Near-infrared fluorescent protein and bioluminescence-based probes for high-resolution in vivo optical imaging

This review presents the recent progress on NIR fluorescent protein and bioluminescence-based probes with high-resolution in vivo imaging techniques.


Abstract
In the last few years, high-resolution near-infrared (NIR) optical imaging has become an indispensable modality for non-invasive visualization of deep tissues both in fundamental life science and preclinical research. This is due to the high tissue permeability, low absorption and low scattering of NIR light as well as the low autofluorescence in the NIR wavelength region (700-1400 nm) in living systems. Compared to magnetic resonance imaging (MRI), X-ray computer tomography (X-ray CT), and positron emission tomography (PET), NIR optical imaging has a high spatiotemporal resolution (~μm) enough to visualize cellular dynamics at the whole-body level. Additionally, NIR optical imaging do not require high-energy ionizing radiation such as X-ray that leads to serious radiation damages on living cells. Furthermore, NIR optical imaging can easily achieve molecular imaging with aid of NIR optical probes, which specifically bind to biomarkers expressing on cell surfaces. Thus, NIR optical imaging has great potential to use for non-invasive optical diagnostics of diseases in medical and clinical fields. For such NIR optical imaging, NIR fluorescent probes with high brightness and biocompatibility are crucial. Although a variety of NIR imaging probes based on nanoparticles such as quantum dots and dye-incorporated polymers have been developed, possible applications of these imaging probes to optical contrast agents are greatly limited due to their cytotoxicity. In contrast, fluorescent proteins and bioluminescence-based probes are highly biocompatible and practical to biomedical applications. During a last few years, a variety of

