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Light-sheet microscopy in the near-infrared II window

Nature Methods volume 16, pages 545–552 (2019)Cite this article

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Abstract

Non-invasive deep-tissue three-dimensional optical imaging of live mammals with high spatiotemporal resolution is challenging owing to light scattering. We developed near-infrared II (1,000–1,700 nm) light-sheet microscopy with excitation and emission of up to approximately 1,320 nm and 1,700 nm, respectively, for optical sectioning at a penetration depth of approximately 750 μm through live tissues without invasive surgery and at a depth of approximately 2 mm in glycerol-cleared brain tissues. Near-infrared II light-sheet microscopy in normal and oblique configurations enabled in vivo imaging of live mice through intact tissue, revealing abnormal blood flow and T-cell motion in tumor microcirculation and mapping out programmed-death ligand 1 and programmed cell death protein 1 in tumors with cellular resolution. Three-dimensional imaging through the intact mouse head resolved vascular channels between the skull and brain cortex, and allowed monitoring of recruitment of macrophages and microglia to the traumatic brain injury site.

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Fig. 1: LSM in various NIR 850- to 1,700-nm emission subregions in glycerol-cleared brain tissues.
Fig. 2: Propagation of light-sheet excitation with progressively longer wavelengths up to 1,319 nm in glycerol-cleared brain tissues.
Fig. 3: Non-invasive in vivo NIR-II LSM of tumors on mice.
Fig. 4: Non-invasive in vivo light-sheet imaging of mouse head using oblique NIR-II LSM.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank members of the Dai group for discussion. This study was supported by the National Institutes of Health through grant DP1-NS-105737.

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Author notes

  1. These authors contributed equally: Feifei Wang, Hao Wan, Zhuoran Ma.

Authors and Affiliations

  1. Department of Chemistry and Bio-X, Stanford University, Stanford, CA, USA

    Feifei Wang, Hao Wan, Zhuoran Ma, Yeteng Zhong, Qinchao Sun, Ye Tian, Haotian Du & Hongjie Dai

  2. Department of Radiology and BRIC, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Liangqiong Qu

  3. State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan, China

    Mingxi Zhang

  4. Palo Alto Veterans Institute for Research, VA Palo Alto Health Care System, Palo Alto, CA, USA

    Lulin Li & Jian Luo

  5. Department of Materials Science and Engineering, South University of Science and Technology of China, Shenzhen, China

    Huilong Ma & Yongye Liang

  6. Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA

    Jian Luo

  7. State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang, China

    Wen Jung Li & Lianqing Liu

  8. Department of Mechanical Engineering, City University of Hong Kong, Kowloon Tong, Hong Kong

    Wen Jung Li

  9. Department of Materials Science and Engineering, Stanford University, Stanford, CA, USA

    Guosong Hong

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Contributions

H. Dai and F.W. conceived and designed the experiments. H. Dai. and F.W. designed the optical system. F.W. set up the optical system. F.W., H.W., Z.M. and Y.Z. performed the experiments. Q.S. did the Monte Carlo simulations. F.W., H.W., Z.M., Y.Z., Q.S., Y.T., L.Q., H. Du, H.M., Y.L., W.J.L., G.H., L. Liu and H. Dai analyzed the data. M.Z. developed the PbS/CdS CSQD dyes. F.W., W.H., Z.M. and H. Dai wrote the manuscript. L. Li and J.L. performed TBI. All authors contributed to the general discussion and revision of the manuscript.

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Correspondence to Hongjie Dai.

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Integrated supplementary information

Supplementary Fig. 1 A schematic of the LSM in the second near-infrared window.

