Highly-multiplexed volumetric mapping with Raman dye imaging and tissue clearing.

Screening and studying tissue clearing methods for immuno-eprSRS Next, we sought to develop RADIANT by extending immuno-eprSRS of MARS probes to volumetric imaging. Two-photon excitation of SRS allows for intrinsic 3D optical sectioning and prevents out-of-focus photo-bleaching ( Extended Fig. 6 ). Our main task is thus to identify proper tissue clearing protocols. While volumetric SRS was recently reported for label-free chemical imaging 29 , a urea-based clearing protocol was used to preserve lipids, resulting in a compromised clearing ability in thick tissues ( Extended Fig. 7a ). Besides, urea is known to disrupt antibody-antigen binding affinity 30 , thus is less ideal for immunostaining. We hence evaluated several immunolabeling-verified and fast clearing approaches including ScaleS 23 , FOCM 24 , Ce3D 25 , 3DISCO 26 and uDISCO 27 . Signal-to-noise ratios (SNR) of immuno-eprSRS on 40 or 100-μm tissue sections were measured after these clearing procedures, reflecting the joint effect of clearing, staining, probe concentration and stability. We found that DISCO-based methods outperformed others ( Fig. 3a ), partly due to probe concentration increase mediated by tissue shrinkage. Therefore, we chose DISCO as our basic protocol. Next, we asked why uDISCO performed better than 3DISCO ( Fig. 3a ), as tissue shrinkage is similar for both methods. In 3DISCO, the RI matching medium DBE is known to be prone to form peroxides 27 ( Fig. 3b and Supplementary Note 2 ). As shown in the absorption spectra of MARS probes freshly dissolved in the RIMS, a substantial intensity decrease was observed in DBE compared to either BABB or BABB-D4 (BABB:DPE=4:1 in uDISCO) ( Fig. 3b , c ). Besides, a bluer absorption band appeared in DBE, especially for C-cored probe ( Extended Fig. 7b ). These results collectively suggested the decomposition of MARS probes, likely induced by peroxides formed in DBE. Going from solutions to tissue samples, uDISCO also generated better SNR in immuno-eprSRS than 3DISCO ( Fig. 3d ) and induced less post-clearing fluorescence background ( Fig. 3e ), in consistent with the solution results. By virtue of the chemical stability of MARS probes in uDISCO, we achieved 3D immuno-eprSRS imaging on 500-μm thick cerebellum sections with good contrast ( Fig. 4a and Extended Fig. 8 ). In comparison, the contrast of uncleared sample decreases rapidly over a shallow depth of 50–100 μm ( Supplementary Fig. 4 ). Such single-color success can be readily extended to multiplex epr-SRS imaging, thanks to the chemical similarity of all the MARS probes ( Fig. 2a ). Notably, 8-target volumetric imaging (six SRS and two fluorescence channels) can be carried out in one shot by RADIANT with expected patterns along the entire depth for each channel ( Fig. 4b – c and Supplementary Fig. 5 , magnified views in Fig. 4c ), which already breaks the color barrier for thick tissues ( Fig. 1 ).

Methods

Protein-MARS probe conjugation NHS-ester-functionalized MARS probes were stored at a concentration of 10 mM in DMSO under −20 °C, protecting from light and moisture. To perform protein-dye conjugation, dye solutions were first diluted in DMSO to a concentration of 2 mg/mL. Conjugation buffer was prepared as 0.1 M NaHCO 3 in PBS buffer with pH adjusted to 8.3. Highly cross-absorbed secondary antibodies were buffer exchanged and concentrated to 2 mg/mL in the conjugation buffer. A 50 μL dye-NHS solution was slowly added to a 0.5 mL secondary antibody solution under stirring. For primary antibody labeling, the protein concentration was adjusted to 1 mg/ml and the molar ratio of dye/protein was usually 10–15. Lectins were first dissolved in conjugation buffer as 2 mg/ml. For Wheat Germ Agglutinin (WGA) labeling, a 10 μL 2 mg/ml dye-NHS solution was added to a 0.5 mL 2 mg/ml WGA solution. For Lycopersicon Esculentum Lectin (LEL) labeling, a 25 μL 2 mg/ml dye-NHS solution was added to a 0.5 mL 2 mg/ml LEL solution. Reactions were all incubated at room temperature for 1 h under constant mild stirring. Labeled proteins were further separated from unreacted dyes by gel permeation chromatography using Sephadex ™ G-25 ( G25150 SIGMA) resins with a column of 1-cm diameter and over 12-cm length. Purified protein solution was centrifuged to remove potential precipitates and further concentrated with Amicon® Ultra Centrifugal Filters (UFC501096, EMD, Millipore). A final concentration of ~2 mg/mL protein solution (for secondary antibodies and lectins) were prepared in stocking buffer (30% glycerol and 5 mM sodium azide in PBS) and stored at −20 °C. The degree of labeling (DOL, i.e. dye-to-protein ratio) were measured with UV-Vis spectrum using Tecan Infinite 200 Reader with a NanoQuant Plate. DOL on secondary antibodies is about 3. Cell culture Human HeLa (ATCC CCL-2), COS-7 (ATCC CRL-1651) cells were cultured in DMEM (Invitrogen 11965) supplemented with 10% FBS (Invitrogen 10082) and 1× penicillin/streptomycin (Invitrogen 15140). Human MCF7 (ATCC HTB-22) cells were cultured in EMEM (ATCC 30–2003) supplemented with 10% FBS and 1× penicillin/streptomycin.

