Abstract
In vivo imaging is one of the ultimate and fundamental approaches for the study of the brain. Two-photon laser scanning microscopy (2PLSM) constitutes the state-of-the-art technique in current neuroscience to address questions regarding brain cell structure, development and function, blood flow regulation and metabolism. This technique evolved from laser scanning confocal microscopy (LSCM), which impacted the field with a major improvement in image resolution of live tissues in the 1980s compared to widefield microscopy. While nowadays some of the unparalleled features of 2PLSM make it the tool of choice for brain studies in vivo, such as the possibility to image deep within a tissue, LSCM can still be useful in this matter. Here we discuss the validity and limitations of LSCM and provide a guide to perform high-resolution in vivo imaging of the brain of live rodents with minimal mechanical disruption employing LSCM. We describe the surgical procedure and experimental setup that allowed us to record intracellular calcium variations in astrocytes evoked by sensory stimulation, and to monitor intact neuronal dendritic spines and astrocytic processes as well as blood vessel dynamics. Therefore, in spite of certain limitations that need to be carefully considered, LSCM constitutes a useful, convenient, and affordable tool for brain studies in vivo.
Highlights
The combination of fluorescence techniques with two-photon laser scanning microscopy (2PLSM) has become the tool of choice for in vivo brain imaging because high-resolution images can be obtained at relatively high depth (>500 μm) from the tissue surface (Theer et al, 2003; Helmchen and Denk, 2005)
In this article we show that the combination of an optimized surgical procedure with intravital staining of astrocytes and laser scanning confocal microscopy (LSCM) represents a suitable approach for imaging in vivo the subcellular structure of astrocytes and neurons, monitoring calcium transients in the astrocytic soma and processes, and visualizing blood vessel dynamics
IN VIVO IMAGING OF ASTROCYTES, NEURONS, AND BLOOD VESSELS USING LSCM To image astrocyte morphology in vivo, we performed a cranial window on anesthetized mice (Figures 1 and 2A) and took advantage of the fluorescent dye Sulforhodamine 101 (SR101) following a slightly modified intravital method recently reported (Appaix et al, 2012; see Methods)
Summary
The combination of fluorescence techniques with two-photon laser scanning microscopy (2PLSM) has become the tool of choice for in vivo brain imaging because high-resolution images can be obtained at relatively high depth (>500 μm) from the tissue surface (Theer et al, 2003; Helmchen and Denk, 2005). It employs ultrashort infrared laser pulses for fluorophore excitation that yield low light scattering by the tissue sample or living brain. Due to the use of shorter light wavelengths the resolution obtained with LSCM is better as the point spread function (e.g., pattern of diffracted light from a subresolution point-source which gives a measure of the smallest objects that can be resolved) is smaller (∼300 nm in xy axis; ∼900 nm in z axis; Abbe, 1873, 1874; Cole et al, 2011)
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