(a) Traditional LSM. The components are as follows: illumination objective (O1), achromatic lenses (L1 – L5), adjustable mechanical slits (S1, S2), cylindrical lens (CL), pinhole (PH), mirrors (M1 – M5), detection objective (O2) and emission filters (F). Three excitation lines can be selected by removable mirrors (M2-M4). A spatial filter was introduced to improve the circularity and quality of the illumination beam and to generate uniform light sheet across the field of view. Before the laser entering a cylindrical lens (CL), a vertically arranged adjustable mechanical slit S2 parallel to the CL was used to adjust the span range of light sheet along Y-axis direction. After magnified by a pair of achromatic lenses (L1, L2), the incident light was focused on the back focal plane of illumination objective (O1). For LSM, the actual illumination NA was adjusted by S1 and the span range along Y was controlled by S2. Fluorescence was collected through a detection objective (O2) and a 200-mm tube lens, then transmitted to a liquid-nitrogen-cooled InGaAs camera (2D-OMA V, Princeton Instruments) after filtered by selected emission filters. The focal lengths of L1, L2, L3, L4 were 60 mm, 100 mm, 30 mm and 60 mm, respectively. (b) Oblique LSM with the optical path the same to the normal LSM as shown in (a) but the illumination objective and detection objective was arranged 45o to horizontal direction. In this configuration, an UV fused silica right-angle prism was used. A cover glass and samples were fixed on scanning stage. The gaps between prism, cover glass and sample were filled with 80% glycerol to compensate refractive index mismatch. A 4× objective (NA = 0.1, Bausch & Lomb Optical Co.), a 5× objective (NA = 0.15, Nikon LU Plan), a 10× objective (NA = 0.25, Bausch & Lomb Optical Co.) and a 50× objective (NA = 0.6, Nikon CF Plan) were selectively used in these experiments (Supplementary Table 2).

Supplementary Fig. 2 An organic nanofluorophore p-FE dye and PEGylated PbS/CdS CSQD (core-shell quantum dot) probe used for this work.

Schematics of (a) p-FE is comprised of organic dyes trapped in amphiphilic polymeric micelles ~ 12 nm in size measured by dynamic light scattering28 and (b) PEGylated PbS/CdS CSQD with a wide range of excitation wavelength spanning from UV to ~ 1300 nm, high brightness, biocompatibility and liver excretion developed recently29. Absorption and emission spectra of (c) p-FE and (d) PEGylated PbS/CdS CSQD. Similar results for n = 5 independent experiments. Taking advantage of these two dyes, we were able to image the cerebral vasculatures as a function of tissue depth Z in three fluorescence emission windows 850–1000 nm (NIR-I, p-FE emission), 1100–1200 nm (NIR-IIa, p-FE emission) and 1500–1700 nm (NIR-IIb, CSQD emission) respectively under the same 785 nm light sheet excitation. This allowed side-by-side comparison of fluorescence LSM imaging in three emission sub-regions of 850–1700 nm under the same 785 nm LS excitation. The wide range of excitation wavelength of CSQD allowed comparing light scattering of different excitations in scattering media.

Supplementary Fig. 3 LSM in various NIR 850- to 1700-nm emission sub-regions in glycerol-cleared brain tissues.

(a) A supplementary figure for Fig. 1c in main text with color scale bars added here. Light-sheet optical sectioning mouse brain vasculatures at various depths in NIR-I, NIR-IIa and NIR-IIb emission regions using the same 785 nm light sheet illumination kept constant power (0.33 mW) at different depths (also see Supplementary Video 1). The glycerol-cleared mouse brain tissue sample was prepared by intravenous injection of p-FE (emission: 850–1000 nm and 1100–1200 nm) and PEGylated PbS/CdS CSQD (emission: 1500–1700 nm) at 5-min interval. The mouse was scarified 30 min post injection of the probes while still in circulation. The mouse brain was taken out, fixed and preserved in glycerol for ex vivo imaging. Similar results for n = 3 independent experiments (C57BL/6, female, 6 weeks old). (b) Cross-sectional normalized fluorescence intensity profiles (scatters) and Gaussian fit (solid lines) along the white-dashed bars in (a) at different imaging depth (Z) in different collection windows. Similar results for n = 3 independent experiments (C57BL/6, female, 6 weeks old).

Supplementary Fig. 4 Compensation of refractive index mismatch for NIR-II LSM imaging.

