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Intravital three-photon microscopy allows visualization over the entire depth of mouse lymph nodes

Abstract

Intravital confocal microscopy and two-photon microscopy are powerful tools to explore the dynamic behavior of immune cells in mouse lymph nodes (LNs), with penetration depth of ~100 and ~300 μm, respectively. Here, we used intravital three-photon microscopy to visualize the popliteal LN through its entire depth (600–900 μm). We determined the laser average power and pulse energy that caused measurable perturbation in lymphocyte migration. Long-wavelength three-photon imaging within permissible parameters was able to image the entire LN vasculature in vivo and measure CD8+ T cells and CD4+ T cell motility in the T cell zone over the entire depth of the LN. We observed that the motility of naive CD4+ T cells in the T cell zone during lipopolysaccharide-induced inflammation was dependent on depth. As such, intravital three-photon microscopy had the potential to examine immune cell behavior in the deeper regions of the LN in vivo.

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Fig. 1: Permissible laser power and pulse energy were determined by monitoring lymphocyte velocity.
Fig. 2: In vivo 3PM of blood vessels through the entire depth of mouse popliteal LNs.
Fig. 3: Comparison of LN blood vessel imaging by 3PM and 2PM.
Fig. 4: In vivo 3PM image of the entire popliteal LN vasculature.
Fig. 5: In vivo 3PM of lymphocyte migration in deep LNs.
Fig. 6: Measurement of T cell motility across the entire depth of popliteal LNs.
Fig. 7: In vivo 3PM of T cell migration in LPS-induced inflamed LNs.
Fig. 8: In vivo 3PM of multicolor GC B cells.

Data availability

Source data of the graphs are provided with this paper. The raw images of this study are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

The MATLAB codes for tiling of multiple stacks and calculating motility coefficients can be found at https://github.com/idglbak87/Tiling-and-Calculating-velocity-and-MC.git.

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Acknowledgements

We thank members of the Xu research group (B. Li, X. Yang, F. Xia, C. Wu, N. Akbari, A. T. Mok, A. K. LaViolette and S. Zhao) and H. M. Isles (Weill Cornell Medicine) for their help with valuable discussions. We thank N. Nishimura (Cornell University) and C.-Y. Eom (Cornell University) for providing the facility for performing lymphocyte isolation and cryosection. We thank K. A. Strednak and the Cornell Center for Animal Resources and Education for their animal care service. This research was supported from Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2019R1A6A3A03033817 to K.C.), NSF DBI-1707312 Cornell NeuroNex Hub (to C.X.), National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases 5R01AI132738–05 (to A.S. and A.M.M.), NIH/National Cancer Institute 1R01CA238745-01A1 (to C.X. and A.S.), the University of Rochester Program for Advanced Immune Bioimaging Pilot (NIH/National Institute of Allergy and Infectious Diseases P01AI102851 to C.X.) and the University of Rochester School of Medicine and Dentistry.

Author information

Authors and Affiliations

Authors

Contributions

K.C. and C.X. conceived the study. K.C. designed, performed the experiments and analyzed the results. K.C., Y.H. and D.G.O. built and maintained the imaging system. T.W. discussed data analysis. E.H. wrote MATLAB codes for calculating lymphocyte motility. K.L., A.S., W.B. and A.M.M. provided Cγ1Cre-Confetti mice with immunization. C.X. supervised the research. K.C. and C.X. wrote the manuscript.

Corresponding authors

Correspondence to Kibaek Choe or Chris Xu.

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The authors declare no competing interests.

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Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Ioana Visan was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Effect of LN temperature on lymphocyte velocity.

a, Comparison of lymphocyte velocities at a low LN temperature of ~28 °C and at a normal LN temperature of ~36.5 °C. b, Changes in lymphocyte velocity when the LN temperature is increased by 1 °C from 35.5 °C to 39.5 °C. ns, not significant; Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Representative of two independent experiments. a-b, eGFP+ lymphocyte velocities at ~300 μm depth in popliteal LN were measured by acquiring a volume (202x202x35 μm3) every 30 seconds for 10 minutes with 1300 nm 3PE at each temperature. The maximum power on the LNs was below 2.7 mW. Each data point indicates an individual lymphocyte track; n = 30 tracks for each condition; the median with the interquartile range.