Mouse sample preparation

Brain tissues.

Animal experimental protocol

(AC-AABD1552) was approved by the Institutional Animal Care and Use Committee (IACUC) at Columbia University. Wild type male and female mice (C57BL/6, 15–25 days old, Jackson Lab) were fully anesthetized using isoflurane, then sacrificed with cervical displacement and immediately perfused with 4% paraformaldehyde (PFA) in PBS transcranially. The brain was extracted and fixed in 4% PFA in PBS at 4 °C for 24 h. After that, the brain was immersed in PBS at 4 °C for 24 h to remove PFA. The cerebellum was embedded in 7% agarose gel and sectioned into 40 μm, 100 μm, 500 μm, and 1 mm thick coronal slices using a vibratome (VT1000S, Leica). Agarose was removed by a tweezer before staining. Pancreas tissues. Animal procedures were performed under protocols approved by the IACUC of Colorado Anschutz Medical campus (#00024). Wild type male and female mice (C57BL/6, 8–12 weeks old, Jackson Lab) were anesthetized by I.P. injection of Ketamine (80 mg/kg) and Xylazine (16 mg/kg) until no longer reactive to toe pinch. Pancreata were dissected and mice were then euthanized by exsanguination and/or Bilateral thoracotomy. Pancreata were fixed in PFA (4% in PBS) at 4 °C rocking for 16–20 h, washed in 4 °C PBS to remove PFA, and embedded in OCT blocks. 8 μm sections were then taken for staining. Stimulated Raman scattering (SRS) and fluorescence integrated imaging platform SRS and fluorescence imaging were performed on an inverted laser scanning microscope (Olympus FV1200) using a 25× water-immersion objective lens (Olympus XLPlan N, 1.05 NA, MP, WD = 2 mm). For SRS imaging, two synchronized 6-ps lasers (called pump and Stokes beams) with 80-MHz repetition rate are provided by a picoEmerald system from APE (Applied Physics & Electronic, Inc.). Pump beam is tunable from 720–990 nm through both temperature control of the nonlinear crystal and a Lyot filter. Stokes beam is fixed at 1064.2 nm. The intensity of the Stokes beam was modulated sinusoidally by a built-in electro-optic modulator (EOM) at 8 MHz with a modulation depth of more than 90%. Spatially and temporally-overlapped pump and Stokes beams were coupled into the laser-scanning microscope. After passing through the specimens, forward-going pump and Stokes beams were collected with an IR-coated oil condenser (1.4 NA, Olympus). Stokes beam were completely filtered with two high-optical-density bandpass filter (890/220 CARS, Chroma Technology) and transmitted pump beam was detected by a large-area (10 mm×10 mm) Si photodiode (FDS1010, Thorlabs). The output current of the photodiode was then sent to a fast lock-in amplifier (HF2LI, Zurich Instruments) for signal demodulation. For immuno-eprSRS imaging, the laser power was set as P pump =17 mW, P Stokes =50 mW for C-cored MARS probes and P pump =17 mW, P Stokes =67 mW for O-cored MARS probes. SRS images were generated through Kalman filtering of 10–30 serial frames with the pixel dwell time of 4 μs. The time constants of lock-in amplifier were chosen as 2–4 μs. Pixel sizes were chosen as about 0.25–1 μm. For volumetric imaging, the step size in z was 5 μm. For two-photon fluorescence, DAPI dye or Alexa Fluor 350 were excited by the SRS pump laser at 760 nm. The backward fluorescence was detected after passing through a 690-nm short-pass filter, reflected by a 570-nm long-pass dichroic with a collection band of 410–490 nm. For confocal fluorescence, green channel is excited by argon laser (488 nm) with a collection band of 505–520 nm; red channel is excited by HeNe(G) laser (543 nm) with a collection band of 560–620 nm; far-red channel is excited by LD laser (635 nm) with a collection band of 655–755 nm. Multichannel photomultiplier tube (PMT) was used for fluorescence detection. Pixel sizes were chosen as about 0.25–1 μm and pixel dwell time were set as 2–4 μs. Immuno-eprSRS staining on thin tissue sections For PFA fixed mouse cerebellum thin sections, 40-μm thick sections from P15-P40 young mice were used. The staining was performed on the floating tissues in a 4-well plate. Samples were permeabilized and blocked with blocking buffer (5% donkey serum, 0.5% Triton X-100 in PBS) at room temperature for 30 min. After blocking, samples were incubated with primary antibody (typical dilution, 1:100) in staining buffer (2% donkey serum, 0.5% Triton X-100 in PBS) at 4 °C for 1–2 d, followed by washing at room temperature for 5–10 min in PBS with 0.5% Triton X-100 (0.5% PBST) three times. Samples were then blocked in blocking buffer for 30 min, followed by incubating with MARS-conjugated secondary antibody (typical dilution, 1:100) in staining buffer at 4 °C for 1–2 d. After staining, the samples were washed at room temperature for 5–10 min in 0.5% PBST three times. Frozen mouse pancreatic tissue slides were stored at −80 °C. Specimens were first equilibrated to room temperature and held in PBS buffer before staining. The staining processes were performed on slides with a similar protocol as described above for PFA tissues, with the following changes. A Triton X-100 concentration of 0.3% was used for blocking buffer, staining buffer and sample washing. Incubation time for both primary and secondary antibodies is 18 h. FFPE human kidney slice used in this study was purchased from Biomax (HuFPT072). FFPE tissue slides were first baked at 60 °C for 10 min. Samples were sequentially placed in following solutions (in a 50 mL Falcon tube) for deparaffinization and rehydration with 3 min each time at room temperature under mild shaking: (1) xylene two times, (2) ethanol two times, (3) 95 vol% ethanol in DI water two times, (4) 70 vol% ethanol in DI water two times, (5) 50 vol% ethanol in DI water one time, (6) DI water one time. For antigen retrieval, the specimen was first transferred into a glass jar filled with 20 mM sodium citrate (pH=8.0) at 100 °C, followed by a quick transfer of the jar to a 60 °C incubation chamber for 45 min. After that, the specimen was washed at room temperature with DI water for 5 min. The staining processes followed the same protocol as the frozen tissues. For imaging specimen preparation, PFA fixed tissue sections were transferred to Superfrost glass slide and all specimens were mounted in ProLong Gold antifade (ThermoFisher, P10144 ) with a glass coverslip, secured with nail polish. Twelve-target mouse cerebellum imaging Staining. See Fig. 2g and Extended Data Fig. 5c – d . The staining processes followed the sample protocol as PFA fixed thin tissue sections above with three rounds of antibody incubation. First round: anti-NeuN antibody (rabbit, dilution 1:50), anti-β-III tubulin antibody (chicken, dilution 1:50), anti-Calbindin antibody (mouse, dilution 1:40), anti-GABBR2 antibody (guinea pig, dilution 1:40), anti-MBP antibody (rat, dilution 1:20), anti-GFAP antibody (goat, dilution 1:50) and MARS2242-conjugated WGA (1:50). Second round: MARS2228-conjugated donkey anti-rabbit IgG antibody, MARS2220-conjugated donkey anti-chicken IgY antibody, MARS2145-conjugated donkey anti-mouse IgG antibody, MARS2212-conjugated donkey anti-guinea pig IgG antibody, MARS2188-conjugated donkey anti-rat IgG antibody and MARS2159-conjugated donkey anti-goat IgG antibody with a dilution of 1:50. Third round: Alexa Fluor 596-conjugated anti-TH antibody (mouse, dilution 1:50), Alexa Fluor 488-conjugated anti-VGluT1 antibody (mouse, dilution 1:50) and MARS2176-conjugated anti-α tubulin antibody (mouse, 0.5 mg/mL, dilution 1:40). Between the second and the third round of antibody incubation, samples were further blocked in 5% normal mouse serum, 0.5% Triton X-100 in PBS at RT for 1 h. The specimens were futher stained with NucBlue and Phalloidin-Alexa Fluor 647 and embedded in the ProLong Gold antifade medium for imaging. Spectral unmixing. A linear-combination algorithm was applied on multiplexed epr-SRS data set to remove potential cross-talks between different channels 16 . For N -channel epr-SRS measurement with N MARS probes ( N =8 here), measured signals ( S ) can be expressed as S = M C , where C is the MARS probe concentrations and M is a N × N matrix determined by Raman cross-sections of MARS probes. Matrix M was measured experimentally on single-color immuno-eprSRS labeled with different MARS probes. MARS probe concentrations were therefore determined using C = M −1 S . Clearing method screening for immuno-eprSRS on MARS probes Staining. Samples were first washed with 0.2% PBST at room temperature on a shaking platform for 1h twice, followed by incubation in 20% DMSO, 0.2% Triton X-100 in PBS at 37 °C on a shaking platform overnight. The tissues were incubated in 0.1% Tween-20, 