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
preclinical level.
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
satisfactory.
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.
KEYWORDS: In vivo imaging, Blood brain barrier, Brain, Blood, Neurons, Neuroimaging, Two photon excitation microscopy, Plasma, Two photon imaging, Tissues
Staining and imaging glial cells in vivo while observing the microvasculature could help understand brain physiology, namely neuronal-glial-vascular communication. Two-photon excitation microscopy provides a means to monitor these interactions at the cellular level in living animals, but the cells of interest must be fluorescent. Injecting dyes intravenously is a rapid and quasi noninvasive method to stain cells in the brain. It necessitates that the dye is soluble in the blood plasma and crosses the blood brain barrier (BBB). We demonstrate here, using two-photon imaging, that sulforhodamine B (SRB) crosses the BBB and stains in vivo, specifically mouse astrocytes. This is confirmed by experiments on primary neurons and astrocytes cultures showing the preferential SRB staining of the latter. SRB is rapidly eliminated from the blood, which allows repeated injections in longitudinal studies.
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.
Brain pathologies, including stroke and tumors, are associated with a variable degree of breakdown of the blood-brain barrier (BBB), which can usefully be studied in animal models. We describe a new optical technique for quantifying extravasation in the cortex of the living mouse and for imaging intraparenchymal tissue. Leakiness of the BBB was induced by microbeam x-irradiation. Two fluorescent dyes were simultaneously infused intravenously, one of high molecular weight (fluorescein-labeled dextran, 70 kDa, green fluorescence) and one of low molecular weight (sulforhodamine B, 559 Da, red fluorescence). A two-photon microscope, directed through a cranial window, obtained separate images of the two dyes in the cortex. The gains of the two channels were adjusted so that the signals coming from within the vessels were equal. Subtraction of the image of the fluorescein-dextran from that of the sulforhodamine B gave images in which the vasculature was invisible and the sulforhodamine B in the parenchyma could be imaged with high resolution. Algorithms are presented for rapidly quantifying the extravasation without the need for shape recognition and for calculating the permeability of the BBB. Sulforhodamine B labeled certain intraparenchymal cells; these cells, and other details, were best observed using the subtraction method.
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.
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