Quantitative assessment of near-infrared fluorescent proteins

doi:10.21203/rs.3.rs-797586/v1

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Quantitative assessment of near-infrared fluorescent proteins

https://doi.org/10.21203/rs.3.rs-797586/v1

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published 04 Sep, 2023

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Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins, which are rapidly becoming popular probes for a variety of imaging applications. To assist end-users with a selection of the right near-infrared fluorescent protein for a given application, we will conduct a quantitative assessment of intracellular brightness, photostability, and oligomeric state of 19 near-infrared fluorescent proteins in cultured mammalian cells. The top-performing proteins will be further validated for in vivo imaging of neurons in C. elegans, zebrafish, and mice. We will also assess the applicability of the selected NIR FPs for expansion microscopy and two-photon imaging.

Structural Biology

Nuclear Medicine & Medical Imaging

fluorescent proteins

NIR FP

in vivo imaging

Fluorescent proteins (FPs) quickly became ubiquitous tools for optical imaging since the cloning of the first green FP from jellyfish Aequorea victoria in 1992¹. Expanding spectral diversity of FPs into near-infrared (NIR) range (~650-900 nm) of the electromagnetic spectrum enabled new imaging capabilities ranging from multiplexed super-resolution imaging of live cells to whole-body in vivo imaging². Red shifted fluorescence spectrum of NIR FPs allows for efficient excitation with standard red lasers (630-640 nm), widely used light sources in microscopy, unlocking standard Cy5 filter set for FPs, which previously were used only for red-shifted dye imaging^3–6. Correspondingly, NIR FPs can be readily imaged with other conventional FPs such as GFPs and RFPs enabling multiplexed structural⁶ and functional imaging^7–9. Further development of monomeric NIR FPs enabled their application as fluorescent tags for subcellular structures in cultured cells under conventional^6,10 and super-resolution microscopy^11–13. In addition, NIR FPs are particularly beneficial for in vivo imaging of model organisms due to reduced autofluorescence, low light scattering, and minimal absorbance of tissue at longer wavelengths^14,15. Unique photophysical properties of NIR FPs make them compatible with multiphoton microscopy^16,17 and photoacoustic imaging thus further increasing the utility of these probes for in vivo imaging of mammals. However, the majority of currently available NIR FPs are derived from bacteriophytochromes, which utilize a linear tetrapyrrole biliverdin IXα (BV) as a chromophore¹⁴. This feature makes the fluorescence of bacteriophytochrome photoreceptor (BphP)-based NIR FPs dependent on the heme metabolism in host cells^6,8,18. As a result, the performance of BphP-based NIR FPs in cultured cells and in vivo preparations cannot be predicted based on the in vitro characteristics measured on proteins purified from bacteria, which is the most common way to select FPs for an imaging experiment^6,13. Furthermore, intracellular brightness of BphP-based NIR FPs in one cell type is not necessarily retained in another cell type⁶. Altogether, this creates a need to test multiple NIR FPs in a particular preparation before selecting the one suitable for the desired application. To assist end-users with selecting the right NIR FPs, we will perform a careful side-by-side quantitative assessment of the most popular NIR FPs in HEK cells and primary neurons, characterizing their intracellular brightness, photostability, and oligomeric state. The top-performing NIR FPs will be expressed in vivo in C. elegans, zebrafish, and mice for a side-by-side comparison of intracellular brightness and photostability. In addition, the proteins exhibiting the highest performance will be further tested for multicolor imaging and validated for expansion microscopy (by quantifying fluorescence retention in cultured cells) and two-photon imaging (by measuring action-cross section spectra).

To perform the quantitative assessment in mammalian cells we selected 19 NIR FPs listed in Table 1. All selected proteins will be characterized in HEK cells and primary cultured mouse neurons for intracellular brightness, intracellular photostability, and in HeLa cells for assessment of oligomeric state. The selected NIR FPs span more than 70 nm of the visible spectrum and thus they can be subdivided into two spectral groups matching the Cy5 or Cy5.5 filter set (see Methods section for the exact specification of the Cy5 and Cy5.5 filters). In this study, we also distinguish Cy5-LP (with long pass emission filter that allows collecting as much NIR fluorescence as possible) and Cy5-BP (with a narrow bandpass filter to improve spectral separation with Cy5.5 filter). We will use all three filter sets for characterizing NIR FPs in cultured cells. The performance of NIR FPs will be quantified as a product of intracellular brightness and photostability for each tested channel. The NIR FPs exhibiting the highest performance in cultured cells will be further tested in vivo in model organisms including C. elegans, zebrafish, and mice. Below we list all proposed experiments, provide pilot data, and describe detailed protocols, which will be used in the course of this study.