Introduction
NIR optical imaging of living system is widely used for tracking of live phenomena, detection of cancerous tumours, and secure handling of surgery. [1][2][3] Currently, NIR optical imaging has become as an indispensable modality for the non-invasive imaging of deep tissues in fundamental life science and preclinical research. Compared to other conventional imaging modalities such as MRI, X-ray CT, and PET, NIR optical imaging has a high spatiotemporal resolution (∼μm) enough to visualize molecular and cellular dynamics at the whole-body level. 4,5 Recent development in the NIR fluorescent proteins (FPs) and bioluminescence-based probes permits non-invasive molecular imaging in vivo, where the conventional imaging modalities such as MRI and X-ray CT are difficult to achieve high-resolution in-vivo molecular imaging.
To date, conventional FPs emitting at the visible region have been commonly used for imaging of live cells. 6 However, visible-emitting FPs are not suitable for tissue imaging at the whole-body level, 7,8 because of the strong tissue absorption and scattering of the visible lights (400-700 nm). In contrast, NIR lights in the wavelengths of 700-1400 nm, so-called " the first and second NIR window" (Fig. 1a) are preferable for deep-tissue imaging. 1,[9][10][11] This is due to the high permeability, low absorption and scattering of the NIR lights in living tissues in addition to the low tissue autofluorescence in the NIR wavelength region (Fig. 1b, 1c). [10][11][12][13][14][15] These superior properties of NIR lights lead to clearer deep-tissue imaging with high signalto-noise (S/N) ratios (Fig. 1d). 15 In the last few years, a variety of NIR FPs have been developed for non-invasive invivo imaging in animals. [16][17][18][19][20][21][22][23][24][25] NIR FPs have several advantages over chemically synthesized fluorescent dyes. NIR FPs can be genetically expressed in living system with cellular protein tags, which help tracking the activities of a gene of interest inside the living cells. So far, many effective NIR FPs have been developed from bacterial phytochromes, leading to various applications in NIR in-vivo imaging. These NIR FPs are categorized in three groups, namely, dimeric NIR FPs (iRFPs, IFPs), 7,17,18 monomeric NIR FPs (miRFPs, mIFP, miRFPnano and Wi-Phy), [19][20][21][22][23][24] and photo-switchable NIR FPs (PAiRFPs). 25 Most of the NIR FPs have high extinction coefficients. Therefore their relatively low quantum yields are compensated by their high extinction coefficients for live imaging applications. Among the available NIR-FPs, miRFP680 is the brightest FP. All NIR-FPs emit fluorescence by utilizing endogenous chromophores in eukaryotic and mammalian cells.
In addition to NIR FPs, bioluminescence-based probes can also be used for noninvasive in-vivo imaging in the NIR window. Unlike NIR FPs, bioluminescent proteins do not need external excitation, leading to high signal to background imaging because of almost no tissue autofluorescence. However, only a limited number of bioluminescent proteins are available for non-invasive in-vivo imaging in the NIR window. Most of bioluminescent protein-substrate systems emit luminescence at the visible region. 26 For example, firefly luciferase/D-luciferin and Renilla luciferase/coelenterazine (CTZ) systems emit bioluminescence around at 480 nm and 560 nm, respectively. 26 However, red-shifted bioluminescent protein-substrate pairs (luciferase /luciferin) have recently been developed for in-vivo imaging by modifications of the chemical structures of luciferins to emit at the longer wavelength region. [27][28][29][30][31][32] To date, a variety of luciferin analogues with extend -conjugation have been synthesized to achieve bioluminescent in-vivo imaging in living systems. [33][34][35] For instance, a red-shifted luciferin analogue (Akalumine) emits in the NIR region over 700 nm, which was used to single-cell bioluminescence imaging in freely moving animals. 34 The redshifted luciferin analogues were also used to NIR in-vivo imaging of cancer cells and cancer metastasis in living mice. 30,35 The use of bioluminescence resonance energy transfer (BRET) is an alternative method to achieve bioluminescence imaging beyond the visible region. 36,37 BRET consists of a luciferase/luciferin system as an energy donor and a NIR fluorophore as an energy acceptor.
Several types of NIR fluorophores such as organic dyes, NIR FPs, and quantum dots (QDs) have been used as energy acceptors in the BRET system. 38 Compared to organic dyes and nanoparticle-based fluorophores such as QDs, NIR FPs can be genetically encoded to cells and tissues in living systems. The genetically encoded NIR FPs are relatively easy to label specific biomolecules and organelles to track functional roles of those in living systems.
Owing to the development of NIR FPs and bioluminescence-based probes, a variety of of high-resolution in-vivo imaging techniques in the NIR region have been developed. These include wide field/confocal NIR fluorescence microscopy, super-resolution microscopy, and light sheet fluorescence microscopy. These imaging techniques have been applied to highresolution in-vivo imaging to visualize three-dimensional structure of molecular and cellular dynamics at sub-micrometer scale. In this review, we present the recent progress on NIR FPs and bioluminescence-based probes as well as high-resolution in-vivo imaging techniques.

NIR FP-based probes for in-vivo imaging
In the past two decades, genetically encoded FPs have been widely used for visualization of a variety of biological functions such as calcium ion fluctuation, protein-protein interaction, and cell-cycle study in living cells. 6 Recently, several types of NIR FPs have been developed for their applications to non-invasive in-vivo imaging. 16 Compared to visible light, NIR light (wavelength: 700-1400 nm) is relatively permeable in living tissues because of its low absorption and low scattering in the tissues. In addition, tissue autofluorescence in the NIR region is very week compared to that in the visible region (wavelength: 400-700 nm) (Fig. 1c).
By combining NIR FPs with advanced imaging techniques such as a two-photon microscopy, non-invasive deep-tissue imaging enables a high spatial resolution of ~ m at millimeter depths. NIR FPs for in-vivo imaging can be categorized in three groups, dimeric NIR proteins, monomeric NIR proteins, and photo-switchable NIR proteins (Table 1 and 2).

