Live imaging of genome has offered important insights into the dynamics of the genome organization and gene expression. The demand to image simultaneously multiple genomic loci has prompted a flurry of exciting advances in multicolor CRISPR imaging, although color-based multiplexing is limited by the need for spectrally distinct fluorophores. Here we introduce an approach to achieve highly multiplexed live recording via correlative CRISPR imaging and sequential DNA fluorescence in situ hybridization (FISH). This approach first performs one-color live imaging of multiple genomic loci and then uses sequential rounds of DNA FISH to determine the loci identity. We have optimized the FISH protocol so that each round is complete in 1 min, demonstrating the identification of seven genomic elements and the capability to sustain reversible staining and washing for up to 20 rounds. We have also developed a correlation-based algorithm to faithfully register live and FISH images. Our approach keeps the rest of the color palette open to image other cellular phenomena of interest, as demonstrated by our simultaneous live imaging of genomic loci together with a cell cycle reporter. Furthermore, the algorithm to register faithfully between live and fixed imaging is directly transferrable to other systems such as multiplex RNA imaging with RNA-FISH and multiplex protein imaging with antibody-staining.

Messenger RNA (mRNA) plays a critical role in cellular growth and development. However, there have been limited methods available to visualize endogenous mRNA in living cells with ease. We have designed RNA-based fluorescence “turn-on” probes that target mRNA by fusing an unstable form of Spinach with target-complementary sequences. These probes have been demonstrated to be selective, stable, and capable of targeting various mRNAs for live E. coli imaging.

A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.

Photoactivatable fluorescent proteins (PA-FPs) are widely used in live single-molecule super-resolution imaging but emit substantially fewer photons than organic dyes do. Herein, we show that in heavy water (D2O) instead of H2O, common PA-FPs emit 26–54% more photons, effectively improving the localization precision in super-resolution imaging.

Super-resolution microscopy techniques are often extremely susceptible to sample drift due to their high spatial resolution and the long time needed for data acquisition. While several techniques for stabilizing against drift exist, many require complicated additional hardware or intrusive sample preparations. We introduce a method that requires no additional sample preparation, is simple to implement and simultaneously corrects for x, y and z drift.We use bright-field images of the specimen itself to calculate drift in all three dimensions: x, y and z. Bright-field images are acquired on an inexpensive CCD. By correlating each acquired bright-field image with an in-focus and two out-of-focus reference images we determine and actively correct for drift at rates of a few Hertz. This method can maintain stability to within 10 nm for x and y and 20 nm for z over several minutes.Our active drift stabilization system is capable of simultaneously compensating x, y and z drift through an image-based correlation method that requires no special sample treatment or extensive microscope modifications. While other techniques may provide better stability, especially for higher frequency drift, our method is easy to implement and widely applicable in terms of both sample type and microscopy technique.

The recent invention of superresolution microscopy has brought up much excitement in the biological research community. Here, we focus on stochastic optical reconstruction microscopy/photoactivated localization microscopy (STORM/PALM) to discuss the challenges in applying superresolution microscopy to the study of developmental biology, including tissue imaging, sample preparation artifacts, and image interpretation. We also summarize new opportunities that superresolution microscopy could bring to the field of developmental biology.

Photoactivatable fluorescent proteins (PA-FPs) are widely used in live single-molecule super-resolution imaging but emit substantially fewer photons than organic dyes do. Herein, we show that in heavy water (D2O) instead of H2O, common PA-FPs emit 26–54% more photons, effectively improving the localization precision in super-resolution imaging.