0.1% Triton X-100, 0.1% deoxycholate, 0.1% NP40, and 20% DMSO in PBS at 37 °C on a shaking platform overnight. The tissues were washed with 0.2% PBST at room temperature on a shaking platform for 1 h twice. The tissues were permeabilized with 0.5% PBST at 37 °C on a shaking platform overnight, and then permeabilized and blocked with blocking buffer (5% donkey serum, 0.5% Triton X-100 in PBS) at 37 °C on a shaking platform for 3 d. The tissues were incubated with anti-MBP antibody (rat, dilution 1:20) or anti-GFAP antibody (goat, dilution 1:40) diluted in staining buffer (2% donkey serum, 0.5% Triton X-100 in PBS) at 37 °C on a shaking platform for 2 d, and then washed with 0.5% PBST at 37 °C on a shaking platform for 1 d (the solution was changed several times in between). Then the tissues were incubated with MARS2200-conjugated donkey anti-rat IgG antibody (dilution 1:50), MARS2228-conjugated donkey anti-goat IgG antibody (dilution 1:40), or MARS2159-conjugate donkey anti-goat IgG antibody (dilution 1:40) diluted in staining buffer at 37 °C on a shaking platform for 2 d. The tissues were washed with 0.5% PBST at 37 °C on a shaking platform for 3 h. The tissues were then incubated in PBS at 4 °C overnight. After that, the tissues were cleared with published tissue clearing methods, including ScaleS 23 , FOCM 24 , Ce3D 25 , 3DISCO 26 and uDISCO 27 as previously described, or with different tissue clearing conditions explored in this work. The cleared tissues were mounted on glass slides in a pool created by dental cement and filled with the corresponding refractive index matching solutions and covered with a glass coverslip for SRS imaging. Quantification. The signal-to-noise ratios of the raw images were quantified as the main criterion to evaluate different tissue clearing methods. Specifically, the signal-to-noise ratio was calculated as (intensity of the labelled structure/cell – intensity of the background) / standard deviation of the background. The background was measured from the same tissue where no stained pattern appears under the same imaging condition. Thus, the noise is composed of both instrumental noise and sample noise. The signal-to-noise ratios of multiple regions of interest were quantified and presented as mean±s.d. Regions of interest were manually selected with correct localization pattern and from a similar depth. rDISCO (Raman DISCO) clearing procedure The immune-labelled tissues were first dehydrated in tert -butanol/DI water series as 30 vol%, 50 vol%, 70 vol%, 80 vol%, 90 vol% tert -butanol; 2 h each at room temperature on a rotator. Then the tissues were incubated in 96 vol% tert -butanol in water at room temperature on a rotator overnight. The tissues were incubated in pure tert -butanol at room temperature on a rotator for 2 h. The tissues were incubated in the refractive index matching solution, which was prepared by mixing BABB (benzyl alcohol and benzyl benzoate was mixed at a ratio of 1:2 and purified by column chromatography using aluminum oxide) with diphenyl ether at a ratio of 4:1 and adding 1% propyl gallate (w/v), at 4 °C for 2 h. The tissues were mounted in the same refractive index matching solution for SRS imaging. Pre-processing and staining of thick tissue sections Sample pre-processing. The pre-processing protocol was adapted from the sample pretreatment procedures in iDISCO 12 . For initial processing, tissue samples were washed with 0.2% PBST at room temperature on a shaking platform for 1 h twice, and then incubated in 20% DMSO in 0.2% PBST at 37 °C on a shaking platform overnight. The tissues were incubated in 0.1% Tween-20, 0.1% Triton X-100, 0.1% deoxycholate, 0.1% NP40, and 20% DMSO in PBS at 37 °C on a shaking platform overnight. The tissues were washed with 0.2% PBST at room temperature on a shaking platform for 1 h twice. The tissues were further permeabilized with 0.5% PBST at 37 °C on a shaking platform overnight and followed by blocking/permeabilization with 5% donkey serum in 0.5% PBST at 37 °C on a shaking platform for 6 d (the solution was changed once after 3 d). After this, the tissue slices were ready for immunostaining. Thick sample staining. The staining processes followed similar procedures as thin tissue with varied time and probe concentrations. Detail descriptions can be found in Supplementary Methods .