As the imaging depth changes, an obvious misalignment of light sheet and the working plane of imaging objective appeared resulting from refractive index mismatch between the tissue and surrounding air and was compensated by a linear movement of detection objective. Comparison of LS imaging of vasculatures (a) before and (b) after objective compensation in glycerol-cleared mouse brain at different depths. Fluorescence signal of PEGylated PbS/CdS CSQD in vasculatures was collected in 1500–1700 nm range under an excitation of 1319 nm. n = 3 independent experiments (C57BL/6, female, 6 weeks old). (c) Compensation movement of detection objective at each depth Z determined for 850–1000 nm, 1100–1200 nm and 1500–1700 nm fluorescence imaging under the same 785 nm excitation. The centre values are mean and error bars representing standard deviation were derived from n = 3 independent experiments (C57BL/6, female, 6 weeks old). The mouse brain was prepared by sequential injection of p-FE (emission: 850–1000 nm and 1100–1200 nm) and PEGylated PbS/CdS CSQD (emission: 1500–1700 nm) at 5-min interval. Then the mouse was scarified at 30 min post injection of the probes and mouse brain was taken out, fixed and preserved in glycerol for ex vivo imaging. A 10× (NA = 0.25) imaging objective and a 5× (NA = 0.15) illumination objective were used in these experiments.

Supplementary Fig. 5 Overlaying different fluorescence-emission channels with objective compensation for NIR-II LSM.

With samples like in Supplementary Fig. 4, three-channel brain vasculature images were recorded by scanning over the same volume of a glycerol-cleared mouse brain tissue sample three times during which fluorescence signal in 850–1000 nm (p-FE), 1100–1200 nm (p-FE) or 1500–1700 nm (PEGylated PbS/CdS CSQD) was recorded under the same 785 nm LS illumination kept constant power (0.33 mW) at different depths. n = 3 independent experiments (C57BL/6, female, 6 weeks old). A 10× detection objective and a 5× illumination objective were used. The overlay images at various depths show accurate alignment of the scanning positions during successive volumetric imaging. To prepare the brain sample, we sequentially injected p-FE and PEGylated PbS/CdS CSQD intravenously into a mouse at an interval of 5 min, sacrificed the mouse at 30 min post injection, fixed the brain and preserved it in glycerol for ex vivo LSM imaging. Scale bars, 100 μm.

Supplementary Fig. 6 High-resolution 3D imaging of vasculatures in glycerol-cleared mouse brain.

(a) Images of fluorescence signal collected in three different spectral windows excited by the same 785 nm laser. A 50× (NA = 0.6) imaging objective was used. (b) FWHM of smallest blood vessels at different depths observed using a 50× (NA = 0.6) imaging objective and a 10× (NA = 0.25) illumination objective. The measurement of FWHM at different depths shows that the smallest vasculatures imaged are in the 2.5–10 μm range, increasing at deeper depth. The centre values are mean and error bars representing standard deviation were derived from analyzing ~ 5 target data at every depth. The resolution is sufficient for imaging single cells up to ~ 2 mm brain depth. (c) 3D rendering of p-FE labelled vasculatures in mouse brain excited by a 785-nm laser and collected in 1100–1200-nm window. The scanning Z incensement was 1 μm. A 50× (NA = 0.6) imaging objective and a 10× (NA = 0.25) illumination objective were used. (d) Selected X-Z cross section of (c). (a,c,d) n = 2 independent experiments (C57BL/6, female, 6 weeks old).The mouse brain was prepared by the way used in Supplementary Fig. 5.

Supplementary Fig. 7 Imaging 658-nm, 785-nm and 1319-nm light-sheet propagation in glycerol to detect light-sheet shape.

