Implementation of phase masking for single molecule tracking in 3D space.
This project implemented techniques to track single molecules in 3D space inside living cells using a specialized microscope called a light sheet fluorescence microscope. One major challenge was extending the depth range over which molecules could be accurately tracked. To address this, we used a li...
- Autores:
-
Castelblanco Villalobos, Alex Artemis
- Tipo de recurso:
- Trabajo de grado de pregrado
- Fecha de publicación:
- 2024
- Institución:
- Universidad de los Andes
- Repositorio:
- Séneca: repositorio Uniandes
- Idioma:
- eng
- OAI Identifier:
- oai:repositorio.uniandes.edu.co:1992/75217
- Acceso en línea:
- https://hdl.handle.net/1992/75217
- Palabra clave:
- PSF
Phase mask
Microscopy
SLM
Diffusion
LSM
SPIM
Tracking
Fluorescence
Molecule
Fourier optics
Optics
Física
- Rights
- openAccess
- License
- Attribution-NonCommercial-NoDerivatives 4.0 International
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Implementation of phase masking for single molecule tracking in 3D space. |
| title |
Implementation of phase masking for single molecule tracking in 3D space. |
| spellingShingle |
Implementation of phase masking for single molecule tracking in 3D space. PSF Phase mask Microscopy SLM Diffusion LSM SPIM Tracking Fluorescence Molecule Fourier optics Optics Física |
| title_short |
Implementation of phase masking for single molecule tracking in 3D space. |
| title_full |
Implementation of phase masking for single molecule tracking in 3D space. |
| title_fullStr |
Implementation of phase masking for single molecule tracking in 3D space. |
| title_full_unstemmed |
Implementation of phase masking for single molecule tracking in 3D space. |
| title_sort |
Implementation of phase masking for single molecule tracking in 3D space. |
| dc.creator.fl_str_mv |
Castelblanco Villalobos, Alex Artemis |
| dc.contributor.advisor.none.fl_str_mv |
Forero Shelton, Antonio Manu |
| dc.contributor.author.none.fl_str_mv |
Castelblanco Villalobos, Alex Artemis |
| dc.contributor.jury.none.fl_str_mv |
Nuñez Portela, Mayerlin |
| dc.contributor.researchgroup.none.fl_str_mv |
Facultad de Ciencias::Biofísica |
| dc.subject.keyword.eng.fl_str_mv |
PSF Phase mask Microscopy SLM Diffusion LSM SPIM Tracking Fluorescence Molecule Fourier optics Optics |
| topic |
PSF Phase mask Microscopy SLM Diffusion LSM SPIM Tracking Fluorescence Molecule Fourier optics Optics Física |
| dc.subject.themes.spa.fl_str_mv |
Física |
| description |
This project implemented techniques to track single molecules in 3D space inside living cells using a specialized microscope called a light sheet fluorescence microscope. One major challenge was extending the depth range over which molecules could be accurately tracked. To address this, we used a liquid crystal spatial light modulator (SLM) to engineer the point spread function (PSF) of the microscope, which is the image produced by a point source of light. We employed a special "double helix" phase mask pattern to project onto the SLM. This modified the PSF in a way that encoded information about the depth position of the molecule into the shape of the PSF image. Careful alignment and sizing of the phase mask on the SLM was required. Additionally we developed software to automatically detect the positions of single fluorescent particles from the modified PSF images and reconstruct their 3D trajectories over time. This allowed us quantify the diffusion dynamics of the particles, which relates to how molecules move around inside cells. To validate our approach, we measured the diffusion coefficients of fluorescent microbeads suspended in a gel, confirming our method worked accurately. In summary, this phase mask technique coupled with the 3D tracking software enabled extending the depth range for precise single molecule tracking in microscopy of living cells. This could help provide insights into the intricate dynamics underlying biological processes at the molecular level. |
| publishDate |
2024 |
| dc.date.accessioned.none.fl_str_mv |
2024-12-02T18:29:21Z |
| dc.date.available.none.fl_str_mv |
2024-12-02T18:29:21Z |
| dc.date.issued.none.fl_str_mv |
2024-12-02 |
| dc.type.none.fl_str_mv |
Trabajo de grado - Pregrado |
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info:eu-repo/semantics/bachelorThesis |
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info:eu-repo/semantics/acceptedVersion |
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http://purl.org/coar/resource_type/c_7a1f |
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Text |
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http://purl.org/coar/resource_type/c_7a1f |
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https://hdl.handle.net/1992/75217 |
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instname:Universidad de los Andes |
