Each species of songbirds has its own unique song. By studying the neural pathways involved in the learning of these songs, researcher Richard H.R. Hahnloser from the University of Zurich and colleagues are gaining insight into how humans learn to speak their primary language. But because neither light microscopy nor electron microscopy could not easily provide answers to their questions, these Swiss scientists used both.
“Correlative microscopy has been around for a while, but I don’t think most people really appreciate the benefits of what correlative microscopy could provide, wholly because of all the technical challenges of getting a sample from one platform to another within a micron, which can be really time consuming,” says Kirk Czymmek, director of the the Bioimaging Center at the University of Delaware.
Now, researchers are beginning to understand that these benefits might outweigh the struggles in correlating microscopy techniques. In response, instrument manufacturers are introducing the first automated tools for 2-D correlative microscopy. But before the results begin pouring in, many aspects of these different microscopy techniques must be brought together, including the instruments, software, and – probably most importantly – the cultures surrounding them.
Although microscopists might have the same goal of imaging samples at resolutions beyond the human eye, how each one accomplishes that can be quite different.
For instance, two popular techniques in the life sciences – electron microscopy and fluorescent microscopy – are very different; each workflow has specialized equipment, sample preparation and fixation requirements, software packages, and applications. As a result, most researchers are highly trained in one technique, which is often the one that’s best suited to solve their exact research question.
“There’s no reason that a confocal microscopy should be driven by a different software package than a scanning electron microscope or a scanning transmission electron microscope, but historically, the developers of each type of system have had different backgrounds and traditions. So, they tackled the same problem in different ways,” says Roger Albert Wepf, the director of the Electron Microscopy Center at the Swiss Federal Institute of Technology in Zurich.
For decades, a cultural divide has existed between light and electron microscopy. This rift is so great that the equipment used to perform these different techniques are often housed in separate facilities at many research universities. This, in turn, makes the transfer of a sample from one system to another rather challenging.
“The idea that facilities need to be merged is something that’s probably not prevalent yet,” says Czymmek. “That requires restructuring the physical location. If you have to go across campus to transfer a sample, then different microscopy centers need to start communicating with each other and sharing protocols.”
Furthermore, this divide is not limited to academia; multiple cultures exist within microscope manufacturers as well. For example, at optic specialist and microscopy manufacturer Carl Zeiss NTS GmbH, the light microscopy and electron microscopy branches of the business have been two distinct organizations within the company, Carl Zeiss Microimaging and Carl Zeiss NTS respectively. Each evolved different products, software platforms, and expertise separately.
But now, times are changing. Modern research demands more results in less time from researchers. To understand what a particular cell or pathway is doing on all levels, scientists need tools to allow them to see how it behaves as well as its structure on the nanoscale. In addition, researchers don’t have the time to master these techniques; they need an easy way to go from one instrument to the other to produce results immediately.
Until recently, this was something correlative microscopy was most certainly not.
Finding Coordinates, Blindfolded
Correlative microscopy has been manual, tedious, and time-consuming at best. It’s an attempt to image the same region of a sample under two different instruments with high precision, to within a micron. And researchers have been doing this essentially blindfolded, with no label or marker that is compatible with both techniques.
Each technique provides completely different perspectives. With the introduction of fluorescent labels in the 1980s, scientists began imaging cells in 3-D with confocal microscopes and following protein expression patterns in living cells. Although electron microscopy provides a higher resolution and more complete pictures of a cell’s structure, it cannot image living material. In the end, fluorescent microscopy provides images of bright signals on a black background on the microscale, while electron microscopy provides grey-scale images of cellular components on the nanoscale.
“Everyone who came into our centralized center wanted to do fluorescence and then asked what is that glowing spot? Over the past 10 years, we’ve gotten more and more reviewers coming back with comments, asking, ‘What are those little fluorescent spots?’ And that requires going to the electron microscopy level,” says Czymmek.