(a) Light sheets formed by three values of NA imaged in glycerol containing uniformly suspended PEGylated PbS/CdS CSQD. The emission was collected in 1500–1700-nm window. w is the waist and b is the double Rayleigh range of light sheet. In this experiment, we rotated the cylindrical lens by 90o and used mechanical slits to control the actual NA and the spanning range along Y (Fig. 1a). By so doing, the illumination plane was also rotated by 90o and the light sheet shape can be recorded in a side view as shown in (a). The light sheet was generated by a 5× illumination objective and observed using a 4× detection objective. n = 3 independent experiments. (b, c) Comparison of experimentally measured light sheet characteristics and theoretically estimated results considering the influence from the performance of the real imaging system, i.e., the convolution of theoretical estimation (w = 2λ/πNA and b = 2πw2/λ) and the point spread function (estimated by Rayleigh criteria, 0.61λ/NA). The centre values are mean and error bars representing standard deviation were derived from n = 3 independent experiments.

Supplementary Fig. 8 Light-sheet propagation in water and scattering intralipid solutions with different intralipid concentrations.

The LS illumination was rotated by the way as described for Supplementary Fig. 7. (a) Experimental results showing 658 nm, 785 nm and 1319 nm light sheets in water, 1.25% intralipid, 2.50% intralipid and 5.00% intralipid containing PEGylated PbS/CdS CSQD. A 5× illumination objective with actual NA = 0.039 and a 4× detection objective were used. The fluorescence signal was collected in 1500–1700 nm window. Light sheet of each wavelength was firstly observed in water containing PEGylated PbS/CdS CSQD. Then water was replaced by intralipid solutions with different concentrations under the same experimental conditions. (b) Monte Carlo simulations33 of light sheet under experimental conditions in (a) and using parameters listed in Supplementary Table 1. The illumination waist measured in water was inputted as initial FWHM of incident light in Monte Carlo simulations. These simulated results were consistent with the experimental observations and demonstrated the optical scattering plays a dominant role for light sheet microscopy in a scattering tissue. Similar results for n = 3 independent experiments.

Supplementary Fig. 9 Comparison of experimentally measured and Monte Carlo-simulated light-sheet FWHM and intensity decay along the incident direction in three intralipid solutions.

(a-i) FWHM and (j-l) normalized intensity in (a-c) 1.25%, (d-f) 2.5% and (g-i) 5% intralipid solutions from experiment in Supplementary Fig. 8a and simulation in Supplementary Fig. 8b by Monte Carlo method33 using parameters summarized in Supplementary Table 1. w0 is the light-sheet waist at initial incident position. Generally, the length over which the light sheet transmits by less than 1.414 times the initial waist (w0) is regarded as the distance useful for imaging55. Given this, (a-i) shows the critical length of the 1319-nm excitation was larger than 1000 μm in 1.25, 2.50 and 5.00% intralipid solutions, much larger than that of 658-nm and 785-nm cases. (j-l) As the intralipid concentration increased, the intensity along propagation direction attenuated faster but the 1319-nm excitation decayed the slowest compared to 658-nm and 785-nm excitations. (a-l) Similar results for n = 3 independent experiments.

Supplementary Fig. 10 Comparison of light-sheet propagating in 2.5% intralipid and glycerol-cleared mouse brain.

Monte Carlo simulation of light sheets of different wavelength (658 nm, 785 nm and 1319 nm) propagating in (a) 2.5% intralipid and (b) brain. (c) Experimentally observed light sheet in mouse brain with vasculatures labelled by PEGylated PbS/CdS CSQD (emission: 1500–1700 nm). The cylindrical lens in the illumination arm was rotated by 90o and a mechanical slit (Supplementary Fig. 1a) was used to control the actual NA = 0.039 as described in Supplementary Fig. 7. A 4× detection objective and a 5× illumination objective were used. Simulated (d) FWHM and (e) intensity decaying of light sheet along the propagation direction when light sheet incidents into 2.5% intralipid and brain corresponding to (a) and (b) respectively. As the brain tissue exhibited larger anisotropy than intralipid, light sheet transmitted longer in the brain than in intralipid. (f-h) Comparison of experimental data in (c) and simulated data (b) of light-sheet FWHM in mouse brain. w0 is the light-sheet waist at initial incident position. The incident photons deviate from initial direction due to scattering and it is more serious for illumination with shorter wavelength. The simulated light propagation in brain was consistent with experimental results of 658-nm, 785-nm and 1319-nm excitations (f-h). Under 1.414w0 definition of uniform light sheet55, the critical distances for uniform illumination were ~ 210 μm, ~ 320 μm and ~ 1000 μm for excitations of 658 nm, 785 nm and 1319 nm in mouse brain, respectively (f-h). The mouse brain tissue was prepared by injection of PEGylated PbS/CdS CSQD intravenously into a mouse, then we sacrificed the mouse at 30 min post injection, fixed the brain and preserved it in glycerol for further ex vivo observations. (a-h) Similar results for n = 3 independent experiments.

