional, since the objective always approaches each depth from the same direction. B. The location of the objective over 100 Z-stack cycles. Each point represents objective position from one cycle of one depth. There are 100 points for each depth. Motions are highly repeatable over time. Colored lines are linear regression fits. C. Correspondence between target and achieved position. D. Achieved position for the Z-depth. Note the data are bimodally distributed. E. Same data as D, but plotted as a function of stimulus repeat. Different symbols distinguish between data obtained in the first and second blocks (10 minute gap between blocks). F. Same as D, but for the depth.

Mentions: Figure 8A shows the location of the fluorescent bar's peak over three cycles of the Z-stack. Each point represents depth position from a single frame. Frames were acquired at 12 FPS and not synchronized to objective motion. Individual steps within each cycle were always approached from the same direction, so these data showcase the unidirectional positioning accuracy of the objective. In this case, the Z-stack was obtained from bottom to top, so at the end of each cycle the objective was re-positioned to a location below the first Z depth. We determined the time periods over which the bar was stationary (stationary epochs at the same depth are colored identically) and extracted the mean position at each step. This allowed us to plot positioning repeatability within each depth (Fig. 8B). We fitted linear regressions to each depth (color codes the same as in Fig. 8A). The slope of the lines indicated drift rates of between and per Z-stack cycle. We plotted the intercepts of the fits as a function of commanded Z position (Fig. 8C). The points are scattered tightly around the blue unity line, indicating a good match between desired and achieved position (RMS of the difference between two is ).


OpenStage: a low-cost motorized microscope stage with sub-micron positioning accuracy.

Campbell RA, Eifert RW, Turner GC - PLoS ONE (2014)

Z-Stack behavior.A. Position of the fluorescent bar as the objective is commanded to upwards in ten steps of . Three cycles of motion are shown. Each point represents data from a single frame. Grey points indicate frames when the Z-stage is moving. Colored points indicate frames when the Z-stage is stationary. Points at the same depth share the same color. Upward motion is indicated by more negative numbers. Positioning accuracy is unidirectional, since the objective always approaches each depth from the same direction. B. The location of the objective over 100 Z-stack cycles. Each point represents objective position from one cycle of one depth. There are 100 points for each depth. Motions are highly repeatable over time. Colored lines are linear regression fits. C. Correspondence between target and achieved position. D. Achieved position for the  Z-depth. Note the data are bimodally distributed. E. Same data as D, but plotted as a function of stimulus repeat. Different symbols distinguish between data obtained in the first and second blocks (10 minute gap between blocks). F. Same as D, but for the  depth.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3935852&req=5

pone-0088977-g008: Z-Stack behavior.A. Position of the fluorescent bar as the objective is commanded to upwards in ten steps of . Three cycles of motion are shown. Each point represents data from a single frame. Grey points indicate frames when the Z-stage is moving. Colored points indicate frames when the Z-stage is stationary. Points at the same depth share the same color. Upward motion is indicated by more negative numbers. Positioning accuracy is unidirectional, since the objective always approaches each depth from the same direction. B. The location of the objective over 100 Z-stack cycles. Each point represents objective position from one cycle of one depth. There are 100 points for each depth. Motions are highly repeatable over time. Colored lines are linear regression fits. C. Correspondence between target and achieved position. D. Achieved position for the Z-depth. Note the data are bimodally distributed. E. Same data as D, but plotted as a function of stimulus repeat. Different symbols distinguish between data obtained in the first and second blocks (10 minute gap between blocks). F. Same as D, but for the depth.
Mentions: Figure 8A shows the location of the fluorescent bar's peak over three cycles of the Z-stack. Each point represents depth position from a single frame. Frames were acquired at 12 FPS and not synchronized to objective motion. Individual steps within each cycle were always approached from the same direction, so these data showcase the unidirectional positioning accuracy of the objective. In this case, the Z-stack was obtained from bottom to top, so at the end of each cycle the objective was re-positioned to a location below the first Z depth. We determined the time periods over which the bar was stationary (stationary epochs at the same depth are colored identically) and extracted the mean position at each step. This allowed us to plot positioning repeatability within each depth (Fig. 8B). We fitted linear regressions to each depth (color codes the same as in Fig. 8A). The slope of the lines indicated drift rates of between and per Z-stack cycle. We plotted the intercepts of the fits as a function of commanded Z position (Fig. 8C). The points are scattered tightly around the blue unity line, indicating a good match between desired and achieved position (RMS of the difference between two is ).

Bottom Line: Home-built multiphoton microscopes are easy to build, highly customizable, and cost effective.We obtain positioning repeatability of the order of 1 μm in X/Y and 0.1 μm in Z.Our "OpenStage" controller is sufficiently flexible that it could be used to drive other devices, such as micro-manipulators, with minimal modifications.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.

ABSTRACT
Recent progress in intracellular calcium sensors and other fluorophores has promoted the widespread adoption of functional optical imaging in the life sciences. Home-built multiphoton microscopes are easy to build, highly customizable, and cost effective. For many imaging applications a 3-axis motorized stage is critical, but commercially available motorization hardware (motorized translators, controller boxes, etc) are often very expensive. Furthermore, the firmware on commercial motor controllers cannot easily be altered and is not usually designed with a microscope stage in mind. Here we describe an open-source motorization solution that is simple to construct, yet far cheaper and more customizable than commercial offerings. The cost of the controller and motorization hardware are under $1000. Hardware costs are kept low by replacing linear actuators with high quality stepper motors. Electronics are assembled from commonly available hobby components, which are easy to work with. Here we describe assembly of the system and quantify the positioning accuracy of all three axes. We obtain positioning repeatability of the order of 1 μm in X/Y and 0.1 μm in Z. A hand-held control-pad allows the user to direct stage motion precisely over a wide range of speeds (10(-1) to 10(2) μm·s(-1)), rapidly store and return to different locations, and execute "jumps" of a fixed size. In addition, the system can be controlled from a PC serial port. Our "OpenStage" controller is sufficiently flexible that it could be used to drive other devices, such as micro-manipulators, with minimal modifications.

Show MeSH
Related in: MedlinePlus