Dimeric NIR FPs
More than twenty NIR FPs are now available. These NIR FPs can be selected based on user's choice and the type of biological imaging applications. Typical NIR FPs are summarised in Table 1. Among them, iRFP720 is a most far-red emitting dimeric FP (34.6 kDa) which emits in the conventional NIR region (Ex max : 702 nm and Em max : 720 nm). 17 This is a dimeric NIR FP based on iRFP713 which is derived from RpBphP2 phytochrome photoreceptor of Rhodopseudomonas palustris 39 (Fig 2). iRFP720 requires the cofactor biliverdin for fluorescence emission. Because of its low acid sensitivity, it can also be utilized for specific applications where the cellular environment is adcidic. 17 iRFP720 works well with multicolor in-vivo imaging with other iRFPs variants fused with several cellular protein tags. 17 However, this protein has limited use for in-vivo tracking and protein dynamics study due to its dimeric nature. Another variant, iRFP702 is one among the highest quantum yield (QY= 8.2 %), 17,18 iRFP720 gives superior brightness in in-vivo imaging owing to the NIR-shifted emission, weak autofluorescence, low scattering and better tissue penetration of the NIR light. [40][41] iRFP720 can also be used as a BRET acceptor for BRET-based in-vivo deep-tissue imaging.
Verkhusha et al. engineered two novel chimeric probes based on RLuc8 luciferase fused iRFP670 and iRFP720 NIR FPs. 14 Due to intramolecular BRET between RLuc8 and iRFP, the chimeric probes show NIR bioluminescence with maxima at 670 nm and 720 nm. These BRET probes enable combined multi-color bioluminescence imaging of the iRFPs, leading to BRETbased highly sensitive NIR detection of cancer cells as well as continuous spatiotemporal analysis of metastatic cells in living mice. IFP1.4 and IFP2.0 are other dimeric NIR FPs with an emission maximum of 708 nm and 711 nm, respectively. IRF1.4 is a 34.8 kDa protein derived from a chromophore-binding domain (DrCBD) of Deinococcus radiodurans. 8 However, exogenous supply of biliverdin cofactor is required to achieve fluorescence imaging of these NIR-FPs in mammalian cells.
IFP2.0, a brighter version of IFP1.4 is developed after insertion of 11 mutations in cDNA of IFP1.4. 18 In some cases, dimeric NIR FPs are preferred over monomer NIR FPs due to the superior brightness of dimeric NIR FPs. They work well with many but not all cellular tag as fusion partner. However, they have limited use in live-cell dynamics and tracking applications due to their dimeric nature.

Monomeric NIR FPs
The development of monomeric NIR FPs makes in-vivo live cell imaging and dynamics study more easily with lower laser power. 1,10,12 Monomeric NIR FP variants have nearly similar photophysical properties to dimeric NIR FPs with slightly compromised brightness (Table 1).
However, they are preferred over dimer NIR FPs for live-cell study and animal imaging with other cellular fusion protein tags because of their monomeric nature. Also, because of their monomeric nature, the localization efficiency of these NIR FPs are better than that of dimer NIR FPs.
The first set of bright monomeric family of NIR FPs was engineered from bacterial phytochromes are mIFP 22 and miRFPs (miRFP670, miRFP670-2, emiRFP670, miRFP680, miRFP682, miRFP703, emiRFP703, miRFP709, miRFP713 and miRFP720) 19.21 (Fig. 3). They work as a good fusion partner with several cellular protein tags like collagen, H2B, vimentin, tubulin, actin and in free form in a living system without showing any aggregation. Molecular weight of mIFPs and miRFPs is 35.1 kDa and 34.6 kDa, respectively. Among them, miRFP680 has the highest QY and superior molecular brightness, this protein expressed very well with many cellular tag-protein and miRFP720. The other bright and photostable NIR-FPs such as, emiRFP670 and emiRFP703 with enhanced photophysical characteristic have been successfully utilized for single and multiplex non-invasive whole body imaging of mice. 42 However, miRFP713 and miRFP720 are more preferable for deep-tissue in-vivo imaging because of their NIR-shifted emission maximum (Em max : 713 and Em max : 720, respectively).
These proteins can be used for dual-colour multiplex imaging with other spectrally far NIR-FP bright variants of miRFPs like miRFP 680, miRFP670 and emiRFP670 for dual-colour in-vivo imaging.
Among the monomeric NIR FPs, miRFP670nano is the another latest and smallest NIR FP (17kDa ) . This protein has an emission maximum at 670nm upon excitation at 645nm excitation with a 95,000 extinction coefficient. 19 The monomeric protein, miRFP670nano has nearly half of the molecular weight of miRFP670, which makes it better a fusion partner candidate with other cellular proteins. In mammalian cells, miRFP670nano exhibits 2.8-and 1.3-fold higher photostability than miRFP670 and miRFP703. It shows precise expression with several fusion tag cellular proteins and behaves as a perfect fluorescence resonance energy transfer (FRET) donor with miRFP720 for in-vitro and in-vivo imaging. 19 NIR FPs also prove to be useful in super-resolution imaging as they generate low autofluorescence signals in living cells. Structured illumination microscopy with miRFP703 and other variants provides superior spatial resolution compared to wide-field fluorescence imaging for tubulin, H2B and various other cellular tags. 9 Enhanced brightness and photostability of the newly developed emiRFP670 and emiRFP703 worked perfectly well in single and dual-colour STED imaging with 11-36 fold decrease STED illumination dose in mammalian cells compare to their previously existing monomer NIR-FPs. 42