Supplementary Fig. 11 Effects of the excitation light-sheet wavelength to optical sectioning along the depth Z direction by NIR-II LSM.

Analysis is for the same data as in Fig. 2e of main text. (a, f) X-Z and Y-Z cross sectional images of vasculatures reconstructed from X-Y images at various depth Z. The scanning step in Z was 5 μm. The fluorescence emission of PEGylated PbS/CdS CSQD in vasculatures was collected in 1500–1700-nm spectral window using a 10×, 0.25-NA detection objective and excitation was generated by a 5×, 0.15-NA illumination objective. (b) and (c) are zoomed areas marked in (a). (d,e) Cross-sectional normalized intensity profiles of the arrow-marked structures shown in (b,c). These data show that feature smearing along Z is lower for longer wavelength light due to reduced scattering of light sheet in Z during propagation in glycerol-cleared brain tissue. The same results were also observed at different imaging depth as shown in (f,g-j). (a-j) Similar results for n = 3 independent experiments (C57BL/6, female, 6 weeks old). Scale bars, 100 μm (a,f), 20 μm (b,c).

Supplementary Fig. 12 Volumetric 1500- to 1700-nm fluorescence imaging of glycerol-cleared mouse brain sectioned by a 1319-nm light sheet.

(a) 3D rendering of PEGylated PbS/CdS CSQD labelled vasculatures in mouse brain. 1500–1700 nm fluorescence was collected at 1319 nm excitation by a 10× detection objective. A 5× illumination objective (with an effective NA = ~ 0.051) was used to generate LS excitation (also see Supplementary Video 4). The scanning increment along Z was 3 μm. The excitation power (~ 1.4 mW) and the exposure time (0.8 s) were kept constant during entire sectioning. (b) Maximum-intensity Y-projection (50 μm in thickness along Y, the maximum-intensity Y-projection took the brightest pixel in X-Z layers through 50-μm Y distance and displayed the maximum intensity values in the final 2D X-Z image) and (c) maximum-intensity Z-projections for a 150 μm-thick volume along Z at Z = 0 μm, 1000 μm and 2000 μm, respectively. (d) 3D rendering of a smaller region in (a). (e) Maximum fluorescence intensity (I) in different emission regions detected at various depths (Z) in the mouse brain. I0 is the fluorescence intensity at Z = 0 μm. In these experiments, the brain was taken out from a mouse intravenously injected with p-FE (excitation: 785 nm, emission: 850–1000 nm and 1100–1200 nm) and PEGylated PbS/CdS CSQD (excitation: 785 nm; emission: 1500–1700 nm) with 5-min interval at 30 min post injection. Then the mouse brain was fixed and preserved in glycerol for ex vivo imaging. (a-e) Similar results for n = 3 independent experiments (C57BL/6, female, 6 weeks old). Scale bars, 100 μm (b,c).

Supplementary Fig. 13 Wide-field imaging of xenograft MC38 tumors expressing immune checkpoint protein PD-L1 on mice injected with anti-PD-L1-TT dye conjugate or renal-excretable free TT dye.

