Fluorescent indicators allow the monitoring of physiological parameters in biological tissues by measuring changes in the brightness of the indicator upon binding of its ligand. Quantitative measurement of these parameters in vivo with high spatio-temporal resolution using multiphoton microscopy often requires the use of fluorescence ratiometric measurements. However, ratiometric measurements can be biased at depth in biological tissues due to absorption and scattering of the photons involved in the process.
Here we have developed a mathematical model that takes into account the chemical properties of the sensors, the spectral optical properties of the biological tissue and the optical system to provide quantitative measurements of the concentration of the sensor ligand. We have also developed a software implementing this model. Both can be used to obtain unbiased measurements of different physiological biomarkers from multiphoton images.
SignificanceAll functional brain imaging methods have technical drawbacks and specific spatial and temporal resolution limitations. Unraveling brain function requires bridging the data acquired with cellular and mesoscopic functional imaging. This imposes the access to animal preparations, allowing longitudinal and multiscale investigations of brain function in anesthetized and awake animals. Such preparations are optimal to study normal and pathological brain functions while reducing the number of animals used.AimTo fulfill these needs, we developed a chronic and stable preparation for a broad set of imaging modalities and experimental design.ApproachWe describe the detailed protocol for a chronic cranial window, transparent to light and ultrasound, devoid of BOLD functional magnetic resonance imaging (fMRI) artifact and allowing stable and longitudinal multimodal imaging of the entire mouse cortex.ResultsThe inexpensive, transparent, and curved polymethylpentene cranial window preparation gives access to the entire mouse cortex. It is compatible with standard microscopic and mesoscopic neuroimaging methods. We present examples of data on the neurovascular unit and its activation using two-photon, functional ultrasound imaging, and BOLD fMRI.ConclusionThis preparation is ideal for multimodal imaging in the same animal.
Laser scanning microscopy is widely used to measure blood hemodynamics with line-scans. Analysis of red blood cell (RBC) velocity involves two types of algorithms. The algorithms of the first kind are the image-processing algorithms such as the shear [1], the LSPIV [2], the radon-transform [3] and the Fourier transform [4, 5] algorithms. Each of them provides the apparent velocity (VRBCapp) of RBCs in the image, which does not consider the scanning conditions. The algorithms of the second kind give the real RBC velocity from VRBC app and the scanning conditions [6]. Here we developed a predictive model that provide the accuracy of one image-processing algorithms. We find that the accuracy of the image-processing algorithm depends on several parameters such as the design of the scanned line, the scanning velocity and the velocity of RBCs. Last we validate our model by testing various types of artificial linescan images. The same strategy will be followed for various image-processing algorithms. Overall, our analysis will allow accurate comparisons of blood velocity from all vessel types in control and pathological animal models.
We investigate tissue and instrument parameters affecting the penetration depth in two-photon microscopy. We show that the temporal redistribution of the same average power into fewer pulses of higher peak energy by means of a regenerative amplifier results in an increase in excitation depth by approximately 2-3 scattering mean free paths. We then measure the excitation scattering mean free path in vitro, using rat brain slices, as a function of the excitation wavelength and tissue age. We find that young-animal tissue (< P18) is two-fold less scattering than adult tissue (P90). We quantify the fall-off of the collected fraction of generated fluorescence in a backward detection geometry, in vivo. At large depths, we observe that the collected fraction scales as the angular acceptance squared (related to the effective field-of-view) of the detection optics. Matching the angular acceptance of the detection optics to that of the objective (63X NA-0.90) results in a factor 3-4 of the collected fluorescence. The collection efficiency can be further increased (10X) by using an objective with large field-of-view and high numerical aperture (20X NA-0.95). These gains translate into approximately 120 micrometers additional depth penetration when working in the rat brain in vivo with a standard Ti:sapphire source.
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