Photo-switchable NIR FPs
Photo-switchable FPs allow illuminating only the area of interest, which drastically improves spatial and temporal resolution and image quality. PAiRFP1 and PAiRFP2 are the only photoswitchable NIR FPs emitting above 700 nm ( Table 2). 25 They are capable of holding multiple dark-bright cycles upon excitation with NIR light before bleaching. 25 The emission peak of PAiRFPs is almost 50 nm red-shifted relative to that of the conventional red-shifted photoswitchable GFP-like protein, PSmOrange, leading to use PAiRFPs for dual-colour imaging.
PAiRFPs are developed from bacterial phytochrome photoreceptor (BphP) known as AtBphP2, 43,44 which utilizes heme-derived biliverdin (ubiquitous in mammalian tissues) as the chromophore. 19 Initially, weakly fluorescent PAiRFPs undergo photoconversion into a highly NIR FPs with two-photon excitation have proven to improve better signal-to-noise contrast and tissue scattering for in-vivo and deep-tissue non-invasive imaging. Several NIR-FPs have been applied to two-photon imaging of primary neuronal cell culture, mouse brain, and brain slice of mouse and monkey. 46-50 Verkhusa group conducted two colour imaging of EGFP and iRFP680 by a low-dose 880 nm two-photon excitation (6.5 mW). 46 They achieved sub-cellular resolution in-vivo imaging of neuron with penetration depth as deep as 285 m from the brain surface. NIR-FP based sensors have been recently developed for high-resolution in-vivo neuroimaging using two-photon excitation in various animal models.
Betzig group achieved wavefront sensing and visualization of mouse cortex up to 700 m depth using two-photon excitation with iRFP713 NIR-FP. 47 Other groups developed NIR-FP based voltage and calcium sensors using mIFP and miRFPs to monitor brain and neuronal activity in mouse, zebrafish, C. elegans and Xaenopus laevis. 48-50 Two-photon neuronal calcium imaging and sensing have been reported by utilizing NIR genetically encoded calcium ion indicator (GECI) namely, NIR-GECO1 and NIR-GECO2 based on mIFP NIR-FPs. 48,49 Campbell group reported multiplex calcium imaging by utilizing 1250 nm two-photon excitation of NIR-GECO1 together with other previously developed calcium sensor such as GCaMP6f and RCaMP1.07 in live brain slice of mouse. 49 Same group reported other improved GECI variants such as NIR-GECO2 and NIR-GECO2G based on miRFP703. 50 Bright NIR-FPs (e.g. miRFP680, emiRFP670 and miRFP720) will be good candidates for the deepertissue calcium and voltage sensors with two-photon excitation technique.
The major advantage of NIR-FPs over chemical dyes is genetically encodability with fusion tag protein of interest. This helps allowing live in-vivo imaging of whole animal. Several such research has been done in the past to develop transgenic animals such as mouse, rat and fruit fly, expressing NIR-FPs. Miwa group developed first iRFP based transgenic animal where he showed iRFP expression in almost every organs such as liver, kidney, lungs, heart, brain, spleen, pancreas, bone, testis, thymus and adipose tissues. 51 In two separate studies, miRFP713 was used to develop transgenic cre-dependent inducible mouse model. In the first study, tumor development and in-vivo recombinase activity were explored. 52 They investigated live tumor progression, metastasis events and recombination event upon condition activation of creinducible system expressing miRFP713. In another study, miRFP713 based mouse transgenic model was used to image neuronal network in-vivo without the supply of exogeneous chromophore biliverdin. 53 A transgenic mouse model expressing Ucp1-iRFP720 was developed by Hisatake groupto study the role of uncoupling protein 1 (UCP1) activity in thermogenesis control in non-invasive in-vivo system. 54 Uncoupling protein1 (UCP1) is a mitochondrial expressing protein to help uncouple electron transport chain from ATP synthesis to produce heat. The mouse model develop in this study is useful for the in-vivo study of pathways, where adipocyte induction and non-shivering thermogenesis involves. 54 Gu et. al. developed a two cell homologues recombination (2C-HR)-CRISPR) method utilizing histone2B-miRFP703 NIR-FP to demonstrate the CRISPR-cas9 gene editing method for the generation of transgenic mouse line and achieved three color imaging with mCherry, Halo-tag and miRFP703. 55 McDole et. al. achieved the fine detail of developing heart of mouse embryo with light-sheet microscopy by utilizing the histone2B-miRFP703 NIR-FP expressing transgenic mouse. 56 They utilized a transgenic mouse embryo expressing histone2B-miRFP703 reported transgenic embryo, where they were able to achieve cellular level resolution up to 600 m depth covering entire linear heart tube, head-fold and foregut pocket region. 56 These studies have demonstrated the efficient use of NIR-FPs for non-invasive deep-tissue imaging and successful expression in transgenic animal system.

