As we known, laser speckle contrast analysis (LASCA) is limited to being a qualitative method for the measurement of blood flow and tissue perfusion as it is sensitive to the measurement configuration. The signal intensity is one of the parameters that can affect the contrast values due to the quantization of the signals by the camera and analog-to-digital converter (ADC). In 2013, Song and Elson deduced the theoretical relationship between signal intensity and contrast values based on the probability density function (PDF) of the speckle pattern and simplify it to a rational function [ 28]. A simple method to correct this contrast error is also suggested [ 28]. The experimental results demonstrate that this relationship can effectively compensate the bias in contrast values induced by the quantized signal intensity and correct for bias induced by signal intensity variations across the field of view [ 28].

He et al. presented a lateral laser speckle contrast analysis method combined with line beam scanning illumination to improve the sampling depth of blood flow imaging [ 29]. Both the phantom and animal experimental results suggest that localized illumination and LSCA can significantly enhance the deep blood flow signal to improve the sampling depth of LSCI compared with the traditional full-field illumination LSCA method [ 29].

Laser speckle spatial contrast analysis (LSSCA) is superior to laser speckle temporal contrast analysis (LSTCA) in monitoring the fast change in blood flow due to its advantage of high temporal resolution. However, the application of LSSCA, which is based on spatial statistics, may be limited when there is nonuniform intensity distribution, such as fibertransmitting laser speckle imaging. In this study, Zhang et al. presented a normalized laser speckle spatial contrast analysis (nLSSCA) to correct the detrimental effects of nonuniform intensity distribution on the spatial statistics [ 30]. Through numerical simulation and phantom experiments, it was found that just ten frames of dynamic laser speckle images are sufficient for nLSSCA to achieve effective correction [ 30]. Furthermore, nLSSCA has higher temporal resolution than LSTCA to respond the change in velocity [ 30]. LSSCA, LSTCA and nLSSCA are all applied in the fiber-transmitting laser speckle imaging system to analyze the change of CBF during cortical spreading depression (CSD) in rat cortex respectively, and the results suggest that nLSSCA can examine the change of CBF more accurately. For these advantages, nLSSCA could be a potential tool for fiber transmitting/ endoscopic laser speckle imaging [ 30].

This system has a frame rate of 10 Hz and can rapidly monitor changes in blood flow in vivo, using the contrast values of the speckle images recorded with 1 ms exposure time [ 31]. In addition, the mean intensities can record the respiration period and can indicate changes in tissue oxygenation [ 31]. This system was tested during an occlusion to a human finger and is being applied in endoscopy [ 31].

LSCI and optical intrinsic signals imaging (OISI) have been used for years in the study of CBF and hemodynamic responses to neural activity. So far, most in vivo rodent experiments are based on the anesthesia model when the animals are in unconscious and restrained conditions. As we known, the anesthesia has influences on the neural activity have been documented. Lu et al. designed a miniature head-mounted dual-modal imager in freely moving animals that could monitor in real time the coupling of local oxygen consumption and blood perfusion of CBF by integrating different imaging modalities of OIS and LSCI [ 32]. The system facilitates the study the cortical hemodynamics and neural-hemodynamic coupling in real time in freely moving animals [ 32].

LSCI has become a widely used technique to image cerebral blood flow in vivo. However, the quantitative accuracy of blood flow changes measured through the thin skull has not been investigated thoroughly. Parthasarathy et al. have recently developed a new multi exposure speckle imaging (MESI) technique to image blood flow while accounting for the effect of scattering from static tissue elements [ 34]. The MESI technique was used to image the blood flow changes in a mouse cortex following photothrombotic occlusion of middle cerebral artery [ 34]. The MESI was found to accurately estimate flow changes due to ischemia in mice brains in vivo [ 34]. These estimates of these flow changes were found to be unaffected by scattering from thinned skull [ 34].

Imaging blood flow or oxygenation changes using optical techniques is useful for monitoring cortical activity in healthy subjects as well as in diseased states. However, in order to gain a better understanding of hemodynamics in conscious, freely moving animals, these techniques must be implemented in a small scale, portable design that is adaptable to a wearable format. Levy et al. have demonstrated a novel system which combines the two techniques of LSCI and intrinsic optical signal imaging simultaneously, using compact laser sources, to monitor induced cortical ischemia in a full field format with high temporal acquisition rates [ 35]. They further demonstrated the advantages of using combined measurements of speckle contrast and oxygenation to establish absolute flow velocities, as well as to statistically distinguish between veins and arteries [ 35]. Levy et al. accomplished this system using coherence reduction techniques applied to vertical cavity surface emitting lasers (VCSELs) operating at 680, 795 and 850 nm. This system uses minimal optical components and can easily be adapted into a portable format for continuous monitoring of cortical hemodynamics [ 35].