Source data

Extended Data Fig. 2 Lymphocyte velocity at 600 μm (a) and 300 μm depth (b) with increasing average power of 1300 nm excitation.

a-b, eGFP+ lymphocytes were imaged at the same site with 4 different average powers by 3PE at 1300 nm. The average power (Power) at surface is proportional to the pulse repetition rate (PRR) while the pulse energy (Pulse E) at focus remains approximately constant. For each depth, four LNs from 3 mice were imaged. The exact range of imaging depths around the nominal imaging depths of 600 μm (a) and 300 μm (b) were 590 μm to 625 μm and 290 μm to 325 μm, respectively. The average power increases with the depth from top (Z1) to bottom (Z2) of the imaging volume. Effective attenuation length (EAL) was calculated by taking 4 images at different depths (150, 300, 450, 600 μm depths for a, 50, 100, 200, 300 μm depths for b). Each data point indicates an individual lymphocyte track; n = 30 tracks for each condition; the median with the interquartile range; ns, not significant; Kruskal-Wallis test followed by Dunn’s multiple comparisons test.

Source data

Extended Data Fig. 3 Lymphocyte velocity at 600 μm with increasing pulse energy and average power at 1300 nm excitation.

a, Schematic of adjusting pulse energy and average power for taking the four 10-min movies sequentially. The average power is proportional to the pulse energy since the repetition rate was kept constant. b-h, eGFP+ lymphocyte velocity was measured at the same site with 4 different pulse energies (at focus) by 3PE at 1300 nm. Pulse repetition rates of 0.66 and 0.33 MHz were used for b-f and g-h, respectively. Power, average power at surface. Seven LNs from 6 mice were imaged. The exact imaging depth was from 590 μm to 625 μm. The average power increases with depth from top (Z1) to bottom (Z2) of the imaging volume. Effective attenuation length (EAL) was calculated by taking 4 images at different depths. Each data point indicates an individual lymphocyte track; n = 30 tracks (except for n = 22 tracks at 1 nJ in h); the median with the interquartile range; ns, not significant; Kruskal-Wallis test followed by Dunn’s multiple comparisons test. The image rapidly darkened within a few minutes when we applied more than 146 mW in f (Supplementary Movie 3). The velocity even at relatively low power and low pulse energy in d-f is lower than 10 μm/min because the imaging site was close to LN boundary (sub-cortical region). This observation is consistent with previous reports that the velocity of both T and B cells in subcortical region is 6–8 μm/min (ref. 30).

Source data

Extended Data Fig. 4 Lymphocyte velocity at 600 μm depth with increasing average power and pulse energy at 1650 nm excitation.

a-f, DsRed+ lymphocytes were imaged at the same site with 3 to 4 different pulse repetition rates by 3PE at 1650 nm. The average power at surface (Power) is proportional to the pulse repetition rate (PRR) while the pulse energy at focus (Pulse E) remains constant. Six LNs from 4 mice were imaged. g-i, DsRed+ lymphocytes velocity was measured at the same site with 4 different pulse energies (at focus). The average power is proportional to the pulse energy while the repetition rate was kept constant (0.33 MHz). Three LNs from 3 mice were imaged. a-i, The exact imaging depth was from 590 μm to 625 μm. The average power increases with depth from top (Z1) to bottom (Z2) of the imaging volume. Effective attenuation length (EAL) was calculated by taking 4 images at different depths. Each data point indicates an individual lymphocyte track; the number of analyzed tracks (n = 21–37) is indicated on the graphs; the median with the interquartile range; ns, not significant; Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Even though most lymphocytes stopped migration at >40mW in a,b and e (Supplementary Movie 3), the measured mean velocity is non-zero due to the uncertainties that occur when determining the cell positions at different times.

Source data

Extended Data Fig. 5 Dependence of fluorescence signal on excitation pulse energy in logarithmic scales.

a, Dependence of fluorescence signal on excitation pulse energy at 1,680 nm in logarithmic scales for Alexa Fluor 647. The slope is 3.03. The signal was measured in Alexa Fluor 647 dye solution with a pulse repetition rate of 0.33 MHz. b, Dependence of fluorescence signal on excitation pulse energy at 1300 nm in logarithmic scales for DsRed. The slope is 2.95, which is in close agreement with the calculated slope value (2.93) at ~0.7 nJ using the 2 P and 3 P cross sections of DsRed (ref. 22). These observations indicate that 3 P excitation was dominant in our imaging condition. The signal was measured at the surface of a LN in an actin-DsRed mouse with a pulse repetition rate of 2 MHz. The average power is proportional to the pulse energy.