In a typical correlative microscopy workflow, researchers first use fluorescent microscopy to find an event on the cellular level. Then, the sample is transferred to an electron microscope to get the specific information about the structure surrounding that event. This allows researchers to scan the tissue for interesting regions with fluorescence microscopy and then zoom in for a more detailed perspective with electron microscopy. Because sample preparation for electron microscopy includes chemical fixation and heavy-metal stains that quench fluorescence, the order of the workflow is almost never reversed.
But the major problem is relocation: that is, moving a sample from one instrument to another and then finding the exact same region at different resolutions, with different imaging additives, with different outputs. In the early days of correlative microscopy, this required researchers to hand-draw coordinate systems based on some internal markers in a sample under a microscope. Then the researcher would attempt to find those markers under the next microscope at a lower resolution, and then slowly zoom in from there.
“A real marker system that you can set in a living cell culture and then find the same marker after you do the prep and imaging with other tools, this doesn’t exist at the moment,” says Wepf. “So, having guiding coordinates could help you be on track with your region of interest.”
And now, some of the first automated stages are being developed by manufacturers. In 2010, Carl Zeiss introduced the Shuttle-and-Find solution for sample transfer between their fluorescence and scanning electron microscopes. The system is simple; it’s a sample carrier that has a common coordinate system that is easily identified by the software on both microscopes.
“The biggest challenge was that we had different software platforms,” says Christian Boeker from Carl Zeiss. “Within these separate organizations, we had different software platforms and products.”
During development of the system, the biggest challenge was the software because both microscopes would have to run the same software to find the coordinate system. For the first time, these two divisions had to talk with one another to resolve this issue. In the end, this pilot project has been so successful that the company announced at Neuroscience 2011 that these two divisions would be merged into one business unit, Carl Zeiss Microscopy.
“These automated systems are available, but it’s become such a recent focus with manufacturers that it’s not readily appreciated and not readily available in most facilities,” says Czymmek.
So now, it becomes a process of bringing people together and bringing them up to speed with one another.
Only the First Step
“It became very obvious that a simple 2-D coordinate transfer between wide field and a scanning electron microscope can only be the first step,” says Boeker. Specifically, three important directions for the development emerged from this meeting: super-resolution, cryo-preparation, and 3-D microscopy.
To correlate super-resolution images with electron microscopy, precision becomes even more important. Since you’re working with images in the nanometer range, the coordinate registration and tracking must be more precise than when working with standard fluorescent microscopy images. If you’re talking single-molecule imaging, you need to make sure the molecule that you’re looking at is the one that is producing the fluorescent signal and not a neighboring molecule.
To work with three dimensions, two things are necessary. First of all, all three axes must be identified in the system. Second, the system must identify this coordinate information even after the distortions that occur during sample prepartion for electron microscopy.
For example, hydrated cells are best for 3-D confocal microscopy, but cells must be dried out prior to electron microscopy. This dehydration results in a change in cell shape and size. While the hardware development for this is relatively straightforward, the software to process these 3-D distortions and identify the coordinate system will be trickier.
Finally, better methods to cryo-prepare samples in shorter time frames would allow researchers to actually pause a biological event of interest instantaneously. Right now, chemical preparations for fixation are standard, but they lead to certain distortions in the cells. When you begin to look at the nanoscale, these distortions could affect the resulting data. While current cryo-preparation techniques can freeze a sample in 5-10 seconds, bringing that time down to the second or sub-second mark would be beneficial for particularly interesting events in cell biology and bring a new level of temporal precision to the study of these events.
Of course, there are loftier goals as well. For example, Wepf sees a day when cryo-electron tomography or pinpointed mass spectrometry could be incorporated into correlative microscopy so that researchers could identify the elements and ions that create the varying shades of gray in electron microscopy images. Meanwhile, Czymmek sees no reason why atomic force microscopy couldn’t provide similar biophysical information if correlated with super-resolution techniques.
“We’re really in the infancy of the whole process. I can imagine in five years, we’ll see a great leap forward,” says Czymmek.
1. Husain, M., C. Thomas, M. Kirschmann, D. Oberti, and R. Hahnloser. 2011. Functional and structural investigation of songbird brain projection neurons with shuttle and find. Microscopy and Microanalysis 17(Supplement S2):242-243.