Why building an infrared confocal system?

Last week, Confocal.nl signed the Eurostars project “DETECtORS” and will lead the multinational research and development team to build the first commercial near-infrared confocal microscope (RCM-NIR). Here we explain in detail, how will this project contribute to the fields neuroscience and cancer research.

Wing of fly. Image obtained during testing of the prototype of RCM-NIR. Excitation 780nm, emission from autofluorescence.

Why is RCM so special?

The RCM (Re-Scan Confocal Microscope) is an evolution of the traditional confocal microscope that has improved the sensitivity and the lateral resolution. The sensitivity is markedly improved by using a sCMOS camera as a detector. The traditional photomultiplier tube (PMT) has a Quantum Efficiency (QE) of 15%. A specialised PMT (GaAsP) has a QE of 35%. The QE of a sCMOS camera ranges from 85% – 95%, depending on the model.

To be able to use the camera instead of PMT as a detector, the RCM includes two sets of XY scanning mirrors. The first set is called “the scanner”, and the second set “the re-scanner”. The scanner scans the specimen with the excitation light while the emitted light is written on the camera chip with the re-scanner. The true breakthrough in RCM design was the increase of the sweep of the re-scanner light beam by a factor of 2. The information that is written on the camera chip is not enlarged by a factor of two; only some motion blur is added. But when the 2 times enlarged re-scanner image is scaled back, the information dot with the added blur is reduced half in size. With this simple trick that is done in real time, the lateral resolution improves from traditional 240nm to 170nm! Most importantly – this improvement is purely opto-mechanical, and no software processing is needed.

So, the key advantages of the RCM are improved sensitivity, and improved lateral resolution (and the price!).

What is deep tissue imaging? 

The light of the confocal microscope can normally penetrate around 300 um into the tissue. This is determined by the physical properties of the tissue (scattering), but also by the wavelength of the light. Longer wavelengths penetrate deeper than shorter ones. In order to penetrate even deeper into the tissue, multi-photon (MP) microscopy was developed. The first MP confocal microscope was invented almost 20 years ago. In MP imaging, two IR photons come together in the focus plane and act like a photon with double the energy (half of the wavelength). Because of the longer wavelength the tissue can be penetrated deeper. Because the reduction (half) of the wavelengths in MP imaging, same visible range dyes can be used as in regular confocal microscopy. The drawback of MP confocal microscopy is the fact that very powerful lasers need to be used (3-5 Watt). These lasers are safety class 4 lasers, which means they can only be operated by trained staff in well shielded environments. With MP imaging, penetration depths of 1000 um = 1mm can be achieved, which can further be extended to about 5 – 10 mm by clearing the tissue.

Why is working in IR so special? 

Long wavelengths penetrate deeper than shorter ones. Because of this fact, the whole animal (mostly mouse) imaging systems are using (Near) Infra Red (NIR / IR) wavelengths. These whole animal imaging systems are used for molecular imaging.  Molecular imaging originated from the field of radio pharmacology due to the need to better understand fundamental molecular pathways inside organisms in a noninvasive manner. As these systems need to image the whole animal, a macro camera is used. The disadvantage of the macro camera is that the resolution is rather low. Only the location of the target within the animal can be viewed, not the content.

With the RCM-NIR using Single-Photon (SP) NIR / IR, we’re going to improve the resolution. It will not be able to scan the whole animal, but the target parts can now be investigated at much higher detail compared to the whole animal imaging systems. Because of the RCM technology, the lower resolution of IR wavelengths compared to the visible light, can be offset. 1,000 nm wavelength results in RCM-NIR resolution of 370nm, which is the same as 700nm regular confocal resolution.

Our aim with this project is to provide new tools for neuro- and cancer research studies that allow them to see more tissue with higher detail, with the equipment that costs significantly less than any current option, which in turn allows more researchers to join the studies. Neuroscience will get a new tool that performs equally to the current MP system, but with less damaging and complicated lasers. Cancer research will get a new tool that allows the use of the same dyes as in whole animal studies, but at much higher resolution that exceeds even the standard confocal microscope.

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