Intravital Imaging of Attenuated Auxotrophic Mycobacterium tuberculosis in Murine Lung during Early Intravascular Infection

Summary

Here, we present a protocol for intravital imaging of an attenuated BSL-2-approved Mycobacterium tuberculosis. strain in the murine lung using a permanent lung window. This approach enables longitudinal, single-cell-resolution analysis of early host-pathogen interactions, including bacillary localization, vascular dynamics, and macrophage uptake within the same lung microenvironment.

Abstract

Intravital microscopy enables direct visualization of dynamic cellular processes within intact tissues. However, its application to Mycobacterium tuberculosis (Mtb) has been limited by Biosafety Level 3 (BSL-3) containment requirements and the technical challenges of stabilizing the lung for high-resolution imaging. Here, we present a protocol that combines the thoracic Window for High-Resolution Imaging of the murine Lung (WHRIL) with mc²7902, a genetically defined triple-auxotrophic Mtb strain approved for use under BSL-2 conditions. We also describe the generation of mc²8471, a tdTomato-expressing derivative of mc²7902, its preparation for intravenous infection, and application for intravital imaging in reporter mice. This system enables real-time visualization of early infection dynamics, including rapid bacillary entry into the pulmonary vasculature, aggregation, dissemination into the lung parenchyma, and macrophage uptake in the same lung microenvironment over the first 3 days post-infection. By enabling longitudinal imaging of bacilli and host–pathogen interactions at single-cell resolution, this approach overcomes key limitations associated with conventional imaging and high-containment models. This protocol provides a practical BSL-2-compatible platform for real-time intravital imaging of attenuated mycobacteria in the lung and establishes a foundation for mechanistic studies of bacterial physiology, host recognition, and immune-mediated clearance.

Introduction

High-resolution intravital microscopy has transformed the study of dynamic cellular behavior within intact tissues by enabling direct visualization of cell migration, vascular interactions, and immune surveillance at single-cell resolution^1,2,3,4. While these approaches are widely used in cancer research^3,5, their application to pulmonary infections has been limited by respiratory motion, tissue fragility, and biosafety constraints. Recent advances in thoracic imaging platforms, incl....

Protocol

All procedures were conducted in accordance with institutional biosafety and animal care guidelines and were approved by the Albert Einstein College of Medicine Animal Care and Use Committee (IACUC). The strain mc²8471 is a pYUB1169 transformant of mc²7902, a triple-auxotrophic Mtb strain approved for BSL-2 use. All work involving live bacteria was performed using standard precautions for handling mycobacteria.

1. Construction of the BSL-2-approved tdTomato-expressing Mtb strain mc²8471

1. Generation of tdTomato-expressing BSL-2-safe mc²8471 by plasmid transformation
   1. Transform the....

Results

Following electroporation and kanamycin selection, isolated colonies of mc²8471 exhibited bright red fluorescence, confirming stable tdTomato expression. mc²8471 bacilli were expanded, prepared as a single-cell suspension, and administered intravenously to mice bearing surgically implanted WHRIL. Intravital imaging was performed to visualize early Mtb infection dynamics in the lung microenvironment. For this study, baseline images of the lung vasculature were acquired before infection (

Discussion

This platform addresses a genuine gap in tuberculosis research by enabling direct visualization of bacilli and host-cell interactions within the intact mammalian lung under BSL-2 conditions. Prior in vivo imaging strategies, including whole-body optical detection using fiber-optic microendoscopic excitation²⁰, reporter enzyme fluorescence imaging²¹, and near-infrared fluorescent protein-expressing Mtb²², enabled sensitive longitudinal monito.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 ml conical tube	Corning	430790
2-0 silk tie	Ethicon, Inc	LA55G
5 mm coverslip	Electron Microscopy Sciences	72296-05
5 mm disposable biopsy punch	Integra	33-35-SH
5-0 silk purse-string suture	Ethicon, Inc	640G
Blunt micro-dissecting scissors	Roboz	RS-5980
Buprenorphine	Hospira	0409-2012-32	Injection of 10 μL buprenorphine (0.3 mg/mL stock solution) diluted in 90 μL of sterile PBS (final dose = 0.1 mg/kg)
Compressed air canister	Falcon	DPSJB-12
Cyanoacrylate adhesive	Loctite	234790
D-calcium pantothenate	Acros	243305000
ECM 630 Electroporation System Complete, with Safety stand	BTX Harvard Apparatus	45-0051
Electroporation cuvette	Fisher Scientific	FB102
FITC-Dextran	Sigma-Aldrich	FD150S-1G	150 kDa
Glycerol	Fisher Chemicals	G33-1
Graefe forceps	Roboz	RS-5135	To grasp and elevate the rib during the lung window implantation procedure
Isoflurane	Pivetal	78949580
Isoflurane vaporizer	SurgiVet	VCT302
Kanamycin	Goldbio.com	K-120-25
L-arginine monohydrochloride	Sigma-Aldrich	A5131-100G
L-leucine	Sigma-Aldrich	L8000
MacBlue mice	Jackson Labs	Strain #: 026051
Middlebrook 7H10 Agar	Difco	262710
Middlebrook 7H9 broth	Difco	271310
Middlebrook OADC Enrichment	Becton Dickinson	212351
Murine ventilator	Kent Scientific	PS-02	PhysioSuite
Nair	Church & Dwight Co., Inc.	40002957
PBS	Corning	21-031-CV
PETG media inkwell bottle	Thermo Scientific	342020
PhysioSuite	Kent Scientific	PS-5623	To monitor the mouse’s tidal volume via intubation during surgery
Plasmid pYUB1169	Custom made plasmid
Rag2 KO mice	Jackson Labs	Strain #: 008449
Small animal lung inflation bulb	Harvard Apparatus	72-9083
Sodium propionate	Thermo Scientific	A17440.36
Sonicator	Fisher Scientific	FB705
Sorval Centrifuge X pro series	Thermo Scientific	75009020
Spectrophotometer Genesys 10 UV	Thermo Spectronic	335903
SurgiSuite	Kent Scientific	SURGI-M02	Multi-Functional Surgical Platform for Mice, with Warming
Tracheal catheter	Exelint International	26746	22G
Tyloxapol	Sigma-Aldrich	T0307-50G
Vacuum pickup system metal probe	Ted Pella, Inc.	528-112