Qin et al. described a newly developed synchronized dual-wavelength LSCI system, which contains two cameras that are synchronously triggered to acquire data [ 36]. The system can acquire data at a high spatiotemporal resolution (up to 500 Hz for 1000 × 1000 pixels) [ 36]. A mouse model of stroke is used to demonstrate the capability for imaging the fast changes (within tens of milliseconds) in oxygenated and deoxygenated hemoglobin concentration, and the relative changes in blood flow in the mouse brain, through an intact cranium [ 36]. This novel imaging technology will enable the study of fast hemodynamics and metabolic changes in vascular diseases [ 36].

CBF is critical for the maintenance of cerebral function by guaranteed constant oxygen and glucose supply to brain. Collateral channels (CCs) are recruited to provide alternatives to CBF to ischemic regions once the primary vessel is occluded during ischemic stroke. However, the knowledge of the relationship between dynamic evolution of collateral flow and the distribution of regional blood flow remains limited. Laser speckle imaging was used by Wang et al. to assess dynamic changes of CCs and regional blood flow in a rat cortex with permanent MCAO [ 37]. It was found that CCs immediately provided blood flow to ischemic territories after MCAO. More importantly, there were three kinds of dynamic changes of CCs during acute stroke: persistent CC, impermanent CC, and transient CC, respectively, related to different distributions of regional blood flow [ 37].

Fig.3

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Fig.3 Spatiotemporal evolution of the distribution of CBF in ischemic cortex by LSCI. After MCAO, the region with low blood perfusion (blue-highlight area) increased with time. And there were dynamic changes in the blood flow of collateral channels (CC). Some of them were persisted (CC1 and CC4); others disappeared with different duration (CC2 and CC3) (A, anterior; L, lateral). This figure was reprinted from Ref. [ 37]

The majority of current preclinical stroke models focus on large-scale ischemic strokes that manifest severe behavioral deficits [ 41]. However, small cortical infarcts are prominent in cases of human small vessel disease and dementia, and are clinically difficult to detect [ 42, 43]. Optical access to the vasculature offers the opportunity for laser-mediated formation of intravascular clots in single vessels, as a model to study the effects of small-scale stroke in rats and mice [ 44– 47].

On the cortical surface, single vessel thrombosis is achieved by irradiating the lumen of the target vessel with a focused green laser to activate the circulating photo- sensitizing dye, Rose Bengal [ 48, 49]. Below the cortical surface, thrombosis is achieved by focusing high-fluence, 100 to 300-fs pulses of near infrared light into the vessel lumen [ 47]. Nonlinear absorption ensures that the laser injury is limited only to the plane of focus. In this case, damage of the vessel wall induces clot formation in the lumen [ 47].

Rose Bengal irradiation was applied to occlude one of many penetrating arterioles visible on the pial surface through a dura-removed cranial window in rat (Fig. 4(a)) or a PoRTS window in a CX(3)CR1 transgenic mouse with green fluorescent protein (GFP)-labeled microglia (Fig. 4(b)).

Fig.4

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Fig.4 Manipulation of blood flow in single-cortical vessels using auxiliary lasers. (a) Rose Bengal-mediated photothrombosis of a single penetrating arteriole on the pial surface. Occlusion was achieved after 1 min of irradiation with a green laser focused in the lumen of the target vessel (filled green circle in left panel). The thrombus formed by irradiation sits stably within the lumen surrounded by stagnant serum (open green circle in right panel); (b) photothrombosis of a single mouse penetrating arteriole through a PoRTS window. The CX(3)CR1 mouse line expresses GFP in microglia and monocytes. This figure was reprinted from Ref. [ 40]

By using Thy1-YFP mice, the fine dendritic structure of deep neuronal populations can be imaged over a period of hours to days following stroke [ 50– 52]. In the example of Fig. 5, blood vessels (red channel in Fig. 5(a)) as well as the dendritic arbors of layer 5 neurons (green channel in Fig. 5(a)) are imaged through a dura intact cranial window [ 53]. Injection of Rose Bengal and irradiation of the vasculature with a broadly focused green laser causes flow to cease in all pial vessels in a region roughly 1 mm² in area (right panel in Figs. 5(a) and 5(b)). In this case, severe ischemia leads to rapid dendritic damage over a period of 30 min, as evidenced by blebbing of the dendritic shafts (Figs. 5(c) and 5(d) [ 54]).