Red-shifted bioluminescent protein-substrate pair
Despite the high sensitivity of bioluminescence imaging, its potential use in bioimaging is mostly restricted to the visible region less than 600 nm. Visible-emitting bioluminescence is strongly absorbed and scattered in biological samples, leading to the attenuation of the bioluminescence signals. To overcome this drawback, red-shifted bioluminescent protein-  A recently developed luciferin analogue, AkaLumine can emit in the NIR region over 700 nm (Fig. 4). 29,32 AkaLumine is a D-luciferin analogue with an extended -conjugation system. This red-shifted luciferin analogue has shown the capability of its NIR bioluminescence for highly sensitive deep-tissue imaging in mice. 30

BRET system
The use of BRET is an alternative method to achieve bioluminescence imaging at the longer wavelengths. BRET consists of a luciferase-luciferin system as a resonance energy donor and a fluorescent acceptor as a resonance energy acceptor. BRET is a special case of resonance energy transfer, where the donor is a luciferase/luciferin system. As BRET acceptors, fluorescent materials such as FPs, 14 where r is the distance between the donor and acceptor and R o is the Förester distance at which the BRET efficiency is 50%. The Förester distance is given by the following equation: 116 where  2 is an orientation parameter depending on the relative orientation of the donor and acceptor dipoles, Q D is the quantum yield of the donor, n is the refractive index of the medium, and J() is the spectral overlap integral between the normalized donor emission and the acceptor excitation spectra. To achieve effective BRET in the combination of the donor (luciferase/luciferin system) and the fluorescent acceptor, two molecules must be close to the distance within 10 nm. In addition, the spectrum of the donor emission should be overlapped with the excitation spectrum of the acceptor.