Three-dimensional reconstruction

Raw volumetric data were unmixed into specific channels using the linear combination algorithm as mentioned above. A median filter was applied to the unmixed images for noise reduction. A Fiji plugin 59 was then applied to the images for attenuation correction. The open radius was set to 0.5. After correction, the images were volume-rendered in the mode of maximal intensity projection using Imaris (Bitplane).

Three-dimensional segmentation and quantitative analyses

Raw volumetric data went through linear unmixing, noise reduction with a median filter, and attenuation correction as mentioned above. Based on the channels of MBP, NeuN, and GABBR2, the image was segmented manually using the “Contour” tool in Imaris into four anatomical layers—white matter, the granular layer, the Purkinje layer, and the molecular layer. The segmentation results were exported from Imaris as four binary masks. After the image was median-filtered using a radius of 2 pixels and thresholded with Otsu’s method, Pearson correlation coefficients were calculated between specific protein channels in the four layers and presented as heatmaps using custom-written MATLAB (MathWorks) scripts. As the MBP channel had no signals in the Purkinje layer and the molecular layer after thresholding, correlation analysis was performed with the rest 10 channels without the MBP channel for these two layers ( Fig. 6b ). Vimentin positive and GFAP positive cells were segmented using Imaris. The GFAP positive cells that did not touch vimentin positive cells were defined as Vim – /GFAP + cells. The vimentin positive cells that did not touch GFAP positive cells were defined as Vim + /GFAP – cells. The GFAP positive cells that touched vimentin positive cells and the vimentin positive cells that touched GFAP positive cells were merged into one channel and segmented as Vim + /GFAP + cells. The proportions of Vim – /GFAP + , Vim + /GFAP – , and Vim + /GFAP + cells in the four layers were quantified using the shortest distances to the segmented four layers (A negative shortest distance meant the cell was inside the layer. Cells that spanned multiple layers were excluded from this analysis). Blood vessels were segmented from the LEL channel using Imaris. Shortest distances from Vim – /GFAP + , Vim + /GFAP – , and Vim + /GFAP + cells to the blood vessels were quantified using Imaris. Correlation-based network graphs were plotted in the four layers using custom-written MATLAB (MathWorks) scripts. Specifically, the “graph” function in MATLAB was used with Pearson correlation coefficients as the input. Self-loops and negative correlation coefficients were discarded. Closeness node centrality was calculated with the “centrality” function and color-coded in each node. The node size was set to be directly proportional to the degree of each node. Network diameters were calculated with the “distances” function. Average clustering coefficients were calculated with the “clusteringcoef” function in the Graph package 60 .

Statistics and Reproducibility

Statistical analysis was carried out using GraphPad Prism 7 and OriginPro 8. Data are presented as mean±s.d. or mean±s.e.m. with statistical significance if required (not significant P ≥0.05, * P