Blood flow tracking deep in the living tissue

Abnormalities in hemodynamics are linked to various disease states, such as stroke, Alzheimer’s, etc., however, to better understand these associations, more precise optical techniques are needed for interrogating blood flow velocity in vivo in 3D. Acousto-optic (AO) multiphoton microscopy is an outstanding technique to perform blood flow tracking at the cellular level, at a high spatiotemporal resolution deep in the living tissue. The unique scanning modes of the FEMTO3D Atlas enable us to record continuously along intricate 3D patterns, and its Dichro extension makes it possible to follow multiple parameters at the same time, like blood flow and cellular activity. These applications can be useful in various fields, such as neuroscience, immunology, dermatology, oncology and more.

FEMTO3D Atlas Dichro with two laser sources for dual excitation

The FEMTO3D Atlas Dichro platform enables fast 3D region of interest (ROI) scanning in a large 3D volume, as well as a quasi-simultaneous detection of neuronal activity and blood flow using two different laser sources. The Femtonics software package provides built-in ROI and curve analysis options, meeting all research needs.

Microscope equipment

• two pairs of acousto-optic crystals for XY and Z rapid focusing (3D imaging)
• 4D Beam Conditioning Unit for the stabilization of the laser beams
• two two-photon laser sources of different wavelengths
• a Dichro system for fast switching between the two laser lines for quasi-simultaneous dual excitation

Technical procedure for acquisition and analysis

Here we injected a red dextran intravenously to make blood vessels appear red. The passing red blood cells (RBCs) show as shadows in the red stream (Fig1.A). This way, the speed of the blood stream can be measured by following RBCs in a time-dependent manner (Fig 1.B).

Fig 1. How to measure blood flow velocity using 3D two-photon imaging. A. An example image and blood stream visualization from one 3D ribbon in a ~200 um depth. B. A montage image showing the stream of individual red blood cells (yellow circles and arrows). The average speed in a certain part of capillaries or vessels can be calculated from the values of the speeds of red blood cells (v₁, v₂…v_n).

Rapid ROI imaging by the state-of the-art FEMTO3D Atlas AO platform enables 3D ribbon scanning, following vessels through multiple cortical layers in a large brain volume. The total length of the ribbons can reach up to ~5 mm, while maintaining a resolution and speed sufficient to measure the velocity of blood flow (Fig 2.)

Fig 2. Maintaining a proper scanning speed for 3D blood stream measurement, the FEMTO3D Atlas is able to scan an up to 5 mm long ribbon network. A. 3D reconstruction of the ribbon pattern laid down to measure blood flow in the mouse cortex. B. Blood stream visualization of a 4,78 mm long 3D ribbon-network; scanning speed: 7 Hz.

Simultaneous measurement of neuronal activity and blood flow velocity

Research of cardiac diseases may be supported by a combined method of simultaneous Ca²⁺ activity and blood flow imaging. This provides a novel opportunity of acquiring local information about neuronal and cardiovascular status. Using the FEMTO3D Atlas Dichro with two laser-sources of different wavelengths, blood cell movement and neuronal activity can be measured at the same time. Fast switching between the two laser sources makes it possible to image quasi-simultaneously on two different emission wavelengths. This method can be useful for studying the detrimental consequences of stroke on vascular condition, neuronal damage, and even regeneration processes (Fig 3.).

Fig 3. Simultaneous imaging of neuronal activity and blood flow. Visual cortex neuron appears in green; red indicates a vessel (as a vertical band).