(a) White-light photograph showing a subcutaneous xenograft MC38 tumor near the hindlimb of a C57BL/6 mouse. Anti-PD-L1-TT or free TT dye was injected into a mouse intravenously and remained circulation for 24 h. 24 h post injection, PEGylated PbS/CdS CSQD was injected and imaged 30 min after injection using a wide-field setup in two channels. (b) A control mouse was intravenously injected with free TT dye (red color, emission: 1000–1200 nm, excitation: 808 nm) without conjugation to any anti-PD-L1. 24 h later, it was injected with PEGylated PbS/CdS CSQD and then imaged in the TT dye and the CSQD channels. CSQDs were still circulating in the blood to give the vasculature images (green channel, 1500–1700 nm fluorescence). However, signal of free TT dye injected 24 h earlier was too weak to be imaged in the tumor (in the region with dashed circle) due to renal excretion. (c) In contrast, much brighter dye signals (red color, emission: 1000–1200 nm, excitation: 808 nm) were observed in a MC38 tumor injected with anti-PD-L1-TT dye due to specific targeting of PD-L1 in the tumor, together with the vasculatures labeled by CSQDs in the 1500–1700 nm green channel. (d) A magnified image of the tumor shown in (c) taken by another pair of achromatic lenses with larger magnification. Unlike LSM, wide-field imaging only provided 2D projected signals and lacked spatial resolution to resolve anti-PD-L1-TT distributions in tumors. (e) In vivo two-color 3D light sheet microscopy of anti-PD-L1-TT (red color, excitation: 785 nm, emission: 1000–1200 nm, exposure: 0.8 s) and vasculatures (green color, PEGylated PbS/CdS CSQD, excitation: 1319 nm, emission: 1500–1700 nm, exposure: 0.8 s) in a MC38 tumor using a 10× detection objective and a 5× illumination objective. The Z scanning increment was 3 μm. The discrete red spots were down to 6 x 6 x 15 μm3 in size, corresponding to PD-L1 expressing cells inside the tumor. No such spots were observed in tumor injected with TT dye without any anti-PD-L1 conjugated (Supplementary Fig. 14a). (a-e) Similar results for n = 3 independent experiments (C57BL/6, female, 6 weeks old).

Supplementary Fig. 14 In vivo NIR-II LSM of tumors and resolution calibration of NIR-II LSM for in vivo imaging.

(a) In vivo 3D light sheet microscopy of free TT (red color, excitation: 785 nm, emission: 1000–1200 nm) and vasculatures (green color, PEGylated PbS/CdS CSQD, excitation: 1319 nm, emission: 1500–1700 nm) in a MC38 tumor before wide-field imaging in Supplementary Fig. 13b. The signal of remained free TT in the tumor was too weak to be observed due to renal excretion without specific binding to the tumor. (b-e) In vivo optical sectioning (green for PEGylated PbS/CdS CSQD labelled vasculatures, red for anti-PD-L1-TT) by LSM of a MC38 tumor (50× detection objective) injected with anti-PD-L1-TT dye conjugate and PbS/CdS CSQD injected 24 h after the injection of anti-PD-L1-TT. Bright discrete red spots corresponding binding of anti-PD-L1-TT dye to PD-L1 expressing cells in the M38 tumor. FWHMs in X-Y and X-Z planes were measured for resolution estimation. (b,d) The discrete anti-PD-L1-TT dye labelled features inside tumors show sub-6-μm FWHM in the lateral X-Y plane and sub-15-μm FWHM in Z under 785 nm excitation and 1000–1200 nm collection, suggesting cellular scale molecular imaging of PD-L1 in vivo. (c,e) Meanwhile, the fluorescence signal of PEGylated PbS/CdS CSQD circulating in the vasculatures shows sub-5 μm x 5 μm x 10 μm volumetric resolution (FWHM) under 1319 nm excitation and 1500–1700 nm collection. (a-e) Similar results for n = 2 independent experiments (C57BL/6, female, 6 weeks old). Scale bars, 200 μm (a); 20 μm (b-e).

Supplementary Fig. 15 Resolution calibration of NIR-II LSM for non-invasive mouse head imaging shown in Fig. 4a.

