Combining near infrared fluorescence and laser speckle imaging with optical tissue clearing for in vivo transcranial monitoring of cerebral blood vessels damaged by photodynamic nanoformulation

In vivo near infrared (NIR) fluorescence imaging and laser speckle contrast imaging (LSCI) are emerging optical bioimaging modalities, which can provide information on blood vessels morphology, volume and the blood flow velocity. Optical tissue clearing (OTC) technique addresses a light scattering problem in optical bioimaging, which is imperative for the transcranial brain imaging. Herein, we report an approach combining NIR fluorescence and LSC microscopy imaging with OTC. A liposomal nanoformulation comprising NIR fluorescent dye ICG and photosensitizer BPD was synthesized and injected intravenously into mouse with OTC treated skull. Transcranial excitation of BPD in nanoliposomes resulted in the localized, irradiation dose dependent photodynamic damage of the brain blood vessels, which was manifested both in NIR fluorescence and LSC transcranial imaging, revealing changes in the vessels morphology, volume and the blood flow rate. The developed approach allows for bimodal imaging guided, localized vascular PDT of cancer and other diseases.


Introduction
The primary advantage of optical imaging lies in its high spatial resolution, reaching the submicron range.This level of resolution is unattainable with biological imaging modalities such as magnetic resonance imaging (MRI), X-ray, and ultrasound imaging.Recently, near-infrared (NIR) fluorescence bioimaging has emerged as a promising and inexpensive in vivo imaging modality, providing high resolution, superior sensitivity, and biosafety [1,2].As a preclinical instrument, it can offer invaluable insights into disease mechanisms and facilitates testing of novel diagnostics and therapeutics before their transition into clinical settings.
On the other hand, laser speckle contrast imaging (LSCI) serves as a wide-field imaging method with several advantages, including its non-invasive nature and label-free approach, along with straightforward.Consequently, it has gained widespread acceptance in blood flowmetry [3][4][5][6].While fluorescence imaging provides information on vascular distribution, LSCI can map relative blood flow.Briefly, when a coherent light source (i.e., laser) is directed onto biological tissue, the backscattering light produces random interference patterns on the detector, known as the speckle pattern.The difference in blood flow for different areas can then be determined using the statistical characteristics of these speckles [7].
The integration of fluorescence imaging with LSCI has been reported for various biomedical applications.In particular, Towle et al. [8] conducted a comparative study between fluorescence imaging and LSCI, focusing on vascular perfusion visualization and quantification.Their findings suggest that combining the two can overcome the constraints associated with indocyanine green (ICG) angiography.Levi et al. [9] concurrently procured maps of both blood flow velocity and vessels distribution, using them to track changes in blood flow velocity during blood-brain barrier (BBB) opening.Their work underscored LSCI as a potentially cost-effective, label-free method for assessing the integrity of the BBB.Furthermore, Kalchenko et al. [10,11] utilized combined LSCI and fluorescence imaging setup and showed the capability of the system to detect permeability reactions in skin blood vessels, indicating its potential in allergen screening.Pathak et al. [12] engineered a microscope integrating fluorescence, LSCI, and intrinsic optical signal (IOS) imaging.The proposed design allowed researchers to efficiently monitor unanesthetized mice brains, further endorsing the use of LSCI imaging in miniaturized devices.
In the aforementioned studies, the predominant use of visible lasers for LSCI imaging limits imaging depth due to a high attenuation of visible light by biological tissues.Similarly, the primary challenge for the progress in vivo fluorescence imaging is the limited penetration of light through the tissues; the extensive scattering and absorption by these tissues compromise the fluorescence imaging quality.Thus, it is important to involve an appropriate optical imaging window, which has lower light absorption and scattering along with negligible autofluorescence.Given that both absorption and scattering of biological tissue decrease from visible to NIR region [13], it becomes intuitive to enhance optical imaging quality of deeper tissues through the in vivo NIR fluorescence imaging [6].NIR fluorescence (photoluminescence) imaging has been already shown to result in enhanced imaging depth and resolution, which makes it a subject of intense interest in recent years.