Table 1. List of the NIR FPs selected for quantitative assessment in cultured cells.

Protein	Ex (nm)	Em (nm)	EC	QY	Molecular Brightness	Ref
mCardinal	604	659	87,000	0.19	16.53	⁴
E2-Crimson	611	646	126,000	0.23	28.98	¹⁹
smURFP	642	670	180,000	0.18	32.4	²⁰
BDFP1.6	642	666	100,000	0.197	19.7	²¹
emiRFP670	642	670	87,400	0.14	12.24	¹⁷
miRFP670-2	643	670	103,000	0.136	14.01	¹⁷
iRFP670	643	670	114,000	0.11	12.54	⁵
miRFP680	661	680	94,000	0.145	13.63	¹⁷
iRFP682	663	682	90,000	0.11	9.9	⁵
emiRFP703	674	703	90,900	0.086	7.82	¹⁷
BDFP1.8	678	702	77,027	0.061	4.7	²²
miRFP2	676	706	55,600	0.043	2.39	⁶
mRhubard713	690	713	113,457	0.0763	8.66	²³
miRFP713	690	713	99,000	0.07	6.93	¹⁷
iRFP713	690	713	105,000	0.06	6.3	²⁴
SNIFP	697	720	149,200	0.022	3.28	¹¹
mRhubarb719	700	719	83,769	0.068	5.7	²³
mRhubarb720	701	720	94,941	0.0646	6.13	²³
miRFP720	702	720	98,000	0.061	5.98	⁹

Proposed experiments

1. Cell culture characterization

1.1. Intracellular brightness will be measured in three NIR channels, namely Cy5-BP, Cy5-LP, and Cy5.5 (see Methods section for exact description) under wide-field excitation. Quantification of intracellular brightness of the selected NIR FPs (see Table 1 for the complete list) will be performed with co-expression of EGFP via P2A self-cleavage peptide to normalize their expression levels. Since we also observe situations when a NIR FP can have a high NIR-to-green fluorescence ratio but at the same time low NIR fluorescence intensity (due to the low expression level) we will also report raw fluorescence intensities for each channel. The following characterization experiments will be further performed:

1.1.1. Brightness of fluorescence in the cytoplasm of live HEK cells as well as live primary mouse neurons with and without administration of exogenous BV.

1.1.2. Brightness of fluorescence in the cytoplasm of PFA fixed HEK cells without administration of exogenous BV.

1.2. Intracellular photostability under Cy5 and Cy5.5 excitation light will be evaluated:

1.2.1. In the cytoplasm of live HEK cells and live primary mouse neurons without administration of exogenous BV.

1.2.2. In the cytoplasm of PFA fixed HEK cells without administration of exogenous BV.

1.3. Establishing oligomeric state using OSER assay in HeLa cells.

1.4. Tubulin, keratin, actin, or H2B fusions imaging in HeLa cells (only for the top 5 most monomeric proteins in each channel based on OSER assay).

1.5. Multicolor imaging in HeLa cells using a pair of best spectrally resolvable NIR FPs using tubulin or keratin and H2B fusions.

1.6. Fluorescence retention in standard ExM protocol (only for top 5 proteins in each channel showing highest fluorescence retention upon 4% PFA fixation).

2. In vitro characterization (only for 5 best performing proteins in each channel).

The following parameters will be measured:

2.1. Absorbance spectrum (in the range of 250-800nm).

2.2. Fluorescence spectrum (in the range of 250-900nm).

2.3. Quantum yield and extinction coefficient.

2.4. pKa (in the range of pH=2.0-11.0).

