Retinal microvascular and neuronal pathologies probed in vivo by adaptive optical two-photon fluorescence microscopy
Curation statements for this article:-
Curated by eLife
eLife assessment
The authors developed a two-photon fluorescence microscope coupled with adaptive optics (AO-2PFM), allowing in vivo imaging of the mouse retinal structure and function. This new imaging system will be important for exploring normal retinal physiology and pathological alterations in retinal disease models.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Neuroscience (eLife)
Abstract
The retina, behind the transparent optics of the eye, is the only neural tissue whose physiology and pathology can be non-invasively probed by optical microscopy. The aberrations intrinsic to the mouse eye, however, prevent high-resolution investigation of retinal structure and function in vivo. Optimizing the design of a two-photon fluorescence microscope (2PFM) and sample preparation procedure, we found that adaptive optics (AO), by measuring and correcting ocular aberrations, is essential for resolving putative synaptic structures and achieving three-dimensional cellular resolution in the mouse retina in vivo. Applying AO-2PFM to longitudinal retinal imaging in transgenic models of retinal pathology, we characterized microvascular lesions with sub-capillary details in a proliferative vascular retinopathy model, and found Lidocaine to effectively suppress retinal ganglion cell hyperactivity in a retinal degeneration model. Tracking structural and functional changes at high-resolution longitudinally, AO-2PFM enables microscopic investigations of retinal pathology and pharmacology for disease diagnosis and treatment in vivo.
Article activity feed
-
-
Author Response
Reviewer #3 (Public Review):
Zhang, Q. et al. developed a two-photon fluorescence microscope (2PFM) by incorporating direct wavefront sensing adaptive optics (AO), which is optimized for mouse in vivo retinal imaging. By using the same 2PFM with the option of using or not using the incorporated AO system, this team compared the in vivo retinal images and convincingly demonstrated that AO correction acquired brighter and higher resolution images of retinal ganglion cells (RGCs) and their axons in both densely and sparse labeled transgenic mouse lines, normal and defected capillary vasculatures, and RGC spontaneous activities detected by genetic Ca2+ sensor. Interestingly and importantly, this team found that a global correction by removing the common aberration from the entire FOV enhances imaging signals throughout …
Author Response
Reviewer #3 (Public Review):
Zhang, Q. et al. developed a two-photon fluorescence microscope (2PFM) by incorporating direct wavefront sensing adaptive optics (AO), which is optimized for mouse in vivo retinal imaging. By using the same 2PFM with the option of using or not using the incorporated AO system, this team compared the in vivo retinal images and convincingly demonstrated that AO correction acquired brighter and higher resolution images of retinal ganglion cells (RGCs) and their axons in both densely and sparse labeled transgenic mouse lines, normal and defected capillary vasculatures, and RGC spontaneous activities detected by genetic Ca2+ sensor. Interestingly and importantly, this team found that a global correction by removing the common aberration from the entire FOV enhances imaging signals throughout the entire large FOV, indicating a preferable AO imaging strategy for large FOVs. The potential applications of the in vivo retinal imaging techniques and strategies developed by this study will certainly inspire further investigation of the dynamic morphological and functional changes of retinal vasculatures and neurons during disease progression and before and after treatments. It would be beneficial to the manuscript and the readers if the authors can elaborate on optic design a little bit more. For example, whether the incorporation of AO adversely affects the 2PFM optic design? If the 2PFM can be further optimized by uncompromised optic design without incorporating AO, the quality of in vivo images will comparable to the AO-2PFM or not?
We thank the reviewer for these thoughtful questions.
Whether the incorporation of AO adversely affects 2PFM optical design may be a matter of perspective. As we demonstrated in the retina and elsewhere, AO substantially improves the achievable spatial resolution. Its incorporation does not reduce the temporal resolution of the system, as the ocular aberrations are temporally stable in the anesthetized mouse due to the lack of eye movement and do not require repeated aberration measurements throughout the imaging session. Signal enhancement by AO can increase the frame rate by reducing pixel dwell time required to achieve desired signal-to-noise ratio (SNR). The deformable mirror used for wavefront correction has high reflectivity, thus does not reduce the power throughput of the 2PFM. Using similar lenses for conjugation of the AO path to those employed by the 2PFM itself, we also maintain the scanning field of view size.