BRET probes using FPs
Although there are many types of visible-emitting FPs, the number of far-red and NIR FPs as BRET acceptors is very limited for bio-imaging. To date, several groups have applied redshifted FPs as BRET acceptors for BRET-based imaging in vitro and in vivo. 14

BRET probes using NIR organic dyes
The use of conjugates between luciferase and NIR fluorescent dyes is a simple and convenient method to construct a BRET system for in-vivo imaging. To date, several types of far-red and

BRET probes using NIR QDs
Compared to NIR FPs and organic dyes, NIR emitting QDs have broad absorption spectra ranging from the visible to NIR regions, which results in the large spectral overlap between the luminescence of luciferase substrates and the absorption of the QDs. Thus, the effective BRET In most of the BRET-based QD probes, luciferase was directly conjugated to the surface of QDs using chemically coupling methods. Since the chemical coupling often results in the decrease in the enzymatic activities of luciferases, mild conjugation methods using the tag technology such as Halo-tag, 94 GST-tag, 106 and His-tag 108 are desirable. Recently, Jin et al.
reported a facile method for preparing of RLuc-EGFP fused protein conjugated NIR-QDs for dual-color molecular imaging of cancer cells (Fig. 5d-g). 115 The major advantage in the use of QDs as BRET acceptors is the bright emission from QDs because of the high extinction coefficients and large stokes shifts. Although most QDs contain heavy metals with cytotoxicity, the improvement of biocompatibility for the QDs should be necessary to develop the biological applications of QD-based BRET probes for NIR in-vivo imaging. In the in-vivo NIR imaging system, imaging microscopes are also important as well as optical NIR probes to achieve high-resolution imaging. Although many types of in vivo imaging instruments are commercially available, most of the NIR imaging instruments are the type of macro fluorescence imaging system, where the spatial resolution is not enough to visualize molecular and cellular dynamics in living tissues. In addition, the excitation and emission wavelengths for the NIR imaging are mostly restricted to less than 900 nm (the first NIR window). In this section, we summarize in-vivo NIR imaging systems consisting of wide field, confocal and super-resolution microscopes for the high-resolution visualization of living tissues at the cellular level.

Wide field epi-NIR fluorescence microscopy
In most of the commercially available in-vivo imaging systems, the NIR wavelength region of 700 to 900 nm (first NIR optical window wavelength) has been employed. This is because the conventional NIR photo-detectors (silicon CCD camera) are sensitive in the first NIR region, and many kinds of NIR emitting probes (e. g. Indocyanine green, Cy 7, and CdSeTe QDs) are easily available. Although NIR fluorescence imaging in the first NIR optical window is useful for the non-invasive visualization of organs and tissues, its spatial resolution is not enough to observe cellular dynamics. Recently, NIR optical imaging in the second NIR window (1000-1400nm) as well as in the first NIR window (700-900 nm) has attracted much attentions to achieve better spatiotemporal-resolution imaging for deep tissue in small animals. 11,119 Dai et al. have reported a high-resolution epi-microscopic system for NIR imaging of brain imaging cerebral vessels (Fig. 6a). [120][121][122][123][124][125] High-magnification intravital imaging of cerebral vessels was carried out in an epifluorescence mode with an 808-nm diode laser (RMPC lasers, 160 mW) as an excitation source and two objective lenses (4× and 10×) (Fig. 6b). 120 The mouse with scalp hair removed was intravenously injected with a solution of single walled carbon nanotubes (SWNTs) and placed in a home-made stereotactic platform fixed on a motorized 3D translational stage that allowed for the digital position adjustment and readout of the mouse relative to the objective. The emitted fluorescence was filtered through a 1000 nm long-pass filter, a 1300 nm long-pass filter and a 1400 nm short-pass filter to ensure only photons in the 1300-1400 nm.
Jin et al. have also reported a wide field epi-NIR fluorescence imaging system which can be used in both in the first and second NIR window (Fig. 6c). 15,126,127 In this imaging system, excitation lasers for 645, 785, and 978 nm excitation, and emission filters of 1100 ± 25 nm, 1300 ± 25 nm, and 1500 ± 25 nm are equipped to the imaging system. A Si EM-CCD camera was employed for VIS and NIR fluorescence imaging in the first NIR window and an InGaAs CMOS camera for NIR fluorescence imaging in the second NIR window. By using the epi-NIR fluorescence microscope, non-invasive imaging of cerebral blood vasculatures in a nude mouse was easily achieved (Fig. 6d).