2.5. Absolute two-photon cross-section spectrum (in the range of 680-1300nm).

2.6. Fluorescence lifetime.

3. In vivo characterization (only for 3-5 best performing proteins in each channel).

3.1. Characterization in neurons in mice via intracranial AAV injection.

3.1.1. Intracellular brightness in acute brain slices under wide-field illumination in Cy5 and Cy5.5 channels (imaging with GFP as a reference).

3.1.2. Intracellular photostability in acute brain slice under wide-field illumination in Cy5 and Cy5.5 channels.

3.1.3. Intracellular brightness in fixed mouse brain under wide-field illumination in Cy5 and Cy5.5 channels (imaging with GFP as a reference).

3.1.4. Intracellular photostability in fixed mouse brain under wide-field illumination in Cy5 and Cy5.5 channels.

3.2. Characterization in liver in mice via tail vein AAV injection.

3.2.1. Whole organ florescence brightness of liver under wide-field illumination in Cy5 and Cy5.5 channels (imaging with GFP as a reference).

3.3. Characterization in neurons in zebrafish larvae.

3.3.1. Intracellular brightness in live zebrafish larvae under 1P with and without HO1 co-expression (imaging with GFP as a reference).

3.3.2. Intracellular photostability in live zebrafish larvae under 1P with and without HO1 co-expression (imaging with GFP as a reference).

3.3.3. Multicolor imaging using one or two best performing NIR FPs.

3.4. Characterization in neurons in C.elegans.

3.4.1. Intracellular brightness in live C. elegans in Cy5 and Cy5.5 channels with HO1 co-expression (imaging with GFP as a reference).

3.4.2. Intracellular photostability in live C. elegans under Cy5 and Cy5.5 illuminations with HO1 co-expression.

Pilot data

In order to verify that all proposed experiments are feasible, we have acquired preliminary data for both cell culture experiments and in vivo experiments. In addition, we published results for some NIR FPs, which were acquired according to the protocols proposed in the present manuscript. For example, cell culture characterization of several NIR FPs included in Table 1 was reported in a recently posted preprint describing the development of the enhanced NIR FPs using directed molecular evolution in mammalian cells⁶. In this preprint, we also demonstrated the ability to perform dual-color imaging with blue-shifted and red-shifted NIR FPs using commercially available lasers installed on the standard wide-field microscope. In past, we reported two-photon action cross-section spectra for multiple NIR FPs^6,25,26. Characterization of NIR FPs in vivo in mice and in C. elegans is also well established in our lab. Our recent preprint posted on BioRxiv reports the characterization of NIR FPs in mice via IUE and in transgenic C. elegans⁶. Zebrafish experiments will be performed by the group of Kazuhiko Namikawa and Reinhard Köster, who have all established infrastructure for making transgenic zebrafish and subsequent in vivo imaging. They co-authored the preprint reporting expression and imaging of NIR FPs in zebrafish larvae⁶. Below we present additional data obtained in the course of this project, which is not published.

OSER assay is a well-established method for quantification of the oligomeric state of GFP-like FPs in mammalian cells²⁷, however, it is not yet validated for BphP-based NIR FPs. Since BphP-based NIR FPs have different dimer unit arrangements than GFP-like FPs, we decided to carry out preliminary experiments for NIR FPs. We selected three BphP-based NIR FPs, which were previously characterized by size exclusion chromatography (SEC) to be either obligated dimer (iRFP713)¹⁰ or monomers (miRFP2 and miRFP720)⁶. The analysis of expression patterns of CytERM fusions in HeLa cells performed according to the previously described protocol revealed that percentage of cells scored without visible OSER whorls was 30% for iRFP713, 70% for miRFP2, and 75% for miRFP720 (Figure 1). These results are in agreement with SEC data thus indicating that OSER assay is suitable for BphP-based NIR FPs.

For assessment of NIR FPs in zebrafish larval neurons, we propose to co-express a NIR FP and mClover3 (for expression level normalization) together with HO1 (for boosting fluorescence intensity of BphP-based NIR FPs) from single plasmid using bidirectional promoter (Figure 2a) allowing for pan-neuronal multicistronic gene expression. To validate the expression vector, we have cloned several plasmids encoding selected NIR FPs listed in Table 1 and expressed them in neurons of zebrafish larvae. Assessment of the obtained larvae demonstrated sufficient level of expression of each transgene, which is comparable to the standard expression system, thus indicating that the constructed expression vector is feasible for the proposed study.