However, the incorporation of AO, including the direct wavefront sensing module (the “L10-L11-SH-sensor” path in Fig. 1A) and the deformable mirror (together with a pair of lenses for optical conjugation), does increase the complexity of the imaging system. Maintaining the optimal performance of AO also requires advanced optical knowledge that may not be possessed by most biological users.
For this reason, we carefully designed the 2PFM path for optimal imaging performance without AO, characterized its performance (“AO two-photon fluorescence microscope (AO-2PFM)” and “System correction” sections of Materials and Methods, Fig. S1), and optimized sample preparation including designing our own contact lens (“In vivo imaging” section of Materials and Methods, Fig. S2). Our efforts, which we believe to have led to the best possible performance of a 2PFM sans AO, allowed us to resolve retinal capillaries and cell bodies (in 2D) in vivo. Therefore, our 2PFM (sans AO) design and sample preparation procedure should benefit users who do not plan to implement AO.
Hypothetically, if the ocular aberrations of all mouse eyes were similar, it would be possible to add a static corrective element to a conventional 2PFM to improve image resolution (in the same spirit as the non-prescription reading glasses for far-sighted human eyes). However, as shown in Fig. S6 (“Zernike decompositions and corrective wavefronts for all experiments”), ocular aberrations are variable. These variabilities may arise from alignment differences (e.g., different angles between the optical axis of the ocular optics and the optical axis of the 2PFM), which can be minimized by establish a procedure to reproducibly position the eyes of different mice in similar ways. In this case, a static corrective element may be designed for substantial aberration reduction. However, the variations also arise from optical differences in the ages [1] or strains [2] of the mice. To have a 2PFM that always performs at the diffraction limit, an adaptive element as employed by AO is necessary to maintain optimal performance regardless of the specifics of the sample.
References
- C. Cheng, J. Parreno, R. B. Nowak, S. K. Biswas, K. Wang, M. Hoshino, K. Uesugi, N. Yagi, J. A. Moncaster, W.-K. Lo, B. Pierscionek, and V. M. Fowler, "Age-related changes in eye lens biomechanics, morphology, refractive index and transparency," Aging (Albany. NY). 11(24), 12497–12531 (2019).
- C. Tan, H. na Park, J. Light, K. Lacy, and M. Pardue, "Strain differences in mouse lens refractive indices when measured with OCT," Invest. Ophthalmol. Vis. Sci. 54(15), 1917 (2013).
-
eLife assessment
The authors developed a two-photon fluorescence microscope coupled with adaptive optics (AO-2PFM), allowing in vivo imaging of the mouse retinal structure and function. This new imaging system will be important for exploring normal retinal physiology and pathological alterations in retinal disease models.
-
Reviewer #1 (Public Review):
The manuscript by Zhang et al. titled "Retinal microvascular and neuronal pathologies probed in vivo by adaptive optical two-photon fluorescence microscopy" reports a custom-designed two-photon fluorescence microscope coupled with adaptive optics (AO-2PFM) that allows in vivo imaging of mouse retinal structures at a lateral resolution of ~0.8 μm and axial resolution of ~6.7 μm. The authors provided two examples of applications for in vivo imaging of mouse retinal structure and function. In the first example, AO-2PFM has been used to visualize capillary lesions in a mouse model of retinal angiomatous proliferation (RAP), a form of age-related macular degeneration characterized by capillary proliferation and focal vascular leakage. Using AO-2PFM, the authors observed capillary disruption, with which dye …
Reviewer #1 (Public Review):
The manuscript by Zhang et al. titled "Retinal microvascular and neuronal pathologies probed in vivo by adaptive optical two-photon fluorescence microscopy" reports a custom-designed two-photon fluorescence microscope coupled with adaptive optics (AO-2PFM) that allows in vivo imaging of mouse retinal structures at a lateral resolution of ~0.8 μm and axial resolution of ~6.7 μm. The authors provided two examples of applications for in vivo imaging of mouse retinal structure and function. In the first example, AO-2PFM has been used to visualize capillary lesions in a mouse model of retinal angiomatous proliferation (RAP), a form of age-related macular degeneration characterized by capillary proliferation and focal vascular leakage. Using AO-2PFM, the authors observed capillary disruption, with which dye leakage was associated. In the second example, the authors performed in vivo functional imaging of Ca2+ signals in RGCs of the rd1 mouse - a model of retinal degeneration with a mutation in the Pde6B gene. They interpreted the elevated Ca2+ signals in RGCs of rd1 mouse as an indication of RGC hyperactivity that has been reported in ex vivo electrophysiological recordings. They further observed dampened Ca2+ signals in RGCs of rd1 mouse upon retro-orbital injection of lidocaine.