Confocal NIR fluorescence microscopy
In order to achieve three-dimensional NIR fluorescence imaging with a high-spatial resolution, one-photon confocal imaging in the second NIR window has been recently reported by Dai group (Fig. 7). 128 the second NIR window. 130 They developed SDCLM for a high-speed optical sectioning of biological tissues in the second NIR window. The SDCLM achieved a lateral resolution of 0.5 ± 0.1 m and an axial resolution of 0.6 ± 0.1 m, showing a ~17% and ~45% enhancement in lateral and axial resolution, respectively.

Photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)
Super-resolution localization methods are based on an indirect approach to achieve diffraction unlimited resolution in light microscopy using high-precision localization of a fluorophore.
Under the optimum concentration of emitted fluorophores, the localized positions of emitting fluorophores can be created from the thousands of images frames to achieve super-resolution imaging. Blinking behavior of both NIR and visible fluorophores is utilized to achieve resolution below diffraction limit of light in PALM and STORM imaging. In these techniques, single molecules localization is determined by photon detection using the poisson process, thus the most crucial factor during imaging is the signal-to-noise ratio. 131,133 Single molecule detection is achieved by utilizing intrinsic blinking or photo-switching of fluorophores.
The controlled photoblinking of a fluorophore is produced with a specific wavelength of light either by switching them on-off or bleaching a subset of fluorophores so that only a few labeled fluorophores emit in each frame. [131][132][133][134][135][136][137][138][139] This method is efficient in achieving lateral resolution up to 10-20 nm, much superior to the diffraction limit in other optical microscopies.
Several visible and NIR photo-switchable fluorophores are in practice to attain live-cell nanoscopy and in-vivo super-resolution imaging, those can be repeatedly or irreversibly switchable between dark and bright states. [133][134][135][136] However, organic dyes labeled with a suitable tag could also be used for various applications in living system. [137][138][139] In the past, several approaches using localization precision-based imaging techniques developed for live imaging like PALM, PALM with independent running acquisition, STORM and direct STORM, etc. [135][136][137][138][139] In the latest development, combining these microscopy techniques with light-sheet microscopy drastically improved resolution for thick sample and small animal in-vivo imaging.
The below equation explains the localization precision efficiency determination as described earlier: [131][132][133] (3) ( where 's' represent standard deviation of the PSF, 'a' is the pixel size in the image, 'N m ' is the total photons emitted from molecule 'm', and 'b m ' is background photons from molecule 'm'.

Stimulated emission depletion (STED) and reversible saturable optical fluorescence transition (RESOLFT) microscopy
Patterned illumination based super-resolution microscopies like STED [140][141][142] and RESOLFT 143-147 uses another donut shape depletion beam to constrain a point-spread function. This second donut shape beam is based on simple but powerful concept to directly erase peripheral fluorescence. 140 Diffraction unlimited spot achieved in STED and RESOLFT by increasing the power of the donut beam. A modified Abbe's equation describes this sub-diffraction resolution as: The concept of RESOLFT emerged from STED after replacing conventional fluorophores with photo-switchable fluorophores. This improvement drastically reduces the excitation and STED beam several folds to achieve long-time super-resolution imaging of live system. RESOLFT is extended advancement of STED microscopy using a special fluorophore, where on-off state controlled by two different wavelengths to achieve super-resolution up to 10-20 nm. [143][144][145][146][147] RESOLFT has the unique advantage of reducing laser power several folds (<10 -6 times) in live-cell imaging. 143,146,147 Recently, NIR FPs such as emiRFP670 and emiRFP703 achieved 50-70 nm spatial resolution in STED imaging of various cellular structures in live-cell imaging (Fig. 8). 42 Development of bright photo-switchable NIR FPs will provide opportunity to users to reduce laser power to extend imaging time and resolution.

