Near-infrared fluorophores for Neuroscience research and engineering
Despite the advent of recent imaging modalities such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET) and ultrasound scanners, imaging of deep tissues has been difficult due to poor photon penetration. In mammalian tissues, which is opaque to light in the visible spectrum (400-700nm), traditional imaging has increasing photon scattering and/or photon absorption the deeper into the tissue the image is taken.
The use of in vivo near-infrared (NIR) fluorophores is an emerging trend in Neuroscience research. This is partly due to its capability in reducing photon scattering, light absorption and auto-fluorescence (Figure 1a). Empirical measurements of scattering in tissues by light with different wavelengths reveal that the degree of scattering for almost all biological tissues follows an inversely proportional wavelength relationship (Figure 1b). Therefore, NIR imaging gives the user high imaging resolution within deep tissue preparations. Together with advances in optical design, which allows for the efficient detection of long-wavelength (NIR-I: 700-900nm and NIR-II: 1000-1700nm) photons, this technology may be used in several functional imaging investigations such as in physiology and clinical diagnostics and may even form a translational link between the two.
Figure 1: Motivation for NIR fluorescence-based biomedical imaging. 1.
In Neuroscience research, the two main approaches to achieve deep tissue imaging have been to:
- Develop new instrumentation, such as two and multi-photon microscopy, light-sheet microscopy and optical coherence tomography.
- Develop new fluorophores with improved quantum yields, biostability and biocompatibility.
During the past 10 years, there has been a steady development of several functional NIR-I fluorophores for highly specific anatomical and molecular imaging 1. This development has now extended into the NIR-II spectrum 2. The NIR-I spectrum is traditionally defined as the ‘biological transparency NIR window’ because at these wavelengths there is low tissue absorption and background fluorescence in vivo (in comparison with tissue absorption and background florescence in the visible spectrum). The expansion of optical fluorescence imaging to the under-explored NIR-II window in the last 5-7 years has only been made possible by the continuous development of biocompatible NIR fluorophores with increasingly longer wavelengths, in conjunction with the improvement of more efficient and affordable photon detectors with adequate sensitivity for in vivo imaging in the NIR-II window 2.
In Neuroscience research, specifically, the development of genetically encoded fluorescence voltage sensors that use fluorescence resonance energy transfer (FRET) to combine the rapid kinetics and substantial voltage-dependence of rhodopsin family voltage-sensing domains with the brightness of genetically engineered protein fluorophores. These FRET-opsin sensors significantly improve upon the spike detection fidelity offered by the genetically encoded voltage sensor, Arclight, while offering faster kinetics and higher brightness.
Genetically encoded fluorescent Ca2+ indicators (GECIs), such as GCaMP (Figure 2) have had a major impact on neuroscience research 3 4. However, Ca2+ imaging fails to reveal individual action potentials in many fast-spiking cell types, poorly captures sub-threshold membrane voltage dynamics and offers insufficient temporal information to permit studies of action potential timing to better than ~50-100 ms.
Figure 2: Genetically encoded calcium indicators (GECIs) 4.
However, genetically encoded voltage indicators (GEVIs) directly sense the transmembrane voltage and thus offer the possibility of faithfully observing action potential waveforms and sub-threshold voltage dynamics. The recent introduction of C1V1 and ArchT, red-shifted opsins with several variants, has addressed this issue directly. Expression of this opsin enables action potential generation in highly expressing neurons via conventional two-photon raster scanning, as performed during standard two-photon imaging 5.
It is important to note that electrical signals by neurons are transformed by the kinetics and the linearity of GEVIs to produce the observed fluorescence responses. For example, GEVIs with activation and inactivation kinetics slower than the 2ms duration of APs show less sensitivity for APs than for slower membrane voltage changes. Thus, faster sensors will produce a less distorted representation of voltage. GEVI improvements have reached a point where, for single AP detection, response amplitudes can be like those of commonly used calcium indicators 6 7.
For two-photon imaging, an opsin with a prolonged voltage-sensitive state may be useful. Improving membrane localization of FRET opsins would be advantageous, so that more of the cellular fluorescence is voltage-responsive. When detection of APs and subthreshold voltage changes is desired but sub-millisecond timing is not necessary, a GEVI exhibiting fast activation but slightly slower inactivation could thus be useful.