Source data

Extended Data Fig. 6 Additional experiments for comparison of LN blood vessel imaging by 3PM and 2PM.

a, In vivo images of fluorescein+ blood vessels using 1,280 nm 3PE and 920 nm 2PE, and Alexa-Fluor-647+ blood vessels using 1280 nm 2PE and 1680 nm 3PE at the same site of the same LN. Three LNs from 3 mice were imaged. The number shown in each image is the average laser power (mW) under the objective lens. Scale bars, 50 μm. b, Signal-to-background ratios (SBRs) were measured at different depths. Each data point is the average and s.d. of SBRs measured in 3 blood vessels in one image. c, Normalized fluorescence signal intensity as a function of imaging depth measured in the same mouse as in a. The fluorescence signal strength at a particular depth is represented by the average value of the brightest 0.5% pixels in the XY image at that depth divided by the square (for 2PE) or cube (for 3PE) of the average power. The effective attenuation length (le) was the inverse of the slope divided by the order of the nonlinear process (that is, 2 for 2PE and 3 for 3PE).

Source data

Extended Data Fig. 7 In vivo 3PM of mouse spleen.

Alexa-Fluor-647+ blood vessels and THG were imaged in spleen of adult mouse by 1650 nm 3PM. A shallow region (red pulp, RP) below the spleen surface contains many THG-generating cells, possibly red blood cells or leukocytes such as monocytes and macrophages. The area of high blood leakage is likely the marginal zone (MZ) where many open-ended blood vessels exist. The area below the MZ is likely white pulp (WP). Scale bars, 50 μm. The maximum average power under the objective lens was 28 mW.

Source data

Extended Data Fig. 8 Naïve CD8+ and CD4+ T cell distribution in LNs.

a, 3D reconstruction of z-stack images (230x800x750 μm3) acquired in a popliteal LN in vivo by 3PM at 1300 nm excitation. b, Naïve eGFP+ CD8+ and DsRed+ CD4 T cell positions of a in yz coordinates. (bottom) Color-maps showing relative T cell density in each 100 × 100 μm2 square area. c, Relative T cell distribution along the z-axis in the dashed boxes in b. Each data point represents the number of cells within the volume from the indicated depth to 50 μm below. a-c, Representative of two independent experiments. d, (1st column) 2D-images were acquired in 50 μm cryosections of popliteal LNs by ex vivo 2PM. Naïve CD8+ T cell and naïve CD+4 T cell were labeled with eGFP and DsRed, respectively for LN1-2. The labeling scheme was switched for LN3, with DsRed and eGFP labeling CD8+ and CD4+ T cells, respectively. C, cortical side. M, medullary side. Scale bars, 200 μm. (2nd column) Naïve CD8+ and CD4+ T cell positions in xy coordinates. Dotted circles are the area presumed to be B cell follicles. (3rd-4th columns) Color-maps showing relative T cell density in each 100 × 100 μm2 square area.

Extended Data Fig. 9 Deep GC of popliteal LN on the imaging axis.

a, A schematic diagram of the deep GC (arrow) on the imaging axis. b, Comparison of the deep GC (~400 μm depth) imaging by 920 nm 2PM and 1300 nm 3PM. The maximum average power under the objective lens was 204 mW (80 MHz repetition rate) and 21 mW (0.33 MHz repetition rate) for 2PM and 3PM, respectively. Scale bars, 50 μm.

