Evaluation of the effects of clearing agents, fixation, and process durations on cardiovascular tissue imaging with second harmonic generation and multi-photon modalities
IntroductionProtocols for tissue clearing have been established and optimized for the central nervous system. However, significant modifications are required for clearing different tissue types. Therefore, effective optical clearing for cardiovascular tissue remains a major challenge. The goal of th...
Saved in:
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2025-07-01
|
| Series: | Frontiers in Bioengineering and Biotechnology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2025.1606425/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849714719652315136 |
|---|---|
| author | Maedeh Makki Zachary A. Molander Sergio A. Pineda-Castillo Devin W. Laurence Shubhra Singhal Yasmin Eltwafsha Gerhard A. Holzapfel Gerhard A. Holzapfel Tingting Gu Chung-Hao Lee |
| author_facet | Maedeh Makki Zachary A. Molander Sergio A. Pineda-Castillo Devin W. Laurence Shubhra Singhal Yasmin Eltwafsha Gerhard A. Holzapfel Gerhard A. Holzapfel Tingting Gu Chung-Hao Lee |
| author_sort | Maedeh Makki |
| collection | DOAJ |
| description | IntroductionProtocols for tissue clearing have been established and optimized for the central nervous system. However, significant modifications are required for clearing different tissue types. Therefore, effective optical clearing for cardiovascular tissue remains a major challenge. The goal of this study is to better understand the responses of porcine left anterior descending artery (LADA) to label-free multiphoton imaging.MethodsTo this end, the effects of different clearing methods (i.e., benzyl alcohol benzyl benzoate–BABB and glycerol), formalin fixation, variations in formalin fixation times (0–240 min), and extended storage in BABB (up to 14 days) are investigated. We compare tissue characteristics under different conditions (e.g., tissue clearing reagent and/or tissue fixation), particularly with regard to tissue preservation and transparency across z-stacks (i.e., imaging depths).ResultsThe glycerol clearing method exhibited relatively lower tissue transparency, whereas BABB increased mean AF-AUC from 0.0035 ± 0.0009 to 0.1205 ± 0.0168 and SHG-AUC from 0.0003 ± 0.0002 to 0.0072 ± 0.0040 (p< 0.001), enabling robust signal intensities at deeper layers of LADA tissue. In addition, we observed that BABB preserves fluorescent signals even after extended tissue storage with no significant loss in integrity over 14 days. Finally, we found that formalin fixation in combination with the glycerol clearing method significantly improved tissue preservation compared to the glycerol clearing method alone. However, in combination with the BABB clearing method, fixation reduced tissue transparency and signal intensity compared to BABB clearing without fixation.DiscussionThese findings establish BABB as the superior, label-free clearing agent for deep 3D multiphoton microscopy/second harmonic generation imaging of cardiovascular tissue and underscore the necessity of tailoring fixation parameters to the chosen clearing method. |
| format | Article |
| id | doaj-art-9d2be43dd0bd4818a8d315de5b0a6552 |
| institution | DOAJ |
| issn | 2296-4185 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj-art-9d2be43dd0bd4818a8d315de5b0a65522025-08-20T03:13:38ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852025-07-011310.3389/fbioe.2025.16064251606425Evaluation of the effects of clearing agents, fixation, and process durations on cardiovascular tissue imaging with second harmonic generation and multi-photon modalitiesMaedeh Makki0Zachary A. Molander1Sergio A. Pineda-Castillo2Devin W. Laurence3Shubhra Singhal4Yasmin Eltwafsha5Gerhard A. Holzapfel6Gerhard A. Holzapfel7Tingting Gu8Chung-Hao Lee9Biomechanics and Biomaterials Design Laboratory (BBDL), Department of Bioengineering, The University of California, Riverside (UCR), Riverside, CA, United StatesBiomechanics and Biomaterials Design Laboratory (BBDL), Department of Bioengineering, The University of California, Riverside (UCR), Riverside, CA, United StatesDepartment of Biomedical Engineering, University of Minnesota, Minneapolis, MN, United StatesDepartment of Anesthesiology and Critical Care, Children’s Hospital of Philadelphia (CHOP), Philadelphia, PA, United StatesBiomechanics and Biomaterials Design Laboratory (BBDL), Department of Bioengineering, The University of California, Riverside (UCR), Riverside, CA, United StatesBiomechanics and Biomaterials Design Laboratory (BBDL), Department of Bioengineering, The University of California, Riverside (UCR), Riverside, CA, United StatesInstitute of Biomechanics, Graz University of Technology, Graz, AustriaDepartment of Structural Engineering, Norwegian University of Science and Technology (NTNU), Trondheim, NorwaySchool of Biological Sciences, The University of Oklahoma, Norman, OK, United StatesBiomechanics and Biomaterials Design Laboratory (BBDL), Department of Bioengineering, The University of California, Riverside (UCR), Riverside, CA, United StatesIntroductionProtocols for tissue clearing have been established and optimized for the central nervous system. However, significant modifications are required for clearing different tissue types. Therefore, effective optical clearing for cardiovascular tissue remains a major challenge. The goal of this study is to better understand the responses of porcine left