As scattering and absorption of light by biological tissues are major challenges for optical bioimaging, optical tissue clearing (OTC) methods have been introduced to render tissues more transparent, thereby enhancing imaging depth and clarity [14][15][16].By reducing light scattering within the tissue and matching the refractive indices of its components, OTC technique allows for improving penetration of light, enabling deep-tissue imaging.Many research teams reported multiple OTC solutions, such as 3DISCO [17][18][19] , CLARITY [20][21][22] and ScaleS [23,24] approaches.When it comes to optical imaging of the brain, light scattering by head tissue, particularly the skull, significantly influences imaging quality.To mitigate the effects of the turbid skull, craniotomy is frequently employed in both experimental and clinical.While opening the skull can eliminate its interference with imaging, it is far from ideal for extended imaging periods, as it alters the brain environment, frequently leading to inflammation.Use optical clearing agents (OCA) for transcranial imaging can effectively preserve the brain environment, allowing for broader field and prolonged periods of imaging [25][26][27].
Photodynamic therapy (PDT) is a photochemistry-based therapeutic approach that utilizes a light-activatable chemical (photosensitizer, PS) and light at appropriate wavelength, to eradicate disease sites via the generation of reactive oxygen species (ROS).PDT has become an established treatment for some diseases, with a long and successful clinical track record for cancer and non-malignancies [28][29][30][31].PDT employs a photosensitizer which, once illuminated by a specific wavelength of light in targeted cells, produces ROS that damage and ultimately destroy these cells [32,33].The precision and minimal invasiveness of PDT support its potential as a consistent and effective treatment in clinical.The advent of nanotechnology has ushered in a new era of clinical treatment, with nanoformulations emerging as a cutting-edge trend.Within the realm of oncology research, various nanoparticles are rapidly gaining prominence as a drug delivery nanovehicles [34].As of now, a diverse range of contrast and therapeutic agents have been encapsulated within nanoliposomes, paving the way for advancements in diagnostic, therapeutic, and preventive medicine, including PDT [35][36][37].Interestingly, liposomal PS have the potential to enhance the efficacy of PDT.This stands in stark contrast to traditional chemotherapy drugs, which necessitate release from the nanocarrier to exert their therapeutic effects.For PDT, the encapsulated PS does not require release.This is because the effectiveness of PDT primarily hinges on the generation of ROS (e.g., singlet oxygen), which can seamlessly diffuse out of the nanoparticles [38].At the same time, to broaden the scope of PDT applications, imaging guided PDT is being developed using multiple nanoformulations, including nanoliposomes [34,[38][39][40][41][42].For PDT guided by NIR fluorescence imaging, nanoliposomal nanoformulation can embrace ICG, which is clinically approved for NIR fluorescence imaging, entrapping it inside liposomes [43][44][45].On the other hand, as demonstrated by Hasan's group, the integration of the water insoluble PS [benzoporphyrin derivative monoacid A (BPD)] [46,47], into lipid bilayers of nanoliposome leads to an enhancement in photocytotoxicity, a reduction in drug efflux from cells, and a capability for spatiotemporal control over drug photorelease [40].
Based on the published studies, we developed a liposomal nanoformulation comprising BPD for photodynamic action and ICG for NIR fluorescence imaging to guide PDT.In this work, we report integration of NIR fluorescence imaging with LSCI and optical clearing technology, which offers significant benefits for transcranial imaging of cerebral vascular distribution and blood flow dynamics.We have built a dual-mode imaging system that combines NIR fluorescence with LSCI, using this system along with OTC technology for transcranial imaging in mice for monitoring dynamic, real-time blood flow and vessel volume changes.While fluorescence imaging allows for cerebral vascular mapping, LSCI facilitates the non-invasive visualization of blood velocity in brain vessels.In PDT experiments, the system is used to induce localized damage of the brain blood vessels in the targeted brain area, successfully detect the damage and monitor recovery of blood vessels after 24 hours.Using the developed comprehensive approach, we are able to effectively monitor the photodynamic damage to identified cerebral vessels and establish laser damage thresholds.