Supplementary information

Reporting Summary

Supplementary Video 1

eGFP+ lymphocyte migration in parenchyma with increasing average power of 1,300 nm excitation. The 30 tracks shown in parenchyma were used for analysis. Imaging volume of 202 × 202 × 35 μm3 was acquired at 590–615 μm depth in popliteal LN every 30 s. Time scale, mm:ss

Supplementary Video 2

DsRed+ lymphocyte migration in parenchyma with increasing average power of 1,650 nm excitation. The 30 tracks shown in parenchyma were used for analysis. Imaging volume of 202 × 202 × 35 μm3 was acquired at 590–615 μm depth in popliteal LN every 30 s. Dead cells (blue) labeled by propidium iodide (PI) were imaged with 0.33 MHz pulse repetition rate. Time scale, mm:ss

Supplementary Video 3

Images rapidly darken under high average power excitation. With 1,300 nm 3PM, we imaged eGFP+ lymphocytes (green), LYVE-1-eFluor615+ lymphatic sinus (red), THG (white). With 1,650 nm 3PM, we imaged DsRed+ lymphocytes (green), LYVE-1-eFluor660+ lymphatic sinus (red), THG (white). Imaging volume of 202 × 202 × 35 μm3 was acquired at 590–615 μm depth in popliteal LN every 30 s. Time scale, mm:ss

Supplementary Video 4

3D reconstruction of multiple z-stack images of fluorescein+ blood vessels in an entire LN. Eight z-stacks were first acquired to cover the entire popliteal LN with 1,280 nm 3PM. The z-stacks were repeated at three regions with low signal by increasing the excitation power. The field of view of the tile indicated by the white box is 600 × 600 μm2. The pulse repetition rate was 0.33 MHz, and the maximum average power under the objective lens was 76 mW

Supplementary Video 5

In vivo 3PM of eGFP+ lymphocyte migration in parenchyma and blood vessel at 8.9 s/volume. Imaging volume of 202 × 202 × 35 μm3 was acquired at 590–625 μm depth in popliteal LN by 1,300 nm excitation. The pulse repetition rate and average power were 0.66 MHz and 11 mW, respectively

Supplementary Video 6

In vivo 3PM of eGFP+ lymphocyte (green) circulating in the blood and crawling on the DsRed+ blood vessel wall (red) at 0.44 s/frame. Imaging frame of 202 × 202 μm2 was acquired at 500 μm depth in popliteal LN by 1,300 nm excitation. The pulse repetition rate and average power were 2 MHz and 70 mW, respectively

Supplementary Video 7

In vivo 3PM of eGFP+ lymphocyte migration in LYVE-1-eFluor615+ lymphatic sinus. Lymphocyte tracks (white lines) in the lymphatic sinuses. Imaging volume of 300 × 300 × 70 μm3 was acquired every 16.8 s at 450–520 μm depth by 1,300 nm excitation. The pulse repetition rate and average power were 0.66 MHz and 21 mW, respectively. Time scale, mm:ss

Supplementary Video 8

In vivo 3PM of naive DsRed+CD8+ T cell and naive eGFP+CD4+ T cell migration at different depths in the paracortical T cell zone of popliteal LN in steady state. Imaging volume of 202 × 202 × 35 μm3 was acquired every 30 s by 1,300 nm excitation with 0.66 MHz pulse repetition rate. Blood vessels were imaged by THG. The maximum average power under the objective lens was 25 mW. Time scale, mm:ss

Supplementary Video 9

In vivo 3PM of naive DsRed+CD8+ T cell and naïve eGFP+CD4+ T cell migration at different depths in the paracortical T cell zone of LPS-induced inflamed popliteal LN. Imaging volume of 300 × 300 × 100 μm3 was acquired every ~29 s by 1,300 nm excitation with 0.66 MHz pulse repetition rate. Blood vessels were imaged by THG. The maximum average power under the objective lens was 71 mW. Time scale, mm:ss

Supplementary Video 10

In vivo 3PM of multicolor GC B cells in shallow and deep DZs of a large GC and in the DZ of a small GC in Cγ1Cre-confetti mouse. Imaging volumes for the large GCs (202 × 202 × 52 μm3) and small GCs (202 × 202 × 39 μm3) were acquired every ~16 s by 1,300 nm excitation with 0.66 MHz pulse repetition rate. Time scale, mm:ss. The maximum average power under the objective lens was 10 mW

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Choe, K., Hontani, Y., Wang, T. et al. Intravital three-photon microscopy allows visualization over the entire depth of mouse lymph nodes. Nat Immunol 23, 330–340 (2022). https://doi.org/10.1038/s41590-021-01101-1

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