List of confirmed consortium members:

Hanbin Zhang, Stavrini Papadaki – cell culture characterization.
Hanbin Zhang, Stavrini Papadaki – in vitro characterization
Hanbin Zhang, Xiaoting Sun – characterization in mice.
Mikhail Drobizhev – two-photon spectra.
Luxia Yao, Stavrini Papadaki, Lianfeng Wu – C. elegans experiments.
Kazuhiko Namikawa, Xinyue Wang, Reinhard W. Köster – zebrafish experiments.
Kiryl Piatkevich – oversees all aspects of the project analyzes the data, and writes the manuscript.

Characterization in cultured mammalian cells. All procedures involving mice at Westlake University will be conducted in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Westlake University Committee on Animal Care. The mammalian codon-optimized genes of NIR FPs listed in Table 1 were cloned into pAAV-CAG-P2A-EGFP vector to enable expression-level independent quantification of intracellular brightness in mammalian cells. The intracellular brightness with and without exogenous BV and photostability will be quantified in HEK cells and in primary hippocampal mouse neurons using a wide-field fluorescence microscope. In addition, fluorescence retention and photostability will be assessed upon chemical fixation with 4% PFA in HEK cells.

To quantify intracellular brightness in live HEK cells, HEK cells will be transfected using standard methods and imaged using fluorescence wide-field microscope 36-48 h post transfection. First, transfected cells will be imaged without exogenous BV, then 25 µM of BV will be administrated to cell medium for 3 h at 37^oC in cell incubator. Before imaging, cells will be washed once with PBS to remove excess of BV and the same FOVs will be imaged again immediately in fresh culture media. Cells will be imaged in green, Cy5-BP (excitation: 635/22nm from SpectraIII LumenCor; emission 679/41 nm), Cy5-LP (excitation: 635/22nm from SpectraIII LumenCor; emission 664LP), and Cy5.5 (excitation: 680/13nm from 680nm CNI laser; emission 710LP) channels. To obtain statistically significant datasets, we will perform 3 independent transfections and analyze at least 100 individual cells for each transfection (at least 300 cells for analysis in total per construct). The mean fluorescence intensity in green, Cy5-BP, Cy5-LP, and Cy5.5 channels for single cells will be extracted and Cy5-BP-to-green, Cy5-LP-to-green, and Cy5.5-to-green fluorescence ratio will be calculated for each construct and used for further comparison of intracellular brightness under corresponding imaging conditions. The data will be excluded from the analysis if cells die after transfection or cell culture is contaminated with bacterial or yeast infection.

To quantify photostability in live HEK cells, HEK cells will be transfected using standard methods and imaged using fluorescence wide-field microscope 36-48 h post-transfection without administration of exogenous BV. To obtain statistically significant datasets, we will photobleach at least 80 cells from 4 independent transfections (at least 20 cells from each transfection) using Cy5 and Cy5.5 wide-field excitation. The photobleaching will be performed for at least 50% fluorescent drop and reported as raw photobleaching curve for each protein (averaged from all individual curves). The data will be excluded from the analysis if cells detach or die during photobleaching.

To quantify fluorescence retention upon 4% PFA fixation, HEK cells will be transfected using standard methods and imaged using fluorescence wide-field microscope 36-48 h post-transfection without administration of exogenous BV. After imaging, cells will be washed with PBS twice and fixed at room temperature for 15 min. Wash cells twice with PBS after fixation, and image again in PBS. Cells will be imaged in the Cy5-LP channel and analyzed as described above. To obtain statistically significant datasets, we will perform 2 independent transfections and analyze at least 100 individual cells for each transfection.