Structured illumination microscopy (SIM) and saturated structured illumination microscopy (SSIM) and
SIM and SSIM are based on the nonlinearity principle. In SSIM imaging, the sample is illuminated with high frequency sinusoidal striped light. [148][149][150][151][152] The requirement of high laser intensity to achieve nonlinearity in SSIM is the limiting factor to achieve high-resolution in three dimension. [148][149][150][151][152] SSIM together with photo-switchable fluorophores emitting in visible and near-infrared region succeed to achieve nonlinearity at very low laser power, because the power of light needed for photoconversion in on-off state could be reduced several folds compared to conventional fluorophores used in SSIM. [150][151][152] Live imaging of dendrites and dendritic spine in zebra-fish brain and mice has been achieved by SIM using green FP (GFP). 152 Combination of NIR photo-switchable FPs with SSIM will further improves the resolution of such imaging condition as proven earlier. In another study, high resolution of the neuronal structure of mouse brain was achieved using patterned illumination-based microscopy in two-photon excitation. 153 This could further be improved by replacing fluorescein with NIR probes. However, the only limitation now is the The resolution of BBPIM could be further improved using photo-controllable or photoswitchable fluorophores in both visible and NIR range. BBPIM combined with a structured illumination approach proved to achieve excellent resolution (both x-y and x-z direction) with reduced phototoxicity. This new nanoscopy approach is known as lattice light sheet nanoscopy region. Thus, the use of NIR-emitting optical probes is crucial to achieve high-resolution invivo imaging. The combination of NIR-emitting optical probes with high-resolution imaging systems enables the non-invasive visualization of living tissues at the cellular resolution level.
Among the NIR FPs designed from the bacterial phytochrome photoreceptor, miRFP680 is the brightest NIR-FP in mammalian cells. 42 The brightness of miRFP613 and miRFP720 are approximately ~30% lower that miRFP680, however their ~33 nm and ~40 nm red-shifted emission spectra would be advantageous for in-vivo imaging and multiplex imaging with other spectrally distinct NIR-FPs. Another recently developed NIR-FP, miRFP670nano has nearly similar brightness to miRFP720 in mammalian cells but its monomeric nature and smallest size is the advantage to use as a fusion tag for live in-vivo imaging. 23 Considering their monomeric nature and brightness, these NIR-FP are suitable for high-resolution in-vivo imaging.
These proteins can also be used for dual-color and FRET imaging in deep-tissues. Photoswitchable NIR FPs, PAmIRFP1 and PAmiRFP2 are unique FPs, and their emission can be controlled by switching on/off with light in a localized region, which is useful for superresolution imaging. The use of photo-switchable FPs for in-vivo imaging avoids the excitation of out-of-focus area, leading to the significant improvement of signal-to-noise ratios by a factor of several folds. After the expansion of NIR photo-switchable FPs, these could also be used with other switchable NIR proteins to develop FRET pairs to improve signal-to noise ratios for the accuracy of NIR based FRET imaging.
Red-shifted bioluminescent protein-substrate pairs as well as NIR FPs can also be used for high-resolution in-vivo imaging. The luciferin analogues modified with a longer conjugation such as AkaLumine enables high-resolution deep-tissue imaging at the cellular level. The use of BRET is an alternative method to achieve in-vivo imaging in the NIR region.
BRET-based molecular imaging has a higher detection sensitivity compared to fluorescencebased molecular imaging due to its low background signals. However, NIR bioluminescence