anterior descending artery (LADA) to label-free multiphoton imaging.MethodsTo this end, the effects of different clearing methods (i.e., benzyl alcohol benzyl benzoate–BABB and glycerol), formalin fixation, variations in formalin fixation times (0–240 min), and extended storage in BABB (up to 14 days) are investigated. We compare tissue characteristics under different conditions (e.g., tissue clearing reagent and/or tissue fixation), particularly with regard to tissue preservation and transparency across z-stacks (i.e., imaging depths).ResultsThe glycerol clearing method exhibited relatively lower tissue transparency, whereas BABB increased mean AF-AUC from 0.0035 ± 0.0009 to 0.1205 ± 0.0168 and SHG-AUC from 0.0003 ± 0.0002 to 0.0072 ± 0.0040 (p< 0.001), enabling robust signal intensities at deeper layers of LADA tissue. In addition, we observed that BABB preserves fluorescent signals even after extended tissue storage with no significant loss in integrity over 14 days. Finally, we found that formalin fixation in combination with the glycerol clearing method significantly improved tissue preservation compared to the glycerol clearing method alone. However, in combination with the BABB clearing method, fixation reduced tissue transparency and signal intensity compared to BABB clearing without fixation.DiscussionThese findings establish BABB as the superior, label-free clearing agent for deep 3D multiphoton microscopy/second harmonic generation imaging of cardiovascular tissue and underscore the necessity of tailoring fixation parameters to the chosen clearing method.https://www.frontiersin.org/articles/10.3389/fbioe.2025.1606425/fullcardiovascular tissueleft anterior descending arterysecond harmonic generation imagingmulti-photon imagingbenzyl alcohol benzyl benzoate (BABB) clearingformalin fixation |
| spellingShingle | Maedeh Makki Zachary A. Molander Sergio A. Pineda-Castillo Devin W. Laurence Shubhra Singhal Yasmin Eltwafsha Gerhard A. Holzapfel Gerhard A. Holzapfel Tingting Gu Chung-Hao Lee Evaluation of the effects of clearing agents, fixation, and process durations on cardiovascular tissue imaging with second harmonic generation and multi-photon modalities Frontiers in Bioengineering and Biotechnology cardiovascular tissue left anterior descending artery second harmonic generation imaging multi-photon imaging benzyl alcohol benzyl benzoate (BABB) clearing formalin fixation |
| title | Evaluation of the effects of clearing agents, fixation, and process durations on cardiovascular tissue imaging with second harmonic generation and multi-photon modalities |
| title_full | Evaluation of the effects of clearing agents, fixation, and process durations on cardiovascular tissue imaging with second harmonic generation and multi-photon modalities |
| title_fullStr | Evaluation of the effects of clearing agents, fixation, and process durations on cardiovascular tissue imaging with second harmonic generation and multi-photon modalities |
| title_full_unstemmed | Evaluation of the effects of clearing agents, fixation, and process durations on cardiovascular tissue imaging with second harmonic generation and multi-photon modalities |
| title_short | Evaluation of the effects of clearing agents, fixation, and process durations on cardiovascular tissue imaging with second harmonic generation and multi-photon modalities |
| title_sort | evaluation of the effects of clearing agents fixation and process durations on cardiovascular tissue imaging with second harmonic generation and multi photon modalities |
| topic | cardiovascular tissue left anterior descending artery second harmonic generation imaging multi-photon imaging benzyl alcohol benzyl benzoate (BABB) clearing formalin fixation |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2025.1606425/full |
| work_keys_str_mv | AT maedehmakki evaluationoftheeffectsofclearingagentsfixationandprocessdurationsoncardiovasculartissueimagingwithsecondharmonicgenerationandmultiphotonmodalities AT zacharyamolander evaluationoftheeffectsofclearingagentsfixationandprocessdurationsoncardiovasculartissueimagingwithsecondharmonicgenerationandmultiphotonmodalities AT sergioapinedacastillo evaluationoftheeffectsofclearingagentsfixationandprocessdurationsoncardiovasculartissueimagingwithsecondharmonicgenerationandmultiphotonmodalities AT devinwlaurence evaluationoftheeffectsofclearingagentsfixationandprocessdurationsoncardiovasculartissueimagingwithsecondharmonicgenerationandmultiphotonmodalities AT shubhrasinghal evaluationoftheeffectsofclearingagentsfixationandprocessdurationsoncardiovasculartissueimagingwithsecondharmonicgenerationandmultiphotonmodalities AT yasmineltwafsha evaluationoftheeffectsofclearingagentsfixationandprocessdurationsoncardiovasculartissueimagingwithsecondharmonicgenerationandmultiphotonmodalities AT gerhardaholzapfel evaluationoftheeffectsofclearingagentsfixationandprocessdurationsoncardiovasculartissueimagingwithsecondharmonicgenerationandmultiphotonmodalities AT gerhardaholzapfel evaluationoftheeffectsofclearingagentsfixationandprocessdurationsoncardiovasculartissueimagingwithsecondharmonicgenerationandmultiphotonmodalities AT tingtinggu evaluationoftheeffectsofclearingagentsfixationandprocessdurationsoncardiovasculartissueimagingwithsecondharmonicgenerationandmultiphotonmodalities AT chunghaolee evaluationoftheeffectsofclearingagentsfixationandprocessdurationsoncardiovasculartissueimagingwithsecondharmonicgenerationandmultiphotonmodalities |