Cross-sectional normalized fluorescence intensity profiles of the smallest vessels (scatters) and Gaussian fit (solid lines) (a) at X-Y plane and (b) along Z direction at different imaging depth. In this experiment, the mouse was intravenously injected with PEGylated PbS/CdS CSQD (excitation: 1319 nm; emission: 1500–1700 nm) and imaged by an oblique NIR-II LSM shown in Fig. 3e. (a,b) Similar imaging were performed at 3 positions of mouse head in each of 2 mice (BALB/c, female, 4 weeks old), for a total n = 6. A 5× illumination objective and a 10× detection objective were used and the scanning increment was 4 μm along the X direction.

Supplementary information

Supplementary Information

Supplementary Figures 1–15, Supplementary Tables 1–3

Reporting Summary

Supplementary Video 1

The influence of emission wavelengths on NIR LSM. An animation constructed with images taken at various depth z in glycerol-cleared mouse brain tissue by NIR LSM under the same 785-nm excitation while detecting emissions in NIR-I (850–1,000 nm, p-FE), NIR-IIa (1,100–1,200 nm, p-FE) and NIR-IIb (1,500–1,700 nm, PEGylated PbS/CdS CSQD probes) (as shown in Fig. 1c). With a 785-nm light sheet, we observed that the brain tissue imaging depth limit increased, background signal decreased and SBR increased at longer detection wavelength from 850–1,000 nm to 1,100–1,200 nm and 1,500–1,700 nm. The imaging depth limit (defined as the tissue depth at which SBR decreased to ~2) increased from zSBR=2 ~1.0 mm to ~2.0 mm and ~2.5 mm as emission wavelength increased from ~850 nm to ~1,100 nm and ~1,700 nm. Similar results for n = 3 independent experiments (C57BL/6, female, 6 weeks old).

Supplementary Video 2

The influence of excitation wavelengths on NIR LSM (xy plane). An animation constructed with NIR-II light-sheet images in the xy plane taken at various depth z in glycerol-cleared mouse brain tissue by detecting 1,500- to 1,700-nm emission of PbS/CdS core-shell quantum dots in vessels under various excitations using 658-nm, 785-nm and 1,319-nm light sheets respectively. Such imaging utilized an important property of quantum dots, i.e., ultra-wide excitation ranges (UV to >1,300 nm; Supplementary Fig. 2). In the recorded FOV, these three light sheets could still allow imaging of small vessels (FWHM < 10 μm). Suppressed scattering of longer-wavelength light sheets was gleaned from the xy images taken at various z, with improved SBR and reduced FWHM of feature sizes especially at deeper depths. Similar results for n = 3 independent experiments (C57BL/6, female, 6 weeks old).

Supplementary Video 3

The influence of excitation wavelengths on NIR LSM (xz plane). An animation of NIR-II light-sheet images in the xz plane (y in the range of 0–640 µm with 5-µm increments) reconstructed from xy images at various depth z in glycerol-cleared mouse brain tissue by detecting 1,500- to 1,700-nm emission (PbS/CdS core-shell quantum dots) under various excitations using 658-nm, 785-nm and 1,319-nm light sheets respectively. Suppressed scattering of longer-wavelength light sheets was gleaned from xz cross-sectional images, with reduced FWHM of feature sizes along the depth z direction, corresponding to higher vertical resolution and better sectioning capability along z. Similar results for n = 3 independent experiments (C57BL/6, female, 6 weeks old).

Supplementary Video 4

Volumetric 1,500- to 1,700-nm fluorescence imaging of glycerol-cleared mouse brain sectioned by a 1,319-nm light sheet. Animated mouse brain video from ex vivo NIR-II LSM images of vasculatures in glycerol-cleared mouse brain. The volumetric imaging was done using an unusually long 1,319-nm excitation and 1,500- to 1,700-nm detection for PbS/CdS core-shell quantum dots in brain vasculatures. The video here shows reconstructed 3D images of brain tissue with various tissue volumes as indicated taken with 3-μm z increment in depth. The excitation power (~ 1.4 mW) and the exposure time (0.8 s) were kept constant during entire sectioning. A 10× detection objective and a 5× illumination objective were used. LSM with both excitation and emission in the 1,000- to 1,700-nm NIR-II window minimized scattering and maximized the resolution, penetration depth and FOV. Similar results for n = 3 independent experiments (C57BL/6, female, 6 weeks old).

