The in-vivo study of physiological and pathological events occurring in inner organs is limited by their intrinsic accessibility. Over time, scientists have engineered ways around this limitation by indirect manipulation. Common approaches combine fluorescent imaging and recording techniques to isolated organs, tissues and cells, mimicking the best "body-like" environments. These strategies capitalize also by innovative genetic and chemogenetic techniques allowing the selective targeting of specific gene products or allowing the on/off-switching of their expression at the embryonal or postnatal age. However, the direct in vivo imaging from inner organs in their physiological context by one-photon absorption based fluorescence microscopy, is not possible. This is because the heterogeneous composition of biological tissues is responsible of a strong light scattering, and the quality of contrast depends linearly on the incident light intensity. The so called non-linear optical microscopy, and in particular the two photon microscopy (2PM), provides the opportunity to overcame this limit, making high-resolution deep imaging possible through long-wavelength laser sources that reduce light scattering. The combination of 2PM and in vivo fluorescence labelling coupled to electrophysiology gives the possibility to study intact tissues in living animals, obtaining imaging at depths of more than 1 mm from organs such as the heart, kidney, lymphatic organs, muscle, and brain, performing functional studies even in awake animals. This represented a revolutionary technical progress for the study of not easily accessible systems such as the CNS, where the 2PM permitted seminal discoveries in the last two decades, from dendritic spine plasticity during learning (Trachtenberg et al. Nature 2002) to homeostatic activity of microglial cells (Davalos et al Nat Neurosci 2005).
Living organisms depend on large-scale cellular interactions, and inter-layer and inter-area communications are crucial for information processing in the mammalian brain, heart, muscles, and in general multilayered tissues, were different layers receive inputs from different areas and send their output signals in different ways. Therefore, to understand the mechanisms of tissue functions, it is essential to simultaneously measure the cellular activity occurring at multiple layers in multiple areas. Two-photon (2P) microscopy is the key instrument for microscopy in living multi-layered tissues by creating the possibility for visualising fine structures and intracellular signalling in single cell in vivo and for collecting data simultaneously from up to several hundred cells.
In order to read out cellular activity in vivo, here we propose a 2P multiple-module, that combines one tunable 2P femtosecond laser with dual output (that can be split in two channels one fixed at 1040 nm for excitation and one tunable for imaging, or viceversa) for simultaneous 2P imaging (i.e. cellular structures or intracellular Ca2+/voltage signals) with 2P optochemistry (i.e. uncaging) or 2P optogenetics (i.e. ChR2 or NpHR) stimulation with an ultrafast resonant scanner (30 fps), with a piezoelectric objective z-actuator (from 2 Hz to 10Hz with 1000 um range) and four high efficiency detector modules (GaAsP detectors). This system is particularly suitable to acquire faster frame rates over the entire field of view in three dimension (3D) and to allow simultaneous readout and manipulation of cellular activity with cellular and subcellular resolution. Indeed, with the addition of a piezo objective positioner, the system is capable of carrying out volume scanning for the acquisition of functional and structural data in three dimensions. The combinations of these optical methods into an "all-optical approach" constitute the stat-of-the art for studying heterogeneous 3D relations of cells with neighbouring structures and to decipher local Ca2+ or voltage signals in single cell with high spatial and temporal resolutions. This option is tailored to the large back aperture objectives so that 30 frames per second can be achieved at a resolution of 512 x 512 pixels.
This enables researchers to monitor activity in a large population of cells or with the high magnification system to a single cell with cellular and subcellular resolution. Resonant scanning systems coupled to piezoelectric objective are a popular choice for 2P Ca2+ or voltage imaging in 3D using genetically encoded indicators (e.g GCaMP for Ca2+ imaging and Archon1 for membrane voltage imaging) or other functional dyes (e.g. OGB-1).
The 2P-multiple-module will be also combined with stages and manipulators that can all be controlled to work together - acting as one and will improve productivity and future proofing for our potential research needs. The manipulators and stages will be used to integrate the electrophysiological equipment in order to study the complex patterns of cellular interaction with imaging and classic electrophysiological studies in vivo. To perform simultaneously in vivo imaging and patch recordings, the system will be equipped with an additional objective (16X, 0.8 NA, 3 mm WD), which allow the proper positioning of recording and stimulating electrodes, thanks to the wide objective angle. This approach will enable researchers to investigate electrical signalling and characteristics, and to measure and control electrical signals with high spatial resolution and cellular specificity. Combining these two techniques is particularly suitable for neuroscience research where it is revolutionising many fields of research.
Finally, this version of the 2P-multiple-module scan head module has been designed to integrate additional equipment without affecting many of the benefits of the original 2P imaging systems. Here we propose to implement in our system a newly developed setup (Neurotar Mobile Cage), in which the head-fixed mouse can move around on an air-lifted mobile home cage, that features a flat floor and tangible walls, and explore the physical environment under stress-free conditions. This setup will be particularly suitable for experiments of 2P optogenetics/optochemistry, 2P imaging, and patch-clamp recordings in tissues of awake behaving mice.
This flexible system, opening new perspectives for the study of multiple tissues in vivo, would be a unique technological platform in Sapienza Research Infrastructures, who recently acquired a new confocal microscope and will be unique in Rome, where the only 2PM is held by IIT, but only permits imaging of in vitro and ex vivo samples.