Optical activity indicators are just one of many transformative optical methods developed over the past decade. These methods include optogenetic control of neuronal excitation and signalling 8, long-term fluorescent labelling of activated neurons 9 and post hoc imaging of optically cleared tissues 10. A powerful aspect of optical methods is that they can be easily combined, so that the same cells are studied by multiple approaches across different phases of an experiment.
Clinical applications of NIR fluorescence imaging
Clinical imaging of human patients is routinely used in hospitals to diagnose and monitor diseases, guide surgical interventions and evaluate treatment efficacy and prognosis 11. Most of the medical-imaging modalities used in clinical practice today fall under the category of tomographic imaging, which relies on deep-penetrating radiation (including both electromagnetic waves and moving subatomic particles) to probe both structural and functional information of the imaged subject. Reconstruction algorithms subsequently visually generate the spatial distribution of signal sources in the context of the anatomy of the human body in 3D. Major limitations of tomographic imaging includes:
- Adverse effects to hazardous ionizing radiation (CT, PET and SPECT)
- Limited spatial resolutions (MRI and PET)
- Poor temporal resolution (CT, MRI, PET and SPECT)
- Lack of both exogenous and endogenous probes for molecular or functional imaging
In contrast, in vivo fluorescence-imaging doesn’t have these limitations, instead it provides the benefits of real-time wide-field image acquisition and diffraction-limited, spatial resolution in living organisms through the interaction of non-hazardous optical radiation with the many available fluorescent labels (as both structural contrast agents and molecular or functional reporters).
Photoactivated drug delivery and bioimaging using NIR light
Currently, the light irradiations at UV (<400nm) and visible (400-700nm) wavelengths are the most commonly excitation sources for the photo-controlled therapy and imaging in vitro and in vivo. However, this short-wavelengths have limited tissue penetrating capability, high auto-fluorescence background, and potential phototoxicity, especially for the light exposure within UV region.
As shown in Figure 3 below, the major light absorbers in living systems including water, lipids, and some intrinsic proteins such as hemoglobin (Hb) and oxyhemoglobin (HbO2), usually demonstrate minimum light absorption in NIR range 12. The light excitation at NIR window may exhibit unique advantages toward deep tissue penetration, low fluorescence background, and limited photo damage, which could thus greatly benefit photoactivated delivery in living systems.
Figure 3: Extinction coefficient value of water, hemoglobin, and oxyhemoglobin. Light absorption of these major absorbers can be avoided by using short-wavelength light 12.
One commonly established strategy of using NIR light in biomedicine is to utilize photosensitive nanoplatforms, which can convert long-wavelength NIR light irradiation into UV or visible emissions 13 and thus spatiotemporally trigger the release of reagents such as ROS species 14 and cancer drugs 15 into living systems.
The demonstrated superior imaging quality (in deep tissues) of long-wavelength fluorescence in the NIR window bodes well for the future of in vivo NIR fluorescence imaging in both basic research and clinical translation. To expand the applications of NIR imaging from its current stage of preclinical anatomical and molecular imaging in experimental animal models, and to increase its clinical translatability and applicability, and for the understanding of complex systems and pathways in living organisms, this technology may evolve in these ways:
- On the basis of NIR-light-controlled physiological modulation, it is also reasonable to propose a non-invasive, fibre-optic-free optogenetic system for neural modulation by fusing NIR-II fluorescent proteins with certain membrane ion channels, where NIR-II light may be used to interrogate and manipulate brain activity at the single-neuron level in a completely non-invasive way 8.
- NIR-II photons may significantly facilitate 3D volumetric imaging of ex vivo tissue samples. Combined with tissue-clearing techniques 10, NIR fluorescence labelling and imaging could potentially afford volumetric imaging of functional connectivity of ex vivo animal tissues with diffraction-limited spatial resolution on the whole-organ or even whole-body level. The current pursuit in brain connectomics heavily relies on optical microscopy and electron microscopy (EM) of mechanically sectioned, thin tissue slices 16, which brings in additional difficulties in the coordinate registration of slices during 3D reconstruction. NIR fluorescence with deep-tissue penetration could provide whole-organ or whole-body imaging of intact biological samples without any slicing-induced artefact to 3D reconstruction.
- Finally, as with the development of any new biomedical imaging technique, in vivo NIR fluorescence imaging is likely to be fuelled by the advent of new, more advanced cameras with higher sensitivity and broader spectral ranges within or even beyond the current NIR window.
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