Fig.5

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Fig.5 Imaging of neuronal structure following targeted stroke. (a) Images from a Thy1-YFP mouse showing Texas red-dextranlabeled vasculature (red) and neuronal dendrites (green). The images are maximal intensity projections of the first 100 mm of the cortex before and 30 min after photoactivation of circulating Rose Bengal; (b and c) quantification of red blood cell (RBC) velocity and dendritic spine number for the animal shown in panel a; (d) dendritic structure was completely lost within 30 min of photothrombosis. Residual blood flow after stroke was zero and reperfusion did not occur. Apparent clotting and breakdown of capillaries were seen 30 min after photothrombosis [ 6]. This figure was reprinted from Ref. [ 40]

The recent application of TPM to biologic specimens has allowed investigators to examine individual synapses within live animals [ 55]. In the case of stroke, TPM imaging has revealed marked swelling of dendrites and loss of spines within minutes of ischemic onset [ 55]. Surprisingly, restoration of blood flow during reperfusion was associated with a return of relatively normal structure [ 55]. Over longer time scales, TPM imaging revealed elevated rates of synaptogenesis within peri-infarct tissues recovering from stroke [ 55]. These results provide an example of how high-resolution in vivo microscopy can be used to provide insight into both the acute pathology and recovery from stroke damage [ 55]. It have been outlined how recent advances in TPM have enabled the study of micrometer-level structures such as synapses to be studied in situ (Fig. 6) [ 55] and to define the vulnerability of the synapse with respect to regional differences in blood flow during stroke and reperfusion (Fig. 6) [ 55].

Fig.6

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Fig.6 Relationship between synaptic circuit damage and local blood flow. Cartoon of a cross-section through the rodent cortex showing the stroke core (black) and penumbra (lighter shades of gray) [ 56] after occlusion of the middle cerebral artery, a common experimental stroke model. The core has<20% of baseline blood flow and fails to regain its fine dendritic structure after reperfusion [ 57]. In the penumbra, blood flow increases moving toward the midline as tissues in this region are supplied by other artery systems that were not blocked during the stroke. Within the penumbra, some loss of dendrite structure will reverse when reperfusion occurs and this is where rewiring over longer time scales will occur to replace connectivity lost due to ischemia [ 57– 59]. This figure was reprinted from Ref. [ 55]

Although tissue death occurs within minutes to hours of the onset of reduced blood flow, functional recovery from stroke-induced brain injury continues weeks and months after the initial insult [ 61]. It has been suggested that this functional recovery occurs via adaptive plasticity in surviving neurons, whereby viable brain tissue reorganizes in order to rebuild or replace damaged synaptic connections and strengthen surviving neuronal networks [ 61– 66]. Indeed, it has been demonstrated in both human stroke patients and animal models of stroke that surviving regions of the cortex can adopt the motor- or sensory-processing functions of regions lost to damage [ 56, 67– 70]. In vivo 2-photon calcium imaging was introduced to determine how the response properties of individual somatosensory cortex neurons are altered during remapping [ 60].

Most stroke survivors exhibit a partial recovery from their deficits. This presumably occurs because of remapping of lost capabilities to functionally related brain areas [ 71]. Functional brain imaging studies suggest that remapping in the contralateral uninjured cortex might represent a transient stage of compensatory plasticity [ 71]. Some postmortem studies have also shown that stroke can trigger dendritic plasticity in the contralateral hemisphere, but the data are controversial [ 71]. Longitudinal in vivo TPM was used in the contralateral homotopic cortex to record changes in dendritic spines of layer 5 pyramidal neurons in green fluorescent protein mice [ 71]. Johnston et al. could not detect de novo growth of dendrites or changes in the density or turnover of spines for up to four weeks after stroke [ 71]. These results suggest that the contralesional cortex may not contribute to functional recovery after stroke and that, at least in mice, the peri-infarct cortex plays the dominant role in postischemic plasticity [ 71].

In the context of blood flow, the TPM technique has enabled depth-resolved measurements of RBC velocities [ 72] and vascular diameters [ 73, 74] routinely at depths of up to 500 mm deep in the cortex and as deep as 1 mm when utilizing advanced laser systems [ 75]. In contrast with LSCI, which utilizes Doppler contrast from RBC motion, TPM velocity measurements typically utilize a fluorescent dye to image the blood plasma and track the RBC ‘shadows’ to estimate velocity in individual capillaries [ 5]. While LSCI can be generally performed through a thin skull, TPM typically requires a cranial window, although thin skull measurements are now being conducted at the expense of depth penetration [ 53]. Velocity and diameter measurements are performed on no more than a few vessel segments at a time. As a result, studies that require measurements throughout the vascular geometry to ascertain the collective behavior are not practical with TPM [ 5].

In vivo TPM and transgenic mouse models expressing cell type specific reporters have been used to examine ischemia-related insults, e.g., perturbations of neuronal process morphology and local blood flow in the MCAO [ 76]. Glia and pericytes can be visualized by selective fluorescent protein expression [ 76]. In these mice, the breakdown of the blood brain barrier and the immediate as well as long-term cellular responses can be monitored [ 76].