To quantify intracellular brightness in live primary mouse hippocampal neurons, cultured neurons will be prepared and transfected as described before²⁵ using pAAV-CAG-NIR-FP-P2A-EGFP plasmids. Briefly, hippocampal neurons will be transfected at DIV 4-5 using the calcium phosphate transfection method and imaged DIV 12-18 using the same imaging setup as described above for HEK cells. Intracellular brightness and photostability will be quantified using the same procedure as describe for HEK cells above. For each NIR FP we will perform at least two independent transfections and analyze at least 20-30 individual neurons for each transfection. The culture will be excluded from the analysis if cells die after transfection or culture is contaminated with bacterial or yeast infection.

Image analysis will be performed using Nikon Elements software for image segmentation and fluorescence mean value calculation. Statistical analysis will be performed in OriginPro software using the Kruskal-Wallis ANOVA test.

To quantify monomeric state of NIR FPs in mammalian cells we will employ OSER assay²⁸ as described before²⁷. Briefly, HeLa cells will be transfected with the pCytERM-NIR-FP-N1 plasmids and imaged 12-18 h post-transfection using the Cy5-LP channel. For each NIR-FP we will perform at least two independent transfections and analyze at least 250 individual cells for each transfection. According to Cranfill et al.²⁷ recommendations, positive cells selected for analysis will have overall similar fluorescence brightness, and cells that are significantly brighter will be excluded. Cells with non-spherical nuclei, ER sheet architectures, or condensed nuclei will be excluded from the assay. Image analysis will be performed by three independent researchers using Nikon Elements software and average value will be reported.

The top 5 NIR FPs in each spectral channel showing the highest monomeric state in OSER assay will be fused with tubulin and H2B for the expression in HeLa cells to assess their localization qualitatively. Pairs of NIR FPs showing the largest spectral separation in Cy5-BP and Cy5.5 channels will be used for two-color imaging in HeLa cells under wide-field microscope.

Based on the quantitative assessment of intracellular brightness and photostability in cultured neurons we will select 5 best performing NIR FPs in each channel for further characterization in vitro and in vivo. To rank the proteins, we will calculate the product of intracellular brightness and photostability (each normalized to its maximum value to ensure the roughly equal contribution of each parameter to the product), and then select the five variants with the highest such products.

In vitro characterization.

The selected NIR FPs will be expressed in E. coli and purified for further characterization in vitro. Expression and purification will be performed as described previously²⁵. Briefly, the genes of the selected NIR FPs will be cloned into pBAD-HisD vector and co-transformed with pWA23h plasmid, encoding heme oxygenase1 from Bradyrhizobium ORS278 (hmuO) under the rhamnose promoter, into the TOP10 E. coli strain (Life Technologies/Invitrogen). The transformed bacterial cells will be grown in RM medium supplemented with ampicillin, kanamycin, 0.002% arabinose, and 0.02% rhamnose for 15–18 h at 37 °C and then for 24 h at 18°C. Proteins will be purified using metal affinity chromatography according to the manufacturer’s protocol with one minor modification: in the wash buffer, 100 mM EDTA was used instead of 400 mM imidazole. The purified proteins will be dialyzed against PBS at pH 7.4 and adjusted to required concentration for spectroscopic measurements. The UV-vis absorption spectra will be measured in the range from 250-800nm using UV-VIS-NIR UV3600Plus spectrophotometer (Shimadzu, Japan). The steady-state and time-resolved fluorescence spectra will be measured in the range of 250-900nm using Photoluminescence Spectrometer FS5 (Edinburgh Instruments, UK). Absolute fluorescence quantum yield will be directly measured using FLS1000 spectrometer equipped with the integrating sphere accessory (Edinburgh Instruments, UK) according to the manufacture protocol. To determine the extinction coefficient, we will compare the absorbance value for the protein at the main peak centered in the red part of the spectrum with the absorbance value of the short-wavelength peak centered at 370-390nm assuming the latter to have the extinction coefficient of the free biliverdin Ixα, which is 39,900M⁻¹ cm⁻¹ (ref.³). pH titrations will be done in the range of pH from 2.0 to 11.0 using a series of commercially available pH buffers (Leagene) and fluorescence will be measured with Varioskan LUX plate reader.

