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Supper-resolution ORCA-Flash Images

Sample: actin bundles in fixed HEK293T cells, labeled with the actin binding peptide Lifeact fused to d2EosFP
Imaging conditions: Exposure time: 50 ms; Gain = 8; Binning = 2; In 375 × 458 pixels after binning; Effective pixel size at sample = 145 nm
Photos courtesy of Dr. Zhen-li Huang (Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology)

	Figure 1. The capability of the Flash 2.8 sCMOS camera in localization microscopy. A stack of 2500 image frames (Exposure time: 50 ms; Gain = 8; Binning = 2; In 375 x 458 pixels after binning; Effective pixel size at sample = 145 nm) was used to construct TIRF images (subset 1 in a, c, e) and localization microscopy images (subset 2 in b, d, f) of actin bundles in fixed HEK293T cells, labeled with the actin binding peptide Lifeact fused to d2EosFP. The data in (f) were cleaned up (g) and further analyzed (h). The projection of all molecules in (g) to an orthogonal direction of the mean contour line is shown in (h). The FWHM of the histogram in (h) was found to be 44 nm by Gaussian fitting. Scale bars: 15 mm for (a, b), 2 mm for (c, d), and 500 nm for (e, f). The total number of localized molecules is 2.3 x 10⁶ in (b).
	Figure 2. Histograms of the height of peak pixel after background subtraction (a), total collected photons (b), signal-noise-ratio (c), signal-background-ratio (d) and localization precision measured in standard deviation (e) from single molecules. The mean values of individual histograms are shown in the right corner of the corresponding figures. Here the same data as those in Figure 1 were used. Note that the ration between the FWHM and standard deviation of the localization precision is 2.35.
	Figure 3. Dependence of spatial resolution and the number of image frames in localization microscopy. Super-resolution images were reconstructed with a total number of 500, 1000, 1500, 2000 and 2500 image frames, respectively, and were used to calculate the total number of localized molecules (LN_mol) from different number of image frames. The super-resolution image constructed from 2500 frames in Figure 1(b) was used to calculate the sample area occupied by actin bundles (S). Labeling density N equals to LN_mol/S, was used to calculate Nyquist resolution and overall resolution . The localization precision measured in FWHM, σ_Localization, is set to be 42 nm.
	Figure4. Original TIRF image frames in the green box were used to build this movie of actin bundles in fixed HEK293T cells, labeled with the actin binding peptide Lifeact fused to d2EosFP. View the movie

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