Imaging system
The Fig. 1 shows the scheme of the advanced dual-mode NIR fluorescence-LSCI imaging system.The fluorescence imaging utilizes an 808 nm laser as an excitation source.After beam expansion and collimation, the laser is directed to the input aperture of the objective.Subsequently, the mouse head receives uniform illumination when the light passes through either a 5x (LU Plan Fluor, Nikon, Japan) or 10x objective (CFI Plan Fluor, Nikon, Japan).The fluorescence/LSC imaging signal is captured by camera (WIDY SWIR 640U-ST, NIT, France).In the LSCI imaging mode, the beam of a 1064 nm laser (MLL-S-1064, Changchun New Industries Optoelectronics Technology, China) is diffused by a ground-glass diffuser after being collimated by collimating (F220APC-1064, Thorlabs, USA).Additionally, a 690nm laser (MRL-III-690, Changchun New Industries Optoelectronics Technology, China) has been integrated into the system.This laser can be focused to a point in the focal plane of the objective, enabling PDT localized at the level of individual blood vessels.

Nanoformulation for NIR fluorescence guided PDT
We have synthesized liposomal nanoformulation (i.e., nanoliposomes, NL) to be employed in bimodal (NIR fluorescence and LSCI) imaging-guided PDT using the developed setup shown in Fig. 1. Figure 2(a) shows a scheme of the proposed study: NL are injected in the blood stream and can be imaged by NIR fluorescence excited by 808 nm light, while the blood stream is monitored by LSCI using 1064 nm laser.At the same time, when exposed to a 690 nm laser, the NL produce ROS (i.e., singlet oxygen), enabling PDT. Figure 2(c) illustrates structure of LBI, which was fabricated by encapsulating ICG in the interior core of the sterically stabilized moderately cationic PEGylated liposomes made from DPPC, DOTAP, PEGylated phospholipids (DSPE-PEG2000) and cholesterol, using procedures described previously [34,[38][39][40].BPD are entrapped in the lipid bilayer, as described in the literature [41,46,47].The synthesized NL is named by us as "LBI", as it combines lipids ("L"), BPD ("B") and ICG ("I").Absorption of LBI nanoformulation and its photoactive components (ICG and BPD) as well as LBI fluorescence are shown in the Fig. 2(b).

Laser speckle contrast imaging
Typically, in LSI experiments, the local dynamics of the observed scattering lead to a blurring of speckle patterns due to limited camera exposure time.Thus, this blurring reduces speckle contrast(K) but allows quantification of flow rates and is defined as where, σ is the standard deviation of intensity, I is the average intensity value.K(x,y) is the average of 20 consecutive spatial contrast map with a window size of 3x3. 1 K 2 can reflect the speed of blood flow.To obtain maximum LSCI imaging quality, the size of the speckle must meet the sampling law [48].

Optical tissue clearing
We have used the method described in [49] to develop a mouse OTC model.To create a durable, transparent skull window, three agents were used, namely S1(saturated supernatant solution of 75% (vol/vol) ethanol and urea), S2(high concentration sodium dodecylbenzenesulfonate solution), and S3(UV curable adhesive).The mouse was first anesthetized, then stabilized and maintained at a constant temperature using a heating pad.The mouse head was treated with depilatory cream; after a 5 minute interval, the cream was removed.The mouse scalp was surgically incised, and a custom-made headgear (mold to be filled with OTC agents, Supplement 1) was securely fixed to the mouse skull using dental cement to ensure a good seal without any leakage.The integrity of the bonding between skull and mold was confirmed by testing with saline.The S1 was introduced into the mold, ensuring full immersion of the exposed skull, and left to act for 10 to 15 minutes.During this period, gentle massaging of the skull with a cotton swab was performed.Following the application of S1, it was removed and replaced with S2, which was carefully removed after 5 min.Finally, S3 was added, filling slightly above the mold level.A cover glass was then placed over the mold, cautiously preventing air bubble formation.The setup was then exposed to ultraviolet light (365 nm from torch UV lamp) for 5 minutes, which resulted in solidifying the transparent window.As a result of the carefully controlled OTC procedure, a clear and accessible cranial window was obtained.