For two-photon spectral characterization, we will use a previously described optical setup and protocol²⁹. Briefly, the setup includes a tunable (680–1300nm) femtosecond InSight DeepSee Dual femtosecond laser (Spectra Physics) and a photon-counting spectrofluorometer, PC1 (ISS). The same measurement protocol²⁹ will be used for a whole series of the selected NIR FPs, which will make a direct comparison of their two-photon absorption properties trustworthy. To measure two-photon excitation spectrum, the laser wavelength is automatically stepped, and the corresponding total fluorescence signal is recorded at each wavelength using our customized LabView program. Well-characterized two-photon standard dye solutions of Coumarin 540A in DMSO and LDS798 in chloroform are used to correct two-photon spectral profile of the sample for variations of wavelength-to-wavelength variations of laser parameters (spatial intensity distribution and pulse duration).

We will use a modification of the previously described method²⁹ of measurement of the two-photon absorption cross section (s₂). In particular, we will collect a total (without monochromator) fluorescence signal of the sample and reference, first upon two-photon excitation at a certain wavelength, and then upon one-photon excitation (with a continuous-wave laser) with a visible wavelength matching absorption spectra of the sample and reference. In these measurements, the sample and the reference solutions are contained in 3x3 mm SOG cuvettes (Starna) and have maximum optical density less than 0.1. The fluorescence is collected at 90⁰ to excitation laser beam through a set of filters that block both the femtosecond near-IR and visible laser wavelengths (typically including FF01-770/SP, or FF01-842/Sp in combination with 561nm EdgeBasic best-value long-pass filters (all Semrock)) using the left emission channel of the spectrofluorometer. We first collect the two-photon excited fluorescence signal I as a function of laser power P for both the sample and reference solutions and fit them to a quadratic function I = aP². From these fits, the coefficients a_S and a_R are obtained for the sample (index S) and reference (index R) solutions, respectively. Second, we measure the one-photon excited fluorescence signals as a function of laser power for the same solutions and in the same registration conditions as in the two-photon experiment and fit them to a linear function: I = bP. The best fits provide coefficients b_S and b_R. The two-photon absorption cross section is then calculated as follows: see formula 2 in the supplementary files section.

Here, l₁ is the wavelength used for one-photon excitation (for example, 561nm), l₂ is the wavelength used for two-photon excitation (1060 or 1064nm), e_R,S(l₁) are the corresponding extinction coefficients, measured at l₁. This approach allows us to automatically correct for the laser beam properties (pulse duration and spatial intensity distribution), fluorescence collection efficiencies for one- and two-photon modes, PMT spectral sensitivity, and differences in quantum yields and concentrations between the sample and reference solutions.

For the measurement of the two-photon absorption cross sections we will use the well characterized standard solutions, Rhodamine 6G in methanol and Rhodamine B in alkaline ethanol. The two-photon absorption cross section of Rhodamine 6G was measured independently in 5 publications at 1060 nm and their averaging provides s₂= 10 ± 1 GM⁵. Rhodamine B has an advantage of more red-shifted absorption spectrum that overlaps better with the NIR FPs. Analysis of available literature data (6 independent measurements) yields s₂= 13 ± 3 GM for Rhodamine B in alkaline ethanol at 1064 nm⁶.

Characterization in mice. All procedures involving mice at Westlake University will be conducted in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Westlake University Committee on Animal Care. The selected NIR FPs will be expressed in brain and liver of mice and assessed for brightness in vivo and ex vivo. We will use C57BL/6 mice supplied by Animal Facility at Westlake University without regard for sex. For expression of NIR FPs in the brain, we will use the neonatal intraventricular injections of custom-made recombinant adeno-associated viruses’ serotype 2/9 (rAAV2/9-CAG-NIRFPs-P2A-GFP, 1-2×10⁹ genome copies per injection) as described previously³⁰. At least five mice will be injected for each NIR FPs. Acute brain slices will be obtained from mice at P20–30 using standard techniques as described before²⁵. For assessment of intracellular brightness and photostability in Cy5 and Cy5.5 channels, we will image at least 9 live brain slices from at least 3 mice. For assessment of intracellular brightness and photostability in the fix brain tissue we will perfuse mice using 4% PFA according to the protocol described before²⁶. For the PFA fix brain tissue assessment, we will use at least 12 slices from at least two mice (age of mice will be P20-30). For expression of NIR FPs in liver, the 3–6-month-old mice will be intravenously injected via tail vein with 1-2×10⁹ genome copies of rAAV2/9 and imaged 5-20 days post injection. The liver will be dissected and imaged using wide-field microscope in Cy5 and Cy5.5 channels as described before³¹. For each construct we will test at least 3 livers. Animals that show no GFP fluorescence will be excluded from the study.

