ArticlePloS one2015
Simple Experimental Methods for Determining the Apparent Focal Shift in a Microscope System.
Article in PloS one, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed.
- Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet.Nature communications · 2024Article
- Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet.bioRxiv : the preprint server for biology · 2024Article
- Three-dimensional structured illumination microscopy with enhanced axial resolution.Nature biotechnology · 2023Article
- Multiscale fluorescence imaging of living samples.Histochemistry and cell biology · 2022Review
- Super-resolved live-cell imaging using random illumination microscopy.Cell reports methods · 2021Article
- Visually precise, low-damage, single-cell spatial manipulation with single-pixel resolution.Chemical science · 2021Article
- Addressing systematic errors in axial distance measurements in single-emitter localization microscopy.Optics express · 2020Article
- Quantitative Image Restoration in Bright Field Optical Microscopy.Biophysical journal · 2017Article
- Quantitative 3D structured illumination microscopy of nuclear structures.Nature protocols · 2017Review
- The effect of antibiotics on protein diffusion in the Escherichia coli cytoplasmic membrane.PloS one · 2017Article
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Authors and funding
2 authors.
Funding
Abstract
Three-dimensional optical microscopy is often complicated by a refractive index mismatch between the sample and objective lens. This mismatch causes focal shift, a difference between sample motion and focal-plane motion, that hinders the accuracy of 3D reconstructions. We present two methods for measuring focal shift using fluorescent beads of different sizes and ring-stained fluorescent beads. These simple methods are applicable to most situations, including total internal reflection objectives and samples very close to the interface. For distances 0-1.5 μm into an aqueous environment, our 1.49-NA objective has a relative focal shift of 0.57 ± 0.02, significantly smaller than the simple n2/n1 approximation of 0.88. We also expand on a previous sub-critical angle theory by means of a simple polynomial extrapolation. We test the validity of this extrapolation by measuring the apparent focal shift in samples where the refractive index is between 1.33 and 1.45 and with objectives with numerical apertures between 1.25 and 1.49.
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Registered trials
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