Photodynamic treatment
When exposed to laser light of specific wavelengths, the BPD contained within LBI generates singlet oxygen [50,51], which can damage the blood vessels, leading to platelet aggregation and thrombus formation, ultimately resulting in ischemic stroke [52][53][54].In our experiment, we utilized a 690 nm laser to irradiate the photosensitizer at an optical power density of 150mw/cm 2 for a duration of 10 minutes.

Animal treatment
All animal studies were carried out in accordance with regulations of the Animal Ethical and Welfare Committee of Shenzhen University.Female C57 mice (8 weeks old) were used for in vivo studies.Mice were anesthetized with a gas anesthesia machine (1.5% isoflurane) during the experiment.The mice were immobilized on the operating table by a head locator and the hair was cleaned with depilatory cream.In the experiment, fluorescent probes were injected into mice through the tail vein, and the mouse was fixed under the microscope while anesthetized with gas (1.5% isoflurane).The mice fixation modality is described in Supplement 1.

Results and discussion
We have assessed an acquisition of NIR fluorescence/LSC imaging using the system shown in Fig. 1 and LBI nanoformulation.With this aim, a water dispersion of LBI and 1% intralipid was pumped through two capillary glass tubes with different diameter, resulting in the higher flow rate in the narrower tube and the lower one in the wider tube.Figure 3 shows fluorescence and LSCI images of these two glass tubes, revealing similar intensity of fluorescence signal from both tubes.In contrast, LSCI, which is inherently sensitive to motion, shows increased signal in the narrower tube on the right, which is associated with the faster-moving dispersion.This enhancement in LSCI is indicative of the higher flow rate, as the motion of particles in the dispersion alters the speckle pattern more rapidly.The contrasting results on NIR fluorescence and LSC imaging of the LBI dispersion flow highlight the different capabilities and complementariness of the two imaging modalities: while fluorescence imaging is advantageous for assessing volume and concentration, LSCI allows for assessing flow dynamics.
In our study, OTC technology was employed to create a sustainable and effective transparent skull window, enhancing the visualization for imaging and PDT experiments.The efficacy of this method is exemplified in Fig. 4, where a marked improvement in skull transparency can be seen, rendering the vascular network within the mouse brain distinctly visible.Figure 4(a) displays a photographic image of the mouse skull prior to the application of OTC.As described in the Methods section, a specialized mold was securely attached to the mouse head.Figure 4(b) presents the transcranial view of the mouse skull post-OTC, showcasing the enhanced clarity achieved through this method.Subsequent to the establishment of the transparent cranial window, mice were transferred to an anesthesia chamber to undergo anesthesia.This process was integral to the preparation for subsequent NIR fluorescence/LSC imaging or PDT experiment.
After skull tissue clearing, mouse was anesthetized and fixed on the microscope stage (Supplement 1) to and the cerebral vasculature was examined using bright-field illumination to select a region rich in vessels and with clear visibility for imaging.Next, the LBI nanoliposomes were administered into the mouse through tail vein.and (f) that despite the fluorescence imaging vividly depicted capillary distribution, the flow rates within these capillaries were so low that they remained unresolved in LSCI.It is worth noting that the obtained in our experiment LSCI images contain interference fringes.We hypothesize that this interference arises from the interaction of light reflected by the outer and inner surfaces of the skull.By eliminating the sinusoidal components in the image spectrum, we successfully removed the interference fringes, as illustrated in the Supplement 1 (Figure S3).Having confirmed that our system can execute both fluorescence imaging and LSCI, we performed imaging guided targeted PDT experiments on selected