Intravital microscopy in historic and contemporary immunology

Abstract

In this review, we discuss intravital microscopy of immune cells, starting from its historic origins to current applications in diverse organs. It is clear from a quantitative review of the literature that intravital microscopy is a key tool in both historic and contemporary immunological research, providing unique advances in our understanding of immune responses. We have chosen to focus this review on how intravital microscopy methodologies are used to image specific organs or systems and we present recent descriptions of fundamental immunological processes that could not have been achieved by other methods. The following target organs/systems are discussed in more detail: cremaster muscle, skin (ear and dorsal skin fold chamber), lymph node, liver, lung, mesenteric vessels, carotid artery, bone marrow, brain, spleen, foetus and lastly vessels of the knee joint.

Figure 1. Overview of the application and importance of Intravital Microscopy.

A. Using the search terms ‘intravital imaging’ or ‘intravital microscopy’ and not ‘in vitro maturation’ in the title or abstract on Pubmed a total of 3766 articles were found. B. To assess the contribution of studies using this technique, the journal impact factor (IF) of IVM articles from the last 5 years is shown. The 5 year IF of journals as published by Reuters Web of Science website was used for analysis. Newer journals (e.g. Intravital, eLife) that do not have a published IF were excluded from the analysis. The proportion of immunological studies using IVM is shown in C. The search terms ‘immunology’, ‘immunity’, ‘immune’, ‘myeloid’, ‘leukocyte’ or ‘lymphoid’ were used to distinguish immunological studies.

Figure 2. Immunological studies using IVM from the last 5 years.

Ranking based on the top 10 journals in Immunology as stated in the Scimago Journal and Country Rank (Scopus). The percentage of IVM studies from 2010-2016 (review articles were excluded from the analysis) in different organs is shown in A. The main cell type relevant to each study is plotted in B. Which imaging method was applied in the various immunological studies is shown in C. In articles where IVM was performed in more than one organ or cell type, each was counted individually. When more than one imaging method was applied each was counted individually.

Figure 3. Lung Intravital microscopy

A. Imaging set-up for IVM on the murine lung as described by Looney et al (2012). Gentle suction is applied via the vacuum port to stabilize the tissue. The anaesthetised and mechanically ventilated mouse is placed on a heat-mat in a right lateral position. Images are acquired using a long working distance water immersion objective. Comparison between precision cut lung slices (B.) and live lung imaging (C.). Live lung imaging can detect distinct changes in cell morphology in vivo (D.1-3). B.-D. PE conjugated Ly6G-Ab (1A8; red, D. 1) and Isolectin B4-Alexa488 (cyan, D. 2) were injected intravenously to label neutrophils and the lung vasculature respectively. D. 3 = pseudocolour merged image.

Figure 4. IVM of hematopoietic cells in the bone marrow.

A. Maximum projection of a z-stack showing two HSCs labelled with DiD (red) observed in the proximity of osteoblastic cells (green) one day after transplantation into irradiated Col2.3GFP transgenic osteoblast reporter recipient mice. B. 3D rendering of a 4D time-lapse image of a T-ALL cell (represented by the red dot) migrating within the bone marrow space. Grey: vasculature highlighted by Cy5-labelled dextran; green: col 2.3GFP+ osteoblastic cells. The colored line shows the track of the cell during the three hour-long imaging period. The white arrow points at the position where the cell underwent mitosis and the two asterisks indicate the position of the two daughter cells at the end of the recording (reproduced from Hawkins et al. 2016 under Creative Commons).