Abstract
To measure nanometric features with super-resolution requires that the stage, which holds the sample, be stable to nanometric precision. Herein we introduce a new method that uses conventional equipment, is low cost, and does not require intensive computation. Fiduciary markers of approximately 1 μm x 1 μm x 1 μm in x, y, and z dimensions are placed at regular intervals on the coverslip. These fiduciary markers are easy to put down, are completely stationary with respect to the coverslip, are biocompatible, and do not interfere with fluorescence or intensity measurements. As the coverslip undergoes drift (or is purposely moved), the x-y center of the fiduciary markers can be readily tracked to 1 nanometer using a Gaussian fit. By focusing the light slightly out-of-focus, the z-axis can also be tracked to < 5 nm for dry samples and <17 nm for wet samples by looking at the diffraction rings. The process of tracking the fiduciary markers does not interfere with visible fluorescence because an infrared light emitting diode (IR-LED) (690 and 850 nm) is used, and the IR-light is separately detected using an inexpensive camera. The resulting motion of the coverslip can then be corrected for, either after-the-fact, or by using active stabilizers, to correct for the motion. We applied this method to watch kinesin walking with ≈8 nm steps.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 12177-12183 |
| Number of pages | 7 |
| Journal | Optics Express |
| Volume | 20 |
| Issue number | 11 |
| DOIs | |
| State | Published - May 21 2012 |
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ASJC Scopus subject areas
- Atomic and Molecular Physics, and Optics
Cite this
Using fixed fiduciary markers for stage drift correction. / Lee, Sang Hak; Baday, Murat; Tjioe, Marco; Simonson, Paul D.; Zhang, Ruobing; Cai, En; Selvin, Paul R.
In: Optics Express, Vol. 20, No. 11, 21.05.2012, p. 12177-12183.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Using fixed fiduciary markers for stage drift correction
AU - Lee, Sang Hak
AU - Baday, Murat
AU - Tjioe, Marco
AU - Simonson, Paul D.
AU - Zhang, Ruobing
AU - Cai, En
AU - Selvin, Paul R
PY - 2012/5/21
Y1 - 2012/5/21
N2 - To measure nanometric features with super-resolution requires that the stage, which holds the sample, be stable to nanometric precision. Herein we introduce a new method that uses conventional equipment, is low cost, and does not require intensive computation. Fiduciary markers of approximately 1 μm x 1 μm x 1 μm in x, y, and z dimensions are placed at regular intervals on the coverslip. These fiduciary markers are easy to put down, are completely stationary with respect to the coverslip, are biocompatible, and do not interfere with fluorescence or intensity measurements. As the coverslip undergoes drift (or is purposely moved), the x-y center of the fiduciary markers can be readily tracked to 1 nanometer using a Gaussian fit. By focusing the light slightly out-of-focus, the z-axis can also be tracked to < 5 nm for dry samples and <17 nm for wet samples by looking at the diffraction rings. The process of tracking the fiduciary markers does not interfere with visible fluorescence because an infrared light emitting diode (IR-LED) (690 and 850 nm) is used, and the IR-light is separately detected using an inexpensive camera. The resulting motion of the coverslip can then be corrected for, either after-the-fact, or by using active stabilizers, to correct for the motion. We applied this method to watch kinesin walking with ≈8 nm steps.
AB - To measure nanometric features with super-resolution requires that the stage, which holds the sample, be stable to nanometric precision. Herein we introduce a new method that uses conventional equipment, is low cost, and does not require intensive computation. Fiduciary markers of approximately 1 μm x 1 μm x 1 μm in x, y, and z dimensions are placed at regular intervals on the coverslip. These fiduciary markers are easy to put down, are completely stationary with respect to the coverslip, are biocompatible, and do not interfere with fluorescence or intensity measurements. As the coverslip undergoes drift (or is purposely moved), the x-y center of the fiduciary markers can be readily tracked to 1 nanometer using a Gaussian fit. By focusing the light slightly out-of-focus, the z-axis can also be tracked to < 5 nm for dry samples and <17 nm for wet samples by looking at the diffraction rings. The process of tracking the fiduciary markers does not interfere with visible fluorescence because an infrared light emitting diode (IR-LED) (690 and 850 nm) is used, and the IR-light is separately detected using an inexpensive camera. The resulting motion of the coverslip can then be corrected for, either after-the-fact, or by using active stabilizers, to correct for the motion. We applied this method to watch kinesin walking with ≈8 nm steps.
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UR - http://www.scopus.com/inward/citedby.url?scp=84861501724&partnerID=8YFLogxK
U2 - 10.1364/OE.20.012177
DO - 10.1364/OE.20.012177
M3 - Article
C2 - 22714205
AN - SCOPUS:84861501724
VL - 20
SP - 12177
EP - 12183
JO - Optics Express
JF - Optics Express
SN - 1094-4087
IS - 11
ER -