brain vessels.With this aim, after obtaining images shown in Fig. 5, we retained 10x objective lens and activated a 690nm laser, irradiating the targeted area for 10 minutes.20 min after the irradiation stopped, the bright-field, fluorescence, and LSCI images of the targeted area were acquired (Fig. 6) displays.Comparing bright field image in Fig. 6(d) with that of the targeted area before irradiation [Fig.5(d)] one can see that there are some noticeable changes with vessels in the left side of the image, although these changes are not very clear.Figure 5(b) and (e) show the NIR fluorescence images, which strikingly reveal bright fluorescent spot attributed to a blood hemorrhage from the damaged vessel.A general reduction of the fluorescence signal has been also observed, which can be associated with a decrease in the LBI concentration in the circulation.On the other hand, LSCI reveals almost complete disappearance of the LSC signal from capillaries within the irradiated region [Fig.5(c) and (f)].This observation suggests that the generation of ROS by LBI in vessels leads to thrombosis and vascular blockage [55,56], which, in turn, strongly decreases the rate of blood flow in the irradiated area (or inhibits it).Comparing LSCI images in Fig. 5 and Fig. 6, one can see a clear preservation of vessels in the non-irradiated area, confirming localized, targeted damage of the selected blood vessels.Figure 7 shows imaging results for the same PDT-treated region as in Fig. 6, but 24 hours post-irradiation.While the bright-field imaging found no significant changes, NIR fluorescence imaging revealed almost complete disappearance of the NIR fluorescence signal from the circulating LBI, suggesting that most of the nanoliposomes had been cleared from the blood stream, leaving only a scant few adhering to the vessel walls [Fig.6(b) and (e)].The only remaining bright fluorescent signal originates from the blood hemorrhage induced by PDT, as the blood seeping from the damaged vessel is not in active circulation but still emits a robust fluorescence signal.On the other hand, LSCI, as showcased in Fig. 6(c) and (f), reveals a recovery of the vessels that had previously been obstructed due to vascular damage.This result stems from the accurate control of the laser irradiation dosage as too high irradiation dose could result in irreversible vascular damage and occlusion of LBI in the targeted area (Figure S4 in Supplement 1).Furthermore, when comparing Fig. 5(f) to Fig. 7(f), a noticeable increase in the blood flow velocity in the existing vessels within field of view could be observed (representative vessel is delineated by green dashed line), along with a visualization of new capillary vessels.Figure 8 shows change of the relative blood flow velocity along the white dashed lines in Fig. 5(c), Fig. 6(c), and Fig. 7(c), illustrating vascular dynamics in response to PDT.Before PDT, the vessels indicated by white dashed line and arrows in Fig. 5(c) are distinctly visible in LSCI image.Right after PDT, the blood vessel disappears in LSCI image, indicating that the blood flow has significantly decreased due to vascular occlusion.24 hours later, the vessels at the location of the arrows in Fig. 7(c) are observed to reappear.However, as one can see in Fig. 8, despite vascular recovery post 24 hours, the blood flow rate velocity has not been restored to the pre-PDT level.This is apparently due to a compensatory blood supply via alternative vascular routes developed as a result of the vascular blockage; an increase in flow velocity within vessels visualized before PDT could be also seen.In summary, our system effectively navigated the injury and subsequent recovery processes of designated vascular regions.It should be noted that both overly high and too low doses of 690 nm laser irradiation can affect the final results of the photoinduced vascular damage experiments.Lower laser irradiation dose could be insufficient to inflict notable damage or occlusion to capillaries.On the contrary, too high dose can obstruct neighboring blood vessels (as it is shown in Figure S4 in Supplement 1) causing extensive ischemia in the brain region.Thus, performing preliminary damage tests to ascertain the optimal PDT dosage is crucial for the success of subsequent studies.