Supplementary Video 5

Non-invasive NIR-II LSM of abnormal blood flow in tumor microcirculation. In vivo time-course LSM imaging of blood perfusion into tumor vasculatures by recording the p-FE nanofluorophore (200 μl, OD = 4 at 808 nm, 785-nm excitation, 1,000- to 1,200-nm detection) signals immediately following intravenous injection into the mouse tail vein. Through imaging, the light sheet was sectioned through the tumor at a fixed plane below the top of the xenograft MC38 tumor (~ 8 mm in diameter) at a depth of z ≈ 300 μm, and imaging was recorded through the same plane at ~ 1 fps. The light-sheet illumination position z was controlled by a 3D motorized translation stage. A 4× detection objective and a 5× illumination objective were used. In this video, blood flows in tumor vasculatures were found to be irregular and intermittent with turning-on and shutting-off behavior, oscillatory/fluctuating flowing patterns and even flow direction reversal in the same vasculature in a tumor. Similar results for n = 3 independent experiments (C57BL/6, female, 6 weeks old).

Supplementary Video 6

Non-invasive NIR-II LSM of abnormal T cell motion in tumor microcirculation.: Noninvasive time-course recording of PD-1-expressing cells in CT26 tumor microcirculation 2 h after intravenous injection of anti-PD-1-PEGylated PbS/CdS CSQD (excitation, 1,319 nm; emission, 1,500–1,700 nm) and 26 h post injection of anti-PD-L1-Er. An oblique light-sheet microscope was used in this observation, in which an illumination and a detection objective were arranged 45° to the samples (Supplementary Fig.1b). The direction of movement of PD-1+ cells changed with the blood flow direction in the tumor microcirculation. Similar results for n = 2 independent experiments (BALB/c, female, 6 weeks old). The exposure time was 40 ms and the frame rate was 20 fps. A 10× detection objective and a 5× illumination objective were used.

Supplementary Video 7

Non-invasive three-color oblique LSM of a CT26 xenograft tumor on a BALB/c mouse. Anti-PD-L1 ErNP molecules were injected intravenously and 24 h later, anti-PD-1 PEGylated PbS/CdS CSQD was injected. In another 29 h, p-FE was injected intravenously for labeling of tumor vessels and three-color LSM imaging of anti-PD-L1 ErNP (magenta), anti-PD-1 PEGylated PbS/CdS CSQD (green) and p-FE (blue) were performed for molecular imaging of PD-L1- and PD-1-expressing cells and vasculatures in tumor. Discrete features labeled by anti-PD-L1 ErNP and anti-PD-1 PEGylated PbS/CdS CSQD dyes labelled features inside tumors were resolved with cellular resolution. Similar results for n = 2 independent experiments (BALB/c, female, 6 weeks old). A 10× detection objective and a 5× illumination objective were used. The lateral increment was 4 μm along the x direction.

Supplementary Video 8

Non-invasive 3D time-course NIR-II light-sheet imaging of meningeal macrophages and microglia dynamics. The recruitment of macrophages/microglia was monitored following brain injury 24 h after intravenous injection of anti-CD11b PEGylated PbS/CdS CSQD (excitation, 1,319 nm; emission, 1,500–1,700 nm) at the boundary of the TBI region. The anti-CD11b PEGylated PbS/CdS CSQD was injected 2 h after injury. The meningeal macrophages and microglia migrated to the injury from the surrounding area. Similar results for n = 2 independent experiments (BALB/c, female, 4 weeks old). The exposure time was 100 ms and the scanning increment was 4 μm along the x direction. A 10× detection objective and a 5× illumination objective were used.

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Wang, F., Wan, H., Ma, Z. et al. Light-sheet microscopy in the near-infrared II window. Nat Methods 16, 545–552 (2019). https://doi.org/10.1038/s41592-019-0398-7

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