The authors carefully documented the technical features of this state-of-the-art in vivo mouse retina imaging system. The manuscript is very well written and, needless to say, the images presented are of superb quality. There is no doubt that the system will be of great value to many retinal researchers studying the normal structure and function of the retina as well as tracking the pathophysiology of retinal disease models longitudinally.
-
Reviewer #2 (Public Review):
This is a technical study by Ji and colleagues that uses adaptive optics to correct for the intrinsic aberrations of the mouse eye to improve the quality of in vivo two-photon retinal imaging. Currently, the most common approach to retinal imaging is to use isolated ex vivo retina preparations for direct access to the tissue. However, in vivo retinal imaging offers the unique advantage of tracking long-term changes in vascular/cellular structure and function in disease or development. The authors describe an optimized adaptive optical two-photon microscope setup for imaging fluorescent markers through the mouse eye and evaluate the effect of the wavefront sensing area on the imaging quality. They further demonstrate the power of this setup by monitoring the focal vascular leakage in a mouse model of …
Reviewer #2 (Public Review):
This is a technical study by Ji and colleagues that uses adaptive optics to correct for the intrinsic aberrations of the mouse eye to improve the quality of in vivo two-photon retinal imaging. Currently, the most common approach to retinal imaging is to use isolated ex vivo retina preparations for direct access to the tissue. However, in vivo retinal imaging offers the unique advantage of tracking long-term changes in vascular/cellular structure and function in disease or development. The authors describe an optimized adaptive optical two-photon microscope setup for imaging fluorescent markers through the mouse eye and evaluate the effect of the wavefront sensing area on the imaging quality. They further demonstrate the power of this setup by monitoring the focal vascular leakage in a mouse model of proliferative vascular retinopathy and by monitoring drug-induced population activity changes using GCaMP6s in a mouse model of photoreceptor degeneration. Together, these results provide a valuable, enabling technical resource for applying AO-two-photo imaging to study outstanding questions in retinal biology that require long-term in vivo imaging. Overall, this is an important development with a broad impact on the investigation of neuronal and vascular functions in the retina.
-
Reviewer #3 (Public Review):
Zhang, Q. et al. developed a two-photon fluorescence microscope (2PFM) by incorporating direct wavefront sensing adaptive optics (AO), which is optimized for mouse in vivo retinal imaging. By using the same 2PFM with the option of using or not using the incorporated AO system, this team compared the in vivo retinal images and convincingly demonstrated that AO correction acquired brighter and higher resolution images of retinal ganglion cells (RGCs) and their axons in both densely and sparse labeled transgenic mouse lines, normal and defected capillary vasculatures, and RGC spontaneous activities detected by genetic Ca2+ sensor. Interestingly and importantly, this team found that a global correction by removing the common aberration from the entire FOV enhances imaging signals throughout the entire large FOV, …
Reviewer #3 (Public Review):
Zhang, Q. et al. developed a two-photon fluorescence microscope (2PFM) by incorporating direct wavefront sensing adaptive optics (AO), which is optimized for mouse in vivo retinal imaging. By using the same 2PFM with the option of using or not using the incorporated AO system, this team compared the in vivo retinal images and convincingly demonstrated that AO correction acquired brighter and higher resolution images of retinal ganglion cells (RGCs) and their axons in both densely and sparse labeled transgenic mouse lines, normal and defected capillary vasculatures, and RGC spontaneous activities detected by genetic Ca2+ sensor. Interestingly and importantly, this team found that a global correction by removing the common aberration from the entire FOV enhances imaging signals throughout the entire large FOV, indicating a preferable AO imaging strategy for large FOVs. The potential applications of the in vivo retinal imaging techniques and strategies developed by this study will certainly inspire further investigation of the dynamic morphological and functional changes of retinal vasculatures and neurons during disease progression and before and after treatments.
It would be beneficial to the manuscript and the readers if the authors can elaborate on optic design a little bit more. For example, whether the incorporation of AO adversely affects the 2PFM optic design? If the 2PFM can be further optimized by uncompromised optic design without incorporating AO, the quality of in vivo images will comparable to the AO-2PFM or not?
-
