Background

Affinity maturation, the process wherein the affinity of serum antibodies towards a specific antigen increases over time, is achieved by selection of B cells bearing high affinity BCRs within germinal centers (GCs). Increase in antibody affinity is mediated through iterative cycles of somatic hyper mutation and affinity-based selection, a process which is orchestrated by T follicular helper (Tfh) cells (Kepler and Perelson, 1993; Oprea and Perelson, 1997; Victora and Nussenzweig, 2012). The GC is comprised of two microanatomical sites; the dark zone, where B cells proliferate and acquire somatic hypermutations, and the light zone, where B cells interact with cognate antigen and T cells. Although intravital imaging techniques were able to define immune cell dynamics in GCs (Allen et al., 2007; Hauser et al., 2007; Schwickert et al., 2007), transition between the GC zones remained in question. This problem was later solved by the generation of mice expressing photoactivatable GFP (PA-GFP) (Victora et al., 2010).

PA-GFP is a GFP variant whose peak excitation wavelength shifts from 415 nm (inactive PA-GFP) to 495 nm (active PA-GFP) upon two-photon irradiation at 830 nm. The non-activated PA-GFP is fluorescent as well, and this property can be used to distinguish between the photoactivated area and the total cells (Patterson and Lippincott-Schwartz, 2002).

The use of mice expressing PA-GFP provided direct evidence for interzonal migration in the GC and for the definition of the major GC exit zone (Victora et al., 2010; Stoler-Barak et al., 2019). Combination of intravital two-photon laser scanning microscopy with in situ photoactivation, allowed the microanatomical labeling of distinct niches within the germinal center, and led to the discovery that T cell help controls the movement between the two GC zones (Victora et al., 2010).

Cell-surface markers are commonly used to define a cell population in a specific niche; however, distinctive markers are not always available, generating a gap between the cellular and spatial information in the studied tissue. Furthermore, unknown cell populations that reside within a specific niche are usually hard to detect and characterize by conventional techniques. To overcome these limitations, photoactivation-based approaches have been used for unbiased identification of tissue-resident immune cells with minimal a priori knowledge of unique cell-surface marker expression. Niche-specific landmarks are often introduced into mice prior to labeling by photoactivation to define the area of interest. For example, adoptive cell transfer of fluorescently labeled B cells mark the B cell area within a tissue and can guide the selection of the region of interest for photoactivation (Medaglia et al., 2017). In the study associated with this protocol and as described here, we specifically photoactivated the subepithelial dome niche within the Peyer’s patch (Biram et al., 2019). This protocol can be adapted to other niches and additional tissues of interest.