It is important to emphasize that while fluorescence and LSCI images may appear similar, they convey distinct information.Fluorescence imaging exploits the fluorescence emission from the nanoliposomes that either circulate within blood vessels or adhered to vascular walls, allowing for assessment of the blood vessels morphology and volume.Conversely, LSCI is based on the dynamic scattering properties of red blood cells: their movement leads to variations in speckle patterns, with faster cells producing a more pronounced signal.As a result, LSCI delivers data about blood flow velocity.When comparing Fig. 5(e) and (f), one can see that the fluorescence signal from the primary blood vessel in the upper left corner of the fluorescence image and the branch vessel below is similar.However, in LSCI, a discernible discrepancy in flow velocities is observed.This situation is similar to that occurring in the modelled NIR fluorescence/LSC imaging shown in Fig. 3. Thus, the integration of NIR fluorescence imaging and NIR LSCI allows for the acquisition of information on blood vessel distribution and velocity, enhancing the complementarity of vascular research and diagnostics, obtaining a more comprehensive understanding of vascular structures and functions, offering invaluable insights for clinical surgery guidance.It is worth noting that the amount of the vessels visualized by fluorescence imaging is significantly higher than that imaged by LSCI.This discrepancy evidently arises from the fact that while the nanoliposomes traverse every capillary (even if the flow rate within these vessels is minimal), LSCI imaging cannot reveal, vessels with very slow flow rate, as they have minimal impact on the speckle pattern.On the other hand, areas within the speckle field where blood vessels are not discernible (e.g., in the parenchyma) still carry blood flow velocity information [7].In LSCI, signals from tiny, slow-moving vessels can easily be overshadowed by more dominant signals, constraining the resolution capabilities of LSCI.It should be also noted that the laser wavelengths typically employed in LSCI span from 632 nm to 808 nm; utilizing a 1064 nm   wavelength laser for LSCI may allow for the imaging depth enhancement.On the other hand, the pixel size of the modern near-infrared cameras tends to be larger than that of visible cameras, which can result in reduced resolution and in speckle patterns that do not adhere to the sampling law.Therefore, when opting for LSCI modality, it is important to maintain a balance between laser wavelength, imaging depth, and camera specifications to suit the demands of intended application.Conversely, with the use of contrast calculation formulas with corrective features, or determining the decorrelation time of speckles using cross-correlation formulas, the linearity and resolution of LSCI can be increased.Such methods offer more precise measurements of blood flow velocity [57][58][59].Yet, this might compromise the real-time imaging of LSCI.Moreover the correlation between blood flow velocity and decorrelation time is intricate, shaped by multiple determinants [59,60].These include the movement traits of red blood cells within vessels and the degree of laser light scattering, whether multiple or singular.Thus, using decorrelation time as a metric to represent blood flow velocity necessitates extensive research and validation to guarantee its precision and dependability.Such efforts mark an active area of exploration in the LSCI domain, with the goal of unraveling these complex interactions.
It is important to note that the developed by us approach, which involves combination of NIR fluorescence/LSC imaging with OTC, has a potential for the imaging guided, targeted treatment of brain tumors and other malignancies.Furthermore, the localized and selective occlusion of the specific blood vessels supplying disease tissues, achieved by the bimodal imaging guided PDT represents an advancement in the targeted and precise PDT of cancer and other diseases.

