# Protocol to analyze marrow B lineage cell dynamics by in vivo three-photon microscopy in intact mouse tibia

## TL;DR

This paper provides a detailed protocol for using three-photon microscopy to study B cell dynamics in mouse tibia marrow, enabling deeper imaging than traditional methods.

## Contribution

A new protocol for 3D time-lapse imaging of B lymphocytes in intact mouse tibia marrow using 3PM at 1,650 nm.

## Key findings

- Steps for verifying 3PM performance and optimizing deep-marrow imaging are described.
- Machine-learning-based denoising improves cell segmentation and tracking accuracy.
- The protocol can be applied to track immune cells in organs inaccessible to two-photon microscopy.

## Abstract

Three-photon microscopy (3PM) allows deep-tissue imaging beyond the capabilities of two-photon microscopy (2PM) owing to infrared excitation. Here, we present a protocol for time-lapse 3D imaging of B lymphocytes in the tibia marrow of fluorescent reporter mice using 3PM at 1,650 nm. We describe steps for verifying microscope performance and tibia imaging. We then detail the cell dynamics analysis, including denoising, cell segmentation, and tracking. This protocol has potential application for immune cell tracking in other optically inaccessible organs in which 2PM fails.

For complete details on the use and execution of this protocol, please refer to Rakhymzhan et al.1

•Guidance on three-photon microscopy performance verification at 1,650 nm•Steps for optimized time-lapse three-photon deep-marrow imaging in intact tibia•Procedure for marrow B lymphocyte dynamics analysis in unperturbed marrow•Instructions on machine-learning-based denoising for cell segmentation and tracking

Guidance on three-photon microscopy performance verification at 1,650 nm

Steps for optimized time-lapse three-photon deep-marrow imaging in intact tibia

Procedure for marrow B lymphocyte dynamics analysis in unperturbed marrow

Instructions on machine-learning-based denoising for cell segmentation and tracking

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Three-photon microscopy (3PM) allows deep-tissue imaging beyond the capabilities of two-photon microscopy (2PM) owing to infrared excitation. Here, we present a protocol for time-lapse 3D imaging of B lymphocytes in the tibia marrow of fluorescent reporter mice using 3PM at 1,650 nm. We describe steps for verifying microscope performance and tibia imaging. We then detail the cell dynamics analysis, including denoising, cell segmentation, and tracking. This protocol has potential application for immune cell tracking in other optically inaccessible organs in which 2PM fails.