Time to focus

September 27, 2013

Life from single molecules to entire populations takes place in four dimensions. Three of which are spatial dimensions and last, but not least, the dimension of time. Interestingly, researchers ignored these hard realities for quite some time. During my PhD project on translational regulation within cells, we would like to master the four dimensions as good as we can. Live-cell imaging is a good method to monitor a single cell over time and to observe what is changing. However, live-cell imaging requires sharp and crisp images in order to be able to track single molecules over longer time spans. The biggest problem with conventional light microscopes are in fact the three spatial dimensions (x, y, z), because all the light from the specimen that you are observing is collected. This means not only the light of a single plane (x,y dimensions) is collected (and later observed), but also the light originating from all other planes above or below (z dimension) (see also Figure 1). Collecting a lot of this so-called “out of focus” light leads to blurred pictures, which means that fine details cannot be distinguished from each other anymore.  A powerful tool to circumvent this problem is a variation of classical light microscopy called CONFOCAL MICROSCOPY. Here, I would like to give a short introduction into this extremely powerful and widely used microscope technique.

ConfocalMicroscopyPlanes

Figure 1: A cell that is observed under a microscope has three dimensions (x, y, z). However, the optics of a microscope dictate that only one z-plane can be “in focus” and not all planes at the same time. A standard microscope collects the light of all planes and therefore often produces blurred images when larger objects such as cells are observed.

In order to make sense of the confocal technique (con-focal = “having the same focus”) I would like to draw your attention to Figure 2. With the help of the steps 1 to 5 I will guide you through the figure. First of all a confocal microscope needs a strong light source. This role is often fulfilled by a short-wavelength laser (Step 1). The laser light is then reflected in a 45° angle by a so-called dichroic mirror (Step 2). This special mirror reflects short wavelengths (such as the green excitation laser), but is permissive for longer wavelengths (such as the emitted red light). The reflected green laser light is focused by the objective lens onto the specimen. Unfortunately, it is impossible to focus the light on only one single z-plane. As a consequence, a number of z-planes are excited by the green light and depending on the fluorescent molecule emit longer wavelength light, here depicted as red, orange, and purple (Step 3). Partly, this emitted light will later form the image that you can observe, but first it needs to travel to your eye: As explained above, the dichroic is permissive for the emitted longer wavelength light. Therefore, the light originating from all z-planes can pass. Since the light is originating from different planes, it also hits the so-called focal lens at different positions resulting in different focal points of this light. And now a small slit, called pinhole comes into play (Step 4): Most light (based on its origin) cannot pass this tiny opening because it is either focused in front of or behind the pinhole. The reason why a confocal microscope produces crisp images is that only light from a single z-plane is able to pass since its focal point is exactly within the pinhole (in this example the red light). Consequently, this light can reach the detector (Step 5) where it is converted into a visible image.

 ConfocalMicroscopy

Figure 2: The setup of a confocal microscope can be described in five simple steps (see text). The pinhole is the central element because it blocks all “out of focus” light originating from non-desired z-planes.

Unfortunately, the seemingly simplistic confocal approach also has two important side effects. First of all, a lot of light is lost because it is shielded by the pinhole. This in turn requires a very strong light source which can damage the sample if applied for a long time. In order to prevent this from happening, the specimen is scanned point-for-point in the x,y dimension. This leads us to the second side effect: Scanning takes a lot of time and this is kind of impractical if you want to observe a live cell. But: Both problems can be (partly) resolved by a variation of confocal microscopy called “spinning-disc confocal microscopy”.

More on this technique in my next post!

Traditionally single-molecule experiments are performed in vitro and therefore in a reduced environment. Recently, it has become possible to combine this single-molecular accuracy with a living single cell and to observe what happens in real time (“live”). For biologists the combination of these three technological ideas creates a lot of possibilities to answer a number of currently unanswered questions. I am very happy to be able to be part of this adventure. In the following I would like to address some aspects of my work:

What am I doing?

Currently I am working on the intriguing and big question how cells translate their DNA into protein. Interestingly, many important sub-questions of this problem still remain unanswered, especially when focusing on the fate of mRNA molecules once they have left the nucleus and are present in the cytoplasm. The quantification of the translation process in time and space, characterization of its steps and major molecular players is our focus area. In order to elucidate what happens to mRNAs in the cellular context we mark them with fluorescent proteins and apply single- and live-cell imaging. In addition, new labeling and detecting technologies allow to study mRNAs at the single-molecular level.

Why study translation live and in single cells?

The so-called central dogma of biology, namely the conversion of information stored in the DNA into proteins, has been dissected by a large number of scientists. However, in most traditional approaches the mRNAs as the central information carriers are isolated from large numbers of cells and therefore removed from their natural cellular context. This results in functional deficits and loss of spatio-temporal information (“Why is this mRNA at this place in this cell at this time?”). In contrast, the combination of single- and live-cell imaging allows to study the fate of mRNAs during translation in their physiologic environment, over a longer period of time and with a minimum of disturbing factors. The use of only single cells also allows to detect differences between cells of the same kind (for example neurons or muscle cells). An organ represents a very heterogeneous environment, so cells have to be different in order to be able to adapt to their local environment. Even 150 years ago Charles Darwin already noted that observable traits can vary widely within a species. Why couldn’t this also be the case for individual cells?

Why single-molecular accuracy?

Next to the advantages that live single-cell analysis has to offer, it is important to keep in mind that most biological processes can be reduced to the level of molecules. When, however, a larger number of molecules is observed (even within a single cell) this automatically leads to an averaging effect. A complicated biological process, like the mRNA translation into protein, involving a number of molecules during specific stages might therefore only be recognized as single event with a “before” and “after” without knowing what really happened in between. By visualizing single molecules it becomes possible to track their role as a puzzle piece within the big picture.

Nice. And how is this done?

There are two major tools. The first one is a microscope (more specific: a light microscope called confocal spinning disc microscope) to observe the single cell with its mRNA molecules. However, the resolution of a light microscope is limited to about 220 nm (1 nm = 1 m / 1,000,000,000). Even though a RNA molecule might be longer, it is also about 1,000 times thinner and therefore not detectable. In order to be able to still detect them we label them with fluorescent proteins. The emitted light results in a so-called “diffraction limited spot” which can be detected by the cameras of our microscope. For the RNA labeling we apply the MS2 and PP7 systems which use specific bacteriophage proteins that are again fused to fluorescent proteins and can bind to specific regions within the mRNA molecule of interest. Importantly, the MS2/PP7 labeling does not harm the biological processes within the observed cell. With this system it is also possible to label a single mRNA molecule in two colors (for example red and green). During the mRNA translation process different parts of the mRNA are targeted by the translation machinery in a sequential manner which has an influence on the binding of the green and red proteins. The appearance of both colors at the same time (yellow), first green and then red, or the other way around, the speed at which this change occurs, and the location within the cell can tell us a lot about the translation process.

In case I could spark your interest for single-molecule live cell imaging please also see our website or check out the following three articles on mRNA labeling and detection:

  1. Hocine et al., Single-molecule analysis of gene expression using two-color RNA labeling in live yeast. Nat Methods. 2013 Feb;10(2):119-21.
  2. Wu et al., Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells. Biophys J. 2012 Jun 20;102(12):2936-44.
  3. Larson et al., Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science. 2011 Apr 22;332(6028):475-8.

More on

  • the Spinning-disc microscope
  • the MS2 and PP7 labeling systems
  • and “diffraction limited spots”

will follow later.