Lung tissue motion arising from breathing and heart beating has been described as the largest annoyance of in vivo
imaging. Consequently, infected lung tissue has never been imaged in vivo thus far, and little is known concerning the
kinetics of the mucosal immune system at the cellular level. We have developed an optimized post-processing strategy to
overcome tissue motion, based upon two-photon and second harmonic generation (SHG) microscopy.
In contrast to previously published data, we have freed the lung parenchyma from any strain and depression in order to
maintain the lungs under optimal physiological parameters. Excitation beams swept the sample throughout normal
breathing and heart movements, allowing the collection of many images. Given that tissue motion is unpredictably, it
was essential to sort images of interest. This step was enhanced by using SHG signal from collagen as a reference for
sampling and realignment phases. A normalized cross-correlation criterion was used between a manually chosen
reference image and rigid transformations of all others. Using CX3CR1+/gfp mice this process allowed the collection of
high resolution images of pulmonary dendritic cells (DCs) interacting with Bacillus anthracis spores, a Gram-positive
bacteria responsible for anthrax disease. We imaged lung tissue for up to one hour, without interrupting normal lung
physiology. Interestingly, our data revealed unexpected interactions between DCs and macrophages, two specialized
phagocytes. These contacts may participate in a better coordinate immune response. Our results not only demonstrate the
phagocytizing task of lung DCs but also infer a cooperative role of alveolar macrophages and DCs.
Lung efficiency as gas exchanger organ is based on the delicate balance of its associated mucosal immune system
between inflammation and sterility. In this study, we developed a dynamic imaging protocol using confocal and twophoton
excitation fluorescence (2PEF) on freshly harvested infected lungs. This modus operandi allowed the collection
of important information about CX3CR1+ pulmonary cells. This major immune cell subset turned out to be distributed in
an anisotropic way in the lungs: subpleural, parenchymal and bronchial CX3CR1+ cells have then been described. The
way parenchymal CX3CR1+ cells react against LPS activation has been considered using Matlab software,
demonstrating a dramatic increase of average cell speed. Then, interactions between Bacillus anthracis spores and
CX3CR1+ dendritic cells have been investigated, providing not only evidences of CX3CR1+ cells involvement in
pathogen uptake but also details about the capture mechanisms.
Multiphoton microscopy has shown a powerful potential for biomedical in vivo and ex vivo analysis of tissue sections
and explants. Studies were carried out on several animal organs such as brain, arteries, lungs, and kidneys. One of the
current challenges is to transfer to the clinic the knowledge and the methods previously developed in the labs at the
For tumour staging, physicians often remove the lymph nodes that are localized at the proximity of the lesion. In case of
breast cancer or melanoma, sentinel lymph node protocol is performed: pathologists randomly realize an extensive
sampling of formol fixed nodes. However, the duration of this protocol is important and its reliability is not always
The aim of our study was to determine if multiphoton microscopy would enable the fast imaging of lymph nodes on
important depths, with or without exogenous staining. Experiments were first conducted on pig lymph nodes in order to
test various dyes and to determine an appropriate protocol. The same experiments were then performed on thin slices of
human lymph nodes bearing metastatic melanoma cells. We obtained relevant images with both endofluorescence plus
second-harmonic generation and xanthene dyes. They show a good contrast between tumour and healthy cells.
Furthermore, images of pig lymph nodes were recorded up to 120μm below the surface. This new method could then
enable a faster diagnosis with higher efficiency for the patient. Experiments on thicker human lymph nodes are currently
underway in order to validate these preliminary results.
Since the early nineties, multiphoton microscopy has become a powerful tool to investigate morphological and
physiological parameters in vivo or on thick ex vivo sections. To stain structures of interest many dyes have been developed and two-photon properties (cross section, excitation and
emission spectra) of existing ones have been characterized.
Recently, our team has shown a new property of sulforhodamine B (SRB). This dye has the ability to bind specifically
elastic fibers. The observation of elastin using its endofluorescence properties was already widely described but required
long exposition delays up to 10s and the imaging depth was limited to approximately 50 μm. With a multiphoton microscope and SRB, it is possible to observe elastic fibers directly in the living animal or on thick tissue sections with a micrometric spatial resolution in less than one second per image with an imaging depth of ~ 200
μm. Moreover, with an appropriate set of filters, we can acquire simultaneously the SRB and the second harmonic generation (SHG) signals of collagen fibers. Here, we report various applications of this new staining method on different arterial rings. The layers of the arterial wall, as well as, the elastic lamellae are observed and are numbered. With the addition of a nuclear stain such as the Hoechst 33342, a more accurate morphological study of the arterial walls can be accomplished. Finally, an intravital observation of the saphenous artery morphology is presented.
Until now, the imaging of elastic fibers was restricted to tissue sections using the endofluorescence properties of elastin or histological dyes. Methods to study their morphology in vivo and in situ have been lacking. We present and characterize a new application of a fluorescent dye for two-photon microscopy: sulforhodamine B (SRB), which is shown to specifically stain elastic fibers in vivo. SRB staining of elastic fibers is demonstrated to be better than using elastin endofluorescence for two-photon microscopy. Our imaging method of elastic fibers is shown to be suitable for simultaneous imaging with both other fluorescent intravital dyes and second-harmonic generation (SHG). We illustrate these findings with intravital imaging of elastic and collagen fibers in muscle epimysium and endomysium and in blood vessel walls. We expect SRB staining to become a key method to study elastic fibers in vivo.