Advanced fluorescence microscopy methods for the real-time study of transcription and chromatin dynamics

Abstract

In this contribution we provide an overview of the recent advances allowed by the use of fluorescence microscopy methods in the study of transcriptional processes and their interplay with the chromatin architecture in living cells. Although the use of fluorophores to label nucleic acids dates back at least to about half a century ago, (1) two recent breakthroughs have effectively opened the way to use fluorescence routinely for specific and quantitative probing of chromatin organization and transcriptional activity in living cells: namely, the possibility of labeling first the chromatin loci and then the mRNA synthesized from a gene using fluorescent proteins. In this contribution we focus on methods that can probe rapid dynamic processes by analyzing fast fluorescence fluctuations.

Keywords: 3D orbital tracking; Fluorescence; Pair Correlation Function; RICS; brightness analysis; fluctuations.

Figure 1. A graphical summary of the principal fluorescence microscopy methods discussed in this manuscript and their application in chromatin and transcription research. (A-C) Different imaging modes. In widefield imaging a large region of the sample is homogeneously illuminated and imaging is performed using a camera. In Point Scanning, a focused laser spot is typically displaced on the sample in a raster pattern. Multiple planes can be imaged to obtain a 3D reconstruction of the sample. (D) In FRAP, a region of the sample is photobleached by intense excitation light, and the recovery of fluorescence is observed over time. (E) SPT allows tracking of an individual particle over time. The reconstructed trajectory can be used to obtain information about the diffusion coefficient, regions confining the motion or interaction with other particles or organelles in a cell. (F) FCS and PCH are complementary approaches that allow extracting diffusion, brightness and concentration information from the sequences of fluctuations in photon numbers detected from a small excitation volume. Autocorrelation of the time sequence provides the fluorescence AutoCorrelation Function. Analysis of the distribution of the photon counts provides brightness information to determine aggregation state of the molecules. (G) pCF can be performed on any microscope able to do fast line (or circular) scans; it measures as a function of the time delay the cross correlations between the fluctuations at two different spatial positions. It can be used to measure diffusion and flow in the presence of obstacles (H) RICS provides a spatiotemporal correlation map of raster scan images. The 2D autocorrelation function can be fit to a model to extract diffusion coefficient and binding rates (I) tICS performs temporal autocorrelation on each pixel of a sequence of images. It can provide a spatial map of slow (> 10 ms temporal resolution) diffusion and binding processes.

Figure 2. mRNA-MS2 fluorescence intensity collected using the 3D orbital tracking method. (A) Fluorescence intensity over time collected using 3D time-lapse imaging in a confocal laser scanning microscope. One point is collected approximately every 20 s (adapted from¹²). (B) Configuration to record mRNA labeled MS2-EGFP fluorescence intensity using the orbital tracking method. A circular orbit above, and one below the particle are used to calculare its x,y,z position. The integrated fluorescence intensity along each orbit cycle is represented as a line in the kymograph in (D). (C) Reconstruction of the 3D trajectory of the lac repressor-mCherry gene array within the nucleus. (D) Kymograph (or intensity carpet) of the fluorescence emission from MS2-EGFP labeled mRNA scanned along a circular orbit locked-in to a lac repressor-mCherry gene array. One line is collected every 32 ms.