This page contains a Flash digital edition of a book.
Plasmonic Gratings Results


Figure 3 : A wide range of dye concentrations were imaged using these silver GLAD gratings (α = 60°): (a) 50 pM and (b) 100 pM DNA/RNA duplex-labeled with Cy3/Cy5; (c) 1 µM and (d) 10 µM Cy5 in PMSSQ matrix. Scale bar = 5 µm for all images. (e–g) Representative time traces for 1 µM Cy5 at hotspots with single/ double/multiple step bleaching behavior using 550 µW 642 nm laser taken with 60× objective in epifl uorescence mode. Reproduced from [ 19 ] with permission, copyright 2016 RSC.


large number of molecules and overlapped signals observed on highly concentrated fl uorescent samples, we recorded the single/ double/multiple step photobleaching behavior on hotspots. T e quantized photobleaching of each Cy5 molecule with a single “on” and “off ” state enables the counting of dye molecules with relative ease by using the number of photobleaching steps, seen as distinct intensity plateaus, to estimate the number of molecules in each time trace [ 30 – 32 ]. Figures 3 e– 3 g illustrate this counting technique with time traces extracted from the sample imaged in Figure 3c .


Beating the diff raction limit. During our localization microscopy analysis, we observed a strong correlation between the density of hotspots in the fl uorescent images and the density of nanoprotrusions observed in atomic force microscope (AFM) scans of the GLAD surface. These data support the idea that nanoprotrusions ( Figure 4a ) are capable of concentrating the E-field as seen in FDTD simulations, which results in the formation of the fluorescence hotspots ( Figure 4b ). Using single-molecule analysis of the fluores- cence images, we observed two hotspots with a separation distance of 211 nm ( Figure 4c ). This distance would be too short to measure in a diff raction-limited image, but resolution obtained here was made possible by the far-fi eld superlensing (FSL) eff ect of the GLAD gratings. Localization microscopy . Despite the sub-diffraction limit resolution of the raw fluorescence images, they still cannot provide the exact size of nanostructures less than ~210 nm without additional processing. T is resolution limit is thought to arise from an overlap of the diff raction-limited, isotropic emission of some single molecules at the 0-order and the sub-diff raction limit ±1-order diff raction from surface plasmon-coupled emission (SPCE). To further improve the resolution, we have used localization microscopy concepts to analyze the time-trace videos from the sample in Figure 3c . In localization microscopy, the coordinates of temporally separated single molecules are found via a fitted, isolated point-spread function (PSF). While PSF fitting is used by many current imaging techniques, those methods often require well-spaced single molecules to obtain accurate sample information [ 33 ]. T is becomes a much larger limitation when applied to imaging fluorescently labeled biological samples where the spatial density of fl uorophores is such that they are too overlapped for single-molecule resolution. T e advantage of the GLAD gratings is that the high SNR (28±9) provided by the nanoprotrusions and nanogaps allows us to better resolve individual molecules even when the signals of several molecules are overlapped. T is in turn allows us to obtain a well-fi tted PSF at concentrations beyond the capabilities of other systems. In addition to PSF analysis we also employed the multiple-


Figure 4 : Comparison of hotspot density between (a) AFM image with the height range (50–90 nm) of the protruding grains highlighted and (b) false-colored fl uorescence image of 10 µM Rhodamine 6G dye in a PMSSQ matrix using a 60× WI objective (NA = 1.2) after background subtraction. (c) Intensity vs. distance plot from fl uorescence image; inset shows two analyzed hotspots and the cross section used for profi le analysis. The fl uorescence images were taken by an ORCA-Flash 2.8 CMOS camera. Reproduced from [ 19 ] with permission, copyright 2016 RSC.


2017 January • www.microscopy-today.com


emitter fi tting analysis (MFA), which is capable of processing a much higher density of emitters than with single-emitter analysis. MFA uses the maximum likelihood estimator to obtain position estimates of many single-molecule emissions over several images and combines them into a single image ( Figure 5 ). T is results in an order of magnitude improvement in the tolerance of the analysis routine with respect to the single-frame active emitter density [ 34 ]. We tested the capabilities of MFA analysis using the sample in Figure 3c and compared the images to the original fluorescence images. We found that the resolution of the super- resolution reconstructed fl uorescence


45


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