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. 2018 May 29;9(1):2125.
doi: 10.1038/s41467-018-04470-8.

Intravital imaging by simultaneous label-free autofluorescence-multiharmonic microscopy

Sixian You  1   2 , Haohua Tu  3 , Eric J Chaney  1 , Yi Sun  1   4 , Youbo Zhao  1 , Andrew J Bower  1   4 , Yuan-Zhi Liu  1 , Marina Marjanovic  1   2 , Saurabh Sinha  5 , Yang Pu  1 , Stephen A Boppart  6   7   8   9
Affiliations

Intravital imaging by simultaneous label-free autofluorescence-multiharmonic microscopy

Sixian You et al. Nat Commun. .

Abstract

Intravital microscopy (IVM) emerged and matured as a powerful tool for elucidating pathways in biological processes. Although label-free multiphoton IVM is attractive for its non-perturbative nature, its wide application has been hindered, mostly due to the limited contrast of each imaging modality and the challenge to integrate them. Here we introduce simultaneous label-free autofluorescence-multiharmonic (SLAM) microscopy, a single-excitation source nonlinear imaging platform that uses a custom-designed excitation window at 1110 nm and shaped ultrafast pulses at 10 MHz to enable fast (2-orders-of-magnitude improvement), simultaneous, and efficient acquisition of autofluorescence (FAD and NADH) and second/third harmonic generation from a wide array of cellular and extracellular components (e.g., tumor cells, immune cells, vesicles, and vessels) in living tissue using only 14 mW for extended time-lapse investigations. Our work demonstrates the versatility and efficiency of SLAM microscopy for tracking cellular events in vivo, and is a major enabling advance in label-free IVM.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Schematic of the SLAM microscopy platform. The high peak-power laser pulses were sent into the PCF to generate a supercontinuum. The pulse shaper is programmed to choose the excitation window (1080–1140 nm) and compensate the dispersion to make the output beam near-transform-limited. Different dichroic mirrors and optical filters are used in the detection system to collect spectrally resolved multimodal multiphoton signals by photomultipliers as specified in the table. DM dichroic mirror, HWP half wave plate, M mirror, OBJ objective, PCF photonic crystal fiber, PM parabolic mirror, PS polarizer splitter
Fig. 2
Fig. 2
Tumor microenvironment of a living rat by SLAM microscopy. Pseudo-color presentation in the overlay image (consistent throughout this paper): green—SHG (collagen fibers); magenta—THG (interface); yellow—2PAF (FAD); cyan—3PAF (NADH). a Full view of a large field (1.5 × 1.5 mm2) at the tumor boundary, with different regions of interest (white dashed squares) magnified in b–e. Tumor cell clusters are highlighted with yellow boundaries and vasculatures with red boundaries. b 10-micron tumor cells (yellow arrows) with co-localized 2PAF (yellow, FAD) and THG (magenta, interface) signals. c Larger (>20-micron) cells (white arrows) with strong 3PAF (cyan, NADH) signals, which are likely to be macrophages due to their irregular cell shape and oval-shaped nucleus. d, e Vascular endothelial cells (red arrows) and adipocytes with THG-strong boundaries and NADH-strong content inside (white dashed arrows). f–h Images acquired from different depths at the center of a, with f displaying a hollow vessel composed of a layer of endothelial cells but no signs of red blood cells and h exhibiting a mature vessel filled with red blood cells. Contrast in h was intentionally brightened to show blood cells in deeper layers. Raw images can be found in Supplementary Fig. 4. Scale bar: 100 µm
Fig. 3
Fig. 3
Two modes of leukocyte arrest captured by SLAM microscopy. a Multi-nucleated neutrophil slow rolling along the vessel wall at an instantaneous velocity of 1.06 µm s−1. b Immediate arrest of a leukocyte with a sudden halt in the movement, followed by adhesion and crawling. Scale bar: 50 µm
Fig. 4
Fig. 4
Leukocyte-swarming visualized and characterized by SLAM microscopy. a, b Images acquired at the beginning and the end of the swarming, respectively. Collagen rearrangement was marked by the white boundary (Cluster 1, C1) while lipid interaction was marked by cyan boundary (Cluster 2, C2). c–e Zoomed-in images of multi-nucleated neutrophils. f Traces of Cells 1–14, which were tracked to travel via similar routes to the same cluster at different time points, as shown in the velocity map j. g Traces of Cells 15–16, which were both tracked for at least 30 min and exhibited different behavior, with Cell 15 migrating towards the cluster with high speed and high directionality throughout the entire time course and Cell 11 mostly making random walk, as shown in the corresponding velocity map k. h Leukocytes leaving the site (Cells 17–18 in Supplementary Movie 5). The series of snapshots were taken every 2 s and shown for every 20 s. The first three snapshots showed the deformation of the leukocyte, changing from a round shape to a stretched cell elongated along the direction of travel, which typically precedes the acceleration process shown in the last three snapshots. i Quantification of collagen clearance, cell accumulation, and lipid deformation within the marked clusters in a and b (C1 and C2). j, k Velocity and directionality map of Cells 1–16. The color of the curves matches the color of the traces in f and g and Supplementary Movie 5. D directionality. Scale bar: 50 µm
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