Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells

Abstract

DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging.

Fig. 1. Multiplexed FL-PAINT imaging of a fixed cell.

a Stack of DNA-PAINT frames with single-molecule localizations. Alexa 555 is depicted in blue color and Atto 550 in orange color. b TCSPC curves of the two fluorophores were obtained by adding all single-molecule localizations of the same type. The colors correspond to the colors used in the single-molecule image in (a). c Lifetime histogram of two-target FL-PAINT of a HeLa cell. The combination of imagers P1-Alexa 555 and P3-Atto 550 allowed for a remarkably good separation between the lifetimes of the two targets, indicated by blue and orange rectangles. d Reconstructed FL-PAINT image of a HeLa cell. The lifetime color bar is shown on the right-hand side of the FL-PAINT image. Scale bar is 5 µm. e Magnification of the white frame in the primary image. A lifetime threshold was used to separate the targets into two images: peroxisomes (f) and mitochondria (g). Scale bars are 1 µm.

Fig. 2. Two- and three-target FL-PAINT of HeLa cells using wide-field FLIM.

a Two-target FL-PAINT image: peroxisomes and mitochondria labeled with imagers P1-Alexa 555 and P3-Cy3b. b Lifetime histogram with two Gaussian fits for the HeLa cell shown in (a). The two peaks in the lifetime histogram correspond to the two targets: mitochondria and peroxisomes. c Two-target FL-PAINT image: peroxisomes and mitochondria labeled with the two imagers P1-Atto 550 and P3-Cy3b. d Lifetime histogram with two Gaussian fits for the HeLa cell shown in (c). e Three-target FL-PAINT image: peroxisomes, endoplasmic reticulum, and mitochondria labeled with the three imagers P1-Alexa 555, P2-Atto 550, and P3-Cy3b, respectively. Bottom: zoom-in of white frames in the primary images with separation into different targets. Zoom-in scale bars are 2 µm. f Lifetime histogram with three Gaussian fits for the FL-PAINT image shown in (e). The blue, green, and orange rectangles show the two or three lifetime ranges associated with the different targets. A lifetime colorbar is shown on the right-hand side of the lifetime images. Scale bars are 10 µm. g–l Validating FL-PAINT with Exchange-PAINT. Exchange-PAINT images of HeLa cell with two targets: peroxisomes (g) and mitochondria (h). i Overlay of the two DNA-PAINT images. j–l FL-PAINT image of the same HeLa cell with two targets. j Reconstructed single-target image of peroxisomes. k Reconstructed single-target image of mitochondria. l Combined two-target FL-PAINT image. Scale bars in (g–l) are 5 µm.

Fig. 3. 2D and 3D FL-PAINT using a CLSM.

a 2D CLSM FL-PAINT image of a HeLa cell with two targets: peroxisomes and mitochondria, labeled with the imagers P1-Atto 550 and P3-Cy3b. b The corresponding lifetime histogram with two Gaussian fits for the cell shown in (a). Colors of fitted Gaussians correspond to the colors of the targets shown in (a). c 2D CLSM FL-PAINT images of a HeLa cell again with two targets: peroxisomes and mitochondria, but now labeled with P1-Alexa 555 and P3-Atto 550 imagers. The images correspond to focal planes at 0, 1, and 2 µm axial distance above the bottom of the cell. Z-projections of the separated images for mitochondria (d) and peroxisomes (e). Here, the color represents the height. Lifetime and height color bars are shown on the right-hand side of the images. Scale bars are 5 µm.