Characterization of NIR FPs in neurons of living zebrafish larvae. All experiments involving zebrafish will be performed according to European Union guidelines and German legislation (EU Directive 2010_63 and license AZ 325.1.53/56.1-TU-BS). The division of Molecular and Cellular Neurobiology at the Zoological Institute of the TU Braunschweig is fully equipped for performing the proposed zebrafish experiments. The zebrafish facility provides zebrafish husbandry, breeding and production of eggs used for the plasmid injection experiments. The laboratories are well-equipped with microinjectors (Eppendorf), fluorescence stereomicroscope (Leica), and confocal laser scanning microscopes (Leica SP8 and Zeiss LSM 880 with Airyscan). See the pilot data acquired by Xinyue Wang, and Kazuhiko Namikawa at the Zoological Institute of the TU Braunschweig.

Intracellular brightness and photostability in neurons of zebrafish larvae.

The top 5-6 NIR FPs that showed the highest performance in cultured cells in each channel will be expressed transiently in zebrafish larvae and assessed for brightness and photostability. The pan-neuronal expression vector will carry two expression components flanked by Tol2 recognition sites. Injection of this construct into zebrafish embryos will allow for Tol2-transposon-mediated early genomic integration of the transgene, which will result in broader expression in neuronal cells of larval zebrafish in a semi-mosaic manner. Pan-neuronal specific Gal4 expression unit encodes a Gal4 variant, KalNFB³² regulated by a compact promoter of 377bp (nbt³⁰⁰2n) which will be composed of an Xenopus neuron-specific β-tubulin (nbt) gene core promoter (accession EF989124, 3182-3478), followed by 2x neuron restrictive silencer element (2x NRSE: 2n) in the 5’ untranslated region, which enables induction of the pan-neuronal specific KalNFB expression. The second expression module will carry 10x upstream activating sequence (10xUAS) flanked on both sides by two E1b minimal promoters, leading to bidirectional co-expression of transgenes in a Gal4-dependent manner.

The mammalian codon-optimized NIR FPs linked to P2A-mClover3 (NIR-FP-P2A-mClover3) as mentioned above will be cloned as one cistron into the bidirectional UAS-co-expression unit. As the other cistrons, zebrafish codon-optimized human HO1, or its inactive form (HO1^H25A) will be inserted. The resultant Gal4/UAS construct will drive multicistronic transgene expression, allowing for the assessment of NIR-FP fluorescence in Gal4 expressing neurons with or without HO1 activity.

The generated expression constructs will be co-injected with Tol2 transposase mRNA into less pigmented brass mutant embryos at the single-cell stage. Injected larvae will be screened for mClover3 fluorescence throughout the nervous system at 2–3 days post fertilization (dpf) using a stereomicroscope (Leica Microsystems, Wetzlar, Germany). Subsequently, alive larval zebrafish will be imaged at 4-5 dpf using a confocal microscope (Leica SP8, Leica Microsystems, Wetzlar, Germany). Fluorescence signals in zebrafish will be detected using a 20x or 40x water immersion objective in a green (excitation: 488nm from an argon laser; emission 495-530nm) and cfCy5-LP (excitation: 633nm from an HeNe laser, emission 660-785nm) channels by confocal microscopy. To quantify intracellular brightness of NIR FPs in neurons, reconstructions and projections from z-stacks of images will be generated using the 3D-projection program included in the Leica LAS X software. Acquired images will be processed with FIJI to measure the fluorescent intensity in green and cfCy5-LP channels, and the mean ratio of each NIR FPs and mClover3 for individual neurons in the hindbrain and spinal cord will be calculated. At least 4 individual larval fish will be imaged to collect the data from at least 80 neurons for analysis in total per construct. The mean fluorescence intensity of each NIR-FP will be calculated, followed by the evaluation of statistically significant differences in brightness for each NIR-FP and compared to their behavior in cultured cells.

