Laser Speckle Contrast Imaging (LSCI) has emerged as a promising imaging modality that offers full field, real time, continuous and agent free monitoring of cerebral blood flow during neurosurgery. Since LSCI does not require the injection of a contrast agent, it has the potential to complement fluorescence-based modalities by providing continuous and dynamic changes in blood flow during critical moments of neurosurgery. We performed a clinical study with LSCI to investigate the clinical utility of the technique intraoperatively. A commercially available Zeiss Pentero 900® microscope was equipped with a λ=785nm laser diode attached to a customized mount. The backscattered laser light was collected by the microscope, producing a laser speckle image on the external camera which was mounted on the microscope side-port. Custom software collected laser speckle images, computed, and then displayed the speckle contrast images in real time throughout the surgery onto the operating room monitors. The images were displayed with custom color maps and thresholding. The robust integration in the surgical workflow of the technology enabled the investigation of the need for dynamic vessel-flow characterization in 20-patients at the Inselspital in Bern, Switzerland. We assessed vessel flow during key time points in the surgery and provided real time and continuous measurements to the surgeon.
Significance: Laser speckle contrast imaging (LSCI) has emerged as a promising tool for intraoperative cerebral blood flow (CBF) monitoring because it produces real-time full-field blood flow maps noninvasively and label free.
Aim: We aim to demonstrate the ability of LSCI to continuously visualize blood flow during neurovascular procedures.
Approach: LSCI hardware was attached to the surgical microscope and did not interfere with the normal operation of the microscope. To more easily visualize CBF in real time, LSCI images were registered with the built-in microscope white light camera such that LSCI images were overlaid on the white light images and displayed to the neurosurgeon continuously in real time.
Results: LSCI was performed throughout each surgery when the microscope was positioned over the patient, providing the surgeon with real-time visualization of blood flow changes before, during, and after aneurysm clipping or arteriovenous malformation (AVM) resection in humans. LSCI was also compared with indocyanine green angiography (ICGA) to assess CBF during aneurysm clipping and AVM surgery; integration of the LSCI hardware with the microscope enabled simultaneous acquisition of LSCI and ICGA.
Conclusions: The results suggest that LSCI can provide continuous and real-time CBF visualization without affecting the surgeon workflow or requiring a contrast agent. The results also demonstrate that LSCI and ICGA provide different, yet complementary information about vessel perfusion.
Cerebral blood flow (CBF) monitoring is crucial during cerebrovascular surgery to inform decision making. In cerebral aneurysm clipping cases, CBF monitoring is routinely used to confirm patency in vessels and determine successful aneurysmal obliteration. Current intraoperative tools for CBF monitoring such as indocyanine green angiography (ICGA) do not provide real-time and continuous assessment of CBF.
We present and validate a forward model for modeling light propagation in brain tissue. The model is a dynamic light scattering Monte Carlo simulation that tracks the dynamic scattering events of a photon through a brain tissue geometry. We use the simulation to create a simulated laser speckle contrast image, and compare the simulated image with experimental images.
Multiphoton microscopy is an essential tool for detailed study of neurovascular structure and function. Wavelength mixing of synchronized laser sources—two-color multiphoton microscopy—increases the spectral window of excitable fluorophores without the need for wavelength tuning. However, implementation of two-color microscopy requires a dual output laser source, which is typically costly and complicated. We have developed a relatively simple and low-cost diamond Raman laser pumped with a ytterbium fiber amplifier. The dual output system generates excitation light at both 1060 nm (pump wavelength) and 1250 nm (first Stokes emission of diamond laser) which, when temporally and spatially overlapped, yield an effective two-color excitation wavelength of 1160 nm. This source provides an almost complete coverage of fluorophores excitable within the range of 1000-1300 nm. When compared with 1060 nm excitation, twocolor excitation at 1160 nm offers a 90% increase in signal for many far-red emitting fluorescent proteins (e.g. tdKatushka2). We demonstrate multicolor imaging of tdKatushka2 and Hoechst 33342 via simultaneous two-color twophoton, and two-color three-photon microscopy in engineered 3-D multicellular spheroids. Additionally, we show that this laser system is capable of in vivo imaging in mouse cortex to nearly 1 mm in depth with two-color excitation. This system can also be used to excite genetically encoded calcium indicators (e.g. RCaMP and GCaMP), which will be paramount in studying neuronal activity.
Deep in vivo imaging of vasculature requires small, bright, and photostable fluorophores suitable for multiphoton microscopy (MPM). Although semiconducting polymer dots (pdots) are an emerging class of highly fluorescent contrast agents with favorable advantages for the next generation of in vivo imaging, their use for deep multiphoton imaging has never before been demonstrated. Here we characterize the multiphoton properties of three pdot variants (CNPPV, PFBT, and PFPV) and demonstrate deep imaging of cortical microvasculature in C57 mice. Specifically, we measure the two- versus three-photon power dependence of these pdots and observe a clear three-photon excitation signature at wavelengths longer than 1300 nm, and a transition from two-photon to three-photon excitation within a 1060 – 1300 nm excitation range. Furthermore, we show that pdots enable in vivo two-photon imaging of cerebrovascular architecture in mice up to 850 μm beneath the pial surface using 800 nm excitation. In contrast with traditional multiphoton probes, we also demonstrate that the broad multiphoton absorption spectrum of pdots permits imaging at longer wavelengths (λex = 1,060 and 1225 nm). These wavelengths approach an ideal biological imaging wavelength near 1,300 nm and confer compatibility with a high-power ytterbium-fiber laser and a high pulse energy optical parametric amplifier, resulting in substantial improvements in signal-to-background ratio (>3.5-fold) and greater cortical imaging depths of 900 μm and 1300 μm. Ultimately, pdots are a versatile tool for MPM due to their extraordinary brightness and broad absorption, which will undoubtedly unlock the ability to interrogate deep structures in vivo.
We perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier laser source tunable between λ=1,100 and 1,400 nm. We demonstrate an imaging depth of 1,200 μm in vasculature and 1,160 μm in neurons. We also demonstrate deep-tissue imaging using Indocyanine Green (ICG), which is FDA approved and a promising route to translate multiphoton microscopy to human applications.
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