There is reduced scattering as well as minimal absorption and auto-fluorescence by tissue when imaging in the second biological window (NIR-II, from 1000 to 1700 nm). As a result, there is a much better image contrast, sensitivity, and penetration depth into tissue at these wavelengths than traditional visible or infrared optical imaging (i.e. 400 - 1000 nm).  Reaching a penetration depth of up to 3 centimeters has a huge impact when imaging small animals like mice. It opens a new window of possibilities since it allows the visualization of full organs as well as cellular processes in real-time with high spatial resolution.


By Stephen Marchant, Jacob Yvon-Leroux and Émilie Beaulieu-Ouellet


In vivo imaging in the second biological window (NIR-II) is in its earlier stages but will undoubtedly push many life science researchers back to the drawing board for their preclinical workflows.

Preclinical optical imaging suffers from the inability to localize signals due to complications associated with light absorption, scattering and autofluorescence in living tissues. In vivo optical imaging can localize a signal well when it is at the surface but not when it is deep in the organism. 

Preclinical biologists still strongly desire the ability to rapidly localize optical signals in vivo, but their discussions with imaging physicists often end up in a standstill. Biologists ask: can I use optical imaging to see my mCherry cancer cells in vivo? What about my luciferase cells? The answer is: it depends on many different factors such as the temperature of the animal, the optical properties of organs, how deep they are and how many photons come out.

NIR-II in vivo imaging is not impacted in the same way by drawbacks of light propagation in living tissues, thus enabling real-time imaging of optical probes much deeper in the organism and with much higher resolutions.

One of the breakthroughs in the field of in vivo SWIR imaging has been the demonstration that both NIR-I and NIR-II probes can work well for this application. There is an abundance of probes for the new imaging modality and many of them remain to be validated. he ball is back in the court for biologists to take. No longer will biologists need to accept the “oh well, I guess it depends” answer when asking an optical imaging physicist if it is possible to localize their probes in vivo.

Monitor Heart and Respiratory Rates Contact-Free

High-speed imaging made available by the fast frame rates of InGaAs cameras allows for contact-free cardiography and respiratory rate measurements in both anesthetized and awake animals.

For instance, if we target the regions of interest (heart and lungs), we can extract the temporal profiles of the signal. Doing so, we can monitor the heartbeats and breathing movements without the need of an electrocardiogram. Every time the heartbeats or the ribcage expands for breathing, the system detects a rise in amplitude of the signal. Suddenly, we can obtain complete cardiography measurements optically. This is a feat in itself. No need for invasive gated MRI nor intrusive CT scan which has always been the standard for experiments. No need for probes or any contact at all for that matter!

Being able to monitor heart and respiratory rate in real-time and contact-free could open all kinds of doors for various fields of study. Could it change how we follow physical tests on a mouse? Perhaps telemetry during drug tests? In addition, what’s stopping us from applying this technology to an active mouse? 


Real-Time Monitoring of In Vivo Targets at Several Locations Simultaneously

NIR-II imaging has the potential to fundamentally impact the development of targeted therapeutics and drug discovery with real-time pharmacokinetic imaging of over a thousand targets simultaneously in a single mouse.

The new NIR-II imaging technologies are now designed to offer the time profiling analysis for each pixel or a region of interest (ROI) in a single click, hence obtaining rapidly the real-time kinetic curves at several locations simultaneously on the mouse (for example, see figure 5). Then, a principal component analysis (PCA) may be applied to a time series of fluorescence imaging to precisely delineate major tissues and organs. 

With one click spectrum extraction, it is possible to measure the kinetics of fluorescent intensity changes allowing scientists to determine the accumulation and elimination of the probes in selected regions or organs and to compare to obtain relative signal ratios between the organs. This information helps understand how the probes are metabolized by the biological system in detail and to obtain values from hepatobiliary elimination and gastrointestinal transit rates.

The fact that you can do this in the same animal now allows for continuing in the same direction as early developments, i.e. further reducing cohort sizes for preclinical studies while increasing the span of applications due to its millisecond acquisition with enhanced spatial resolution and depth penetration. 
One could also imagine that this could start to play a role in compound screening and allow researchers to use in vivo imaging earlier in the process of probe validation. 



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