Conclusions
In conclusion, this work introduces a transcranial imaging and therapy approach that synergizes optical tissue clearing technology with bimodal (fluorescence and LSC) near-infrared microscopy imaging system and a nanoformulation that enables imaging guided PDT.The optical tissue clearing technology maintains the skull integrity, ensuring a stable internal environment within the mouse brain for extended observations.The microscopy imaging system enables in vivo bimodal imaging of visualizing brain blood vessels and conducting their damage assessment.This capability was demonstrated in OTC treated mice, which were intravenously injected with nanoliposomal formulation that contained NIR fluorescent dye ICG excitable with 808 nm laser and photosensitizer BPD, providing photodynamic action under 690 nm laser irradiation.Moreover, the recovery of blood vessels in the targeted area was observed 24 hours after 690 nm laser irradiation at a lower dose, while the damage was found to be irreversible at a higher irradiation dose.Armed with the developed approach, we can target precise elimination of disease tissues by occluding specific blood vessels in the process of imaging guided vascular PDT.

Fig. 2 .
Fig. 2. (a) Schematic presentation of principal mechanisms involved, LSCI, PDT.Under 808 nm laser irradiation, nanoliposomes serves for fluorescence imaging and produce ROS under 690nm irradiation, which causes vascular damage.A 1064nm laser is used to irradiate blood vessels, generating a LSCI signal.(b) Absorption spectra of ICG and BPD which are included in the nanoliposomes, Absorption and emission spectrum of LBI.(c) Schematic diagram of LBI with ICG and BPD in core.

Figure 5 (
a) to (c) represent bright-field, fluorescence and LSC imaging of the mouse head under 5x objective, while Fig.5(d) to (f) illustrate the same imaging with 10x magnification.Compared to the bright-field, fluorescence images in Fig.5(b) and (e) offer a more distinct visualization of brain vessel distribution, proving beneficial when pinpointing appropriate vessels for further PDT experiments.It should be noted that, as illustrated by Fig.3, regions with similar fluorescence imaging signals can exhibit substantial variations in capillary flow rates revealed by LSCI.Notably, it can be seen in Fig.5(e)

Fig. 3 .
Fig. 3. Demonstration of the complementariness of fluorescence imaging and LSCI using LBI.The top panel displays fluorescence images, and the middle panel shows LSCI image of the glass tubes with flowing LBI dispersion.Bottom panel presents a plot of the fluorescence (purple line) and LSCI (red line) signals distributions along the lines shown in the fluorescence and LSCI images.

Fig. 4 .
Fig. 4. Using optical tissue clearing technology to make the skull transparent.(a) Photographic image of the mouse skull before OTC treatment.A custom-made headgear is securely fixed to the mouse skull using dental cement to ensure a good seal without any leakage.The diameter of the headgear in the image is 9mm.(b) The mouse skull after OTC treatment becomes transparent, with the brain blood vessels clearly visible.

Fig. 5 .
Fig. 5. Mouse brain transcranial microscopy imaging using 5x and 10x objective lenses, where (a), (b), (c) represent bright-field, fluorescence and LSCI microscopy images obtained using 5x objective; and (d), (e), (f) represent bright-field, fluorescence and LSCI microscopy images obtained using 10x objective.White dashed line and arrows in (c) show the location, where the blood flow rate is evaluated for further comparison.Scale bar: 200 µm (a-c) or 100 µm (d-e).

Fig. 6 .
Fig. 6.Photodamage of the selected blood vessels induced by 690 nm laser light and visualized using bright-field imaging, fluorescence imaging, and LSCI with 5x and 10x microscope objectives.(a) and (d): Blood vessels with visible hemorrhages in the upper left corner are visualized in bright-field images.(b) and (e): PDT induced vascular disruption and blood leakage is seen in fluorescence images.(c) and (f): Vascular damage led to the formation of thrombi, causing capillary occlusion and blood flow stoppage, visualized by LSCI.Red dashed lines in (c) and (f) delineate single blood vessel that disappeared in LSCI but remained visible in NIR fluorescence imaging [(b) and (e)].White dashed line in (c) shows the location where the blood flow rate is evaluated for further comparison (see Fig. 8).Scale bar: 200 µm (a-c) or 100 µm (d-e).

Fig. 7 .
Fig. 7. Mouse brain transcranial microscopy imaging 24 h after PDT using 5x and 10x objective lenses, where (a), (b), (c) represent bright-field, fluorescence and LSCI images obtained using 5x objective; and (d), (e), (f) represent bright-field, fluorescence and LSCI images obtained using 10x objective.White dashed line in (c) shows the location where the blood flow rate is evaluated for further comparison (see Fig. 8).Green dashed line delineates blood vessel with blood flow increased after PDT.Scale bar: 200 µm (a-c) or 100 µm (d-e).

Fig. 8 .
Fig. 8. Relative blood flow rates evaluated along the white dashed lines in Fig. 5(c), Fig. 6(c), and Fig. 7(c).The black line shows distribution of the cerebral vascular blood flow index along the white dashed line in Fig. 5(c), before PDT; the red line shows distribution of the vascular blood flow index along the white dashed line in Fig. 6(c), right after PDT; the blue line shows along the white dashed line in Fig. 7(c), 24 hours later.