To quantify photostability of NIR FPs fluorescence in zebrafish neurons, at least 4 independent 4-5 dpf larvae coexpressing HO1 and the respective NIR FP to be tested will be subjected to confocal microscopy image recording using a 40x objective in cfCy5-LP, and a region of interest will be selected encompassing 5-10 neurons labeled with the respective NIR FP. To collect photobleaching data from at least 15 neurons (more than 5 cells from 3 independent larvae), a single plane image (optical section: 3-4 µm) will be recorded continuously for 3-5 minutes with approximately 40-70% laser power to eventually reduce fluorescence intensity below 50% of the initial value. Acquired images will be processed with FIJI to measure the fluorescent intensity in individual neurons for each time point. The data will be excluded from the analysis if zebrafish moved during image recording periods. Statistically significant differences in brightness will be evaluated as described for cultured cells above.

Multi-color imaging in live zebrafish larvae

To examine the use of NIR FPs for in vivo multicolor imaging, the best one or two NIR FPs will be chosen based on the results of intracellular brightness and photostability analysis in zebrafish neurons. To further enhance the expression level, the zebrafish codon-optimized genes of the selected NIR FPs will be synthesized. To achieve multicolor imaging, cDNAs of additional FPs including mTurquoise2 (cyan FP), mGreenLantern (green FP), and mScarlet (red FP) will be synthesized also with a zebrafish codon usage optimization. These cDNAs will be fused to sequences from endogenous proteins targeting FPs to different subcellular, and neuronal compartments, which will include Histone 2B (H2B, nucleus), N-terminal fragment of growth-associated protein-43 (GAP43, plasma membrane), microtubule-associated protein 1 light chain 3 (LC3, autophagosome and autolysosome), lysosomal-associated membrane protein 1 (Lamp-1, lysosome), synaptophysin2 (pre-synapse), and postsynaptic protein 95 (PSD95, post-synapse). For some reporters, two of them will be encoded together in a bicistronic transgene separated by a T2A peptide. These reporter gene cassettes will be assembled into the pTol2-MCS³² in which the insert is composed of two bidirectional expression units regulated by 10xUAS, allowing for quadruple gene expressions. When one of the BphP-based NIR FPs is expressed, the HO1 cDNA will be included in the expression unit to enhance the brightness of NIR fluorescence. The generated UAS constructs will be co-injected into brass mutant embryos together with Tol2 mRNA and an additional plasmid containing KalNFB expressed under the control of nbt³⁰⁰2n, or cpce⁹ to achieve pan-neuronal, or Purkinje neuron-specific expression, respectively. Two or three days after injection, larval fish expressing each reporter will be sorted as mentioned above, followed by confocal microscopy image recording at 4-5 dpf (Leica SP8, or Zeiss LSM 880 with Airyscan) using either a 40, or 63x water immersion objective. Green and NIR fluorescent emission signals will be recorded simultaneously (mGreenLantern, excitation: 510nm using an argon laser; emission 520-545nm) (NIF-FP, excitation: 633nm using a HeNe laser, emission 660-785nm), followed by image recording in cyan (mTurquoise2, 458nm using an argon laser; emission 465-490nm, excitation) and red (mScarlet, DPSS 561nm laser, emission 570-600nm) emission ranges. Images will subsequently be rendered from optical z-stack sections using the Leica LAS X, or Zeiss ZEN software, followed by image processing using FIJI. At least 4 individual larval fish will be used for collecting images.

Funding

This work is supported by start-up funding from the Foundation of Westlake University, National Natural Science Foundation of China grant 32050410298, 2020 BBRF Young Investigator Grant, and MRIC Funding 103536022023 to K.D.P., the German Research Foundation (DFG, K1949/7-2) Project 241961032 to Reinhard W. Köster, the Foundation of Westlake University and the National Natural Science Foundation of China grant 32071151 to L.W., and NIH BRAIN program U24NS109107 grant to M.D.

Data availability

We will share all raw data as source data associate with this manuscript and/or deposit to a public open access domain.

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Quantitative assessment of near-infrared fluorescent proteins

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