CLIC to Enhance

The CLIC imaging step-by-step process

1. Flow chamber is assembled sing two glass coverslips separated by a custom adhesive spacer, with a thickness of between 10 and 100 µm. This is mounted within the CLIC device, where it is held tight and sealed.
   
   
2. The sample is loaded using a pipette and then air is used to push the liquid from the reservoir into the flow chamber. Usually, the imaging buffer is inserted before the sample; this creates a good environment for biomolecules and minimizes nonspecific adsorption.
   
   
3. Imaging chamber geometry is formed by reshaping the planar flow cell into a thin volume suitable for imaging. A convex push-down lens is lowered onto the top of the flow chamber, gradually bringing it into contact with the bottom surface. This creates a graduated chamber. The bottom surface can also contain embedded nanostructures designed to manipulate the shape of molecules once they are squeezed into the chamber.
   
   
4. Imaging is performed by acquiring high-resolution fluorescence images, usually at a series of locations within the chamber, thereby allowing the user to optimize image settings.

We’ve been able to visualize single biomolecules for years, but the techniques we use are not without their limitations: keeping molecules in focus, background fluorescence interference, inadequate resolution, and DNA loading and viewing challenges, can make the entire process pretty difficult and the end result an inaccurate one. And that’s without taking into account the risk of losing sight of valuable interactions between protein and DNA – these often disappear from view when using traditional methods (1).

Although confocal and total internal reflection fluorescence (TIRF) microscopy techniques, for example, have supported our molecular imaging efforts well so far, I feel we need to take it a step further. It’s particularly pertinent as the importance of molecular imaging continues to increase at an astounding pace.

In light of this rising interest, we are hearing about a lot of exciting new technological developments, but many are considered out of reach to the normal lab, for one main reason – cost. The other reasons? Workflow changes, training needs, lab space challenges. Recognizing the limitations of current microscopes, my colleagues and I set about finding a solution that was both easy to implement and cost-effective. And we think we have. Convex-lens induced confinement, or CLIC.

CLIC imaging relies on a simple principle: molecules are forced into a well-defined nanoscale space, which can be used to confine the molecules to the focal plane of your existing inverted fluorescence microscope. The thinness of this space means that background fluorescence is reduced (so image quality is higher), molecule conformation is easier to change and manipulate (an advantage which has implications for genome mapping), and importantly, CLIC imaging chambers can be assembled by you, which means you can customize the process to your own needs.

Based on our own studies of using the CLIC imaging system, which we incorporated onto an inverted fluorescence microscope, we have found several key advantages relative to other single-molecule techniques (which are also typically employed on inverted microscopes), including: enhanced observation of single molecules, reduced background fluorescence, and over a thousand-fold increase in observation time (2).

How can it help molecular pathologists?

In a recent article by my colleagues and I (2), we discuss a number of applications for CLIC, one being in genome mapping of long DNA strands.

Open-face nanochannels on the bottom surface of the chamber make it possible to observe molecules as they load into the chamber. They are able to fully extend along the channels without breaking, which is important as it allows the mapping of long-range structural rearrangements of the genome. In contrast, conventional methods, such as nanofluidic technologies, often use large applied fields or pressure to load the DNA, which may cause the strands to break into smaller pieces and clog the channels.

I also see the potential for CLIC to be used as a detection method for a panel of cancer biomarkers, for example, because of the sensitive imaging chamber which can detect molecules over a range of volumes.

We are now engaging in collaborations to combine CLIC with other nanotechnologies to create platforms for optimal sensing of biomolecules – something I think many molecular pathologists will be excited about.

How can it help clinical pathologists?

In addition, clinical laboratories which already have an inverted fluorescence microscope could incorporate CLIC, and our belief is that this could be used in clinical diagnostics should our follow-up project yield the results we expect it to.

As part of a recent collaboration (2), we aim to develop a single-cell imaging device which first lyses a cell, next purifies the DNA and then loads it into a CLIC imaging chamber. We expect to have a basic prototype within one year. This will enable faster and cheaper diagnostics of single cells, in particular when compared with current methods used for prenatal (where few cells are available to work with) and cancer diagnoses.

If we consider cancer; the genome for each cell can be very different, so the ability to map cancer genomes one cell at a time may be important in understanding disease onset. Most current techniques work with the average of a population of cells, which might cause essential information to be missed. It is possible for a small fraction of cells among a population to be virulent and persistent, and I believe single-cell diagnostics are needed to understand this behavior.

Geometry glitches

I think CLIC imaging could provide a leap forward in microscopic imaging, but as with any new innovation, there were challenges during its development. Difficulties included loading samples and caring for surfaces. We also had to create an approach to control and measure imaging geometry. However, these technical problems were interesting to solve along the way, and I believe we have succeeded in creating a user-friendly device that overcomes the issues we have faced. Currently, we are augmenting the microfluidic capabilities of our CLIC device to allow us to insert multiple reagents in a controlled fashion, and temporally resolve their interactions. We are pushing the buffer exchange capabilities, and the flow chamber design and material is being optimized for imaging quality and control over the imaging geometry.

Where to next?

The modular CLIC device (Figure 1) my team has built is now ready for distribution and use in laboratory settings, and we are looking into commercialization. More long-term (over many years), we hope to create a miniature, hand-held CLIC device which is diagnostic specific.

I believe that current microscopy techniques and equipment do not always provide high enough quality imaging, and I think CLIC could provide better-quality results. Importantly, using CLIC imaging would not require a laboratory overhaul or the installation of large or complex new pieces of equipment.

I hope to establish CLIC as a helpful tool for tackling a wide range of challenges in pathology, biology, medicine and biophysics, and to me, the best way to do this is to get CLIC out there and into the hands of the scientists who could use it in exciting and ingenious ways.  So far, the overall response to our technology has been positive, and is growing, which I find very exciting. We also encourage any scientists who are finding their own applications for CLIC imaging to get in touch with us.

Sabrina Leslie is an assistant professor in the Department of Physics at McGill University Montreal, Canada.