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reponame:Repositorio Institucional Séneca |
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eng |
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eng |
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J. J. H. Ackerman and J. J. Neil, “Biophysics of Diffusion in Cells,” in Diffusion MRI: Theory, Methods, and Applications, P. Jones Derek K., Ed., Oxford University Press, 2010, p. 0. doi: 10.1093/med/9780195369779.003.0008. “Adaptive optical microscopy: the ongoing quest for a perfect image | Light: Science & Applications.” Accessed: Apr. 02, 2024. [Online]. Available: https://www.nature.com/articles/lsa201446 Y. Shechtman, S. J. Sahl, A. S. Backer, and W. E. Moerner, “Optimal Point Spread Function Design for 3D Imaging,” Phys Rev Lett, vol. 113, no. 13, p. 133902, Sep. 2014. K. Hampson et al., “Adaptive optics for high-resolution imaging,” Nature Reviews Methods Primers, vol. 1, p. 68, Oct. 2021, doi: 10.1038/s43586-021-00066-7. V. Lakshminarayanan and A. Fleck, “Zernike polynomials: a guide,” Journal of Modern Optics, vol. 58, no. 18, pp. 1678–1678, Oct. 2011, doi: 10.1080/09500340.2011.633763. R. J. Mathar, English: Zernike polynomials. 2010. Accessed: Feb. 22, 2024. [Online]. Available: https://commons.wikimedia.org/wiki/File:Zernike_polynomials3.pdf “Digital Imaging Processing.” Accessed: Mar. 31, 2024. [Online]. Available: https://www.pearson.com/en-us/subject-catalog/p/digital-imageprocessing/P200000003224/9780137848560 J. W. Goodman, Introduction to Fourier Optics, 2nd ed. McGraw-Hill, 1996. C. Williams and O. A. Becklund, Introduction to the Optical Transfer Function, 1st ed. Bellingham: SPIEóThe International Society for Optical Engineering, 2002. R. Phillips, J. Kondev, and J. Theriot, Physical Biology of the Cell, 2nd, illustrated ed. New York: Garland Science, 2013. W. Hong et al., “Adaptive light-sheet fluorescence microscopy with a deformable mirror for video-rate volumetric imaging,” Applied Physics Letters, vol. 121, p. 193703, Nov. 2022, doi: 10.1063/5.0125946. M. Lelek et al., “Single-molecule localization microscopy,” Nat Rev Methods Primers, vol. 1, no. 1, Art. no. 1, Jun. 2021, doi: 10.1038/s43586-021-00038-x. A. Martins, “ngmsoftware/blender_wavefront_sensor.” /dev/.mind, Jun. 27, 2023. Accessed: Apr. 02, 2024. [Online]. Available: https://github.com/ngmsoftware/blender_wavefront_sensor C. Manzo and M. F. Garcia-Parajo, “A review of progress in single particle tracking: from methods to biophysical insights,” Rep Prog Phys, vol. 78, no. 12, p. 124601, Dec. 2015, doi: 10.1088/0034-4885/78/12/124601. T. Kuhn, J. Hettich, R. Davtyan, and J. C. M. Gebhardt, “Single molecule tracking and analysis framework including theory-predicted parameter settings,” Sci Rep, vol. 11, no. 1, Art. no. 1, May 2021, doi: 10.1038/s41598-021-88802-7. I. Wohl and E. Sherman, “ATP-Dependent Diffusion Entropy and Homogeneity in Living Cells,” Entropy (Basel), vol. 21, no. 10, p. 962, Oct. 2019, doi: 10.3390/e21100962. S. C. Weber, A. J. Spakowitz, and J. A. Theriot, “Nonthermal ATP-dependent fluctuations contribute to the in vivo motion of chromosomal loci,” Proceedings of the National Academy of Sciences, vol. 109, no. 19, pp. 7338–7343, May 2012, doi: 10.1073/pnas.1119505109. E. J. Gualda, H. Pereira, G. G. Martins, R. Gardner, and N. Moreno, “Threedimensional imaging flow cytometry through light-sheet fluorescence microscopy,” Cytometry Part A, vol. 91, no. 2, pp. 144–151, 2017, doi: 10.1002/cyto.a.23046. T. F. Holekamp, D. Turaga, and T. E. Holy, “Fast Three-Dimensional Fluorescence Imaging of Activity in Neural Populations by Objective-Coupled Planar Illumination Microscopy,” Neuron, vol. 57, no. 5, pp. 661–672, Mar. 2008, doi: 10.1016/j.neuron.2008.01.011. R. A. Abello Verbel, “Implementación de un microscopio de fluorescencia con una máscara de fase para ampliar su profundidad de campo,” Universidad de los Andes, Física, Facultad de Ciencias, Departamento de Física, 2022. “Shearing Interferometers.” Accessed: Dec. 13, 2023. [Online]. Available: https://www.thorlabs.com A. D. Chandra and A. Banerjee, “Rapid phase calibration of a spatial light modulator using novel phase masks and optimization of its efficiency using an iterative algorithm,” Journal of Modern Optics, vol. 67, no. 7, pp. 628–637, Apr. 2020, doi: 10.1080/09500340.2020.1760954. M. J. Townson, O. J. D. Farley, G. O. de Xivry, J. Osborn, and A. P. Reeves, “AOtools: a Python package for adaptive optics modelling and analysis,” Opt. Express, OE, vol. 27, no. 22, pp. 31316–31329, Oct. 2019, doi: 10.1364/OE.27.031316. B. Dong, D. Ren, and X. Zhang, “Stochastic parallel gradient descent based adaptive optics used for high contrast imaging coronagraph,” Res. Astron. Astrophys., vol. 11, no. 8, pp. 997–1002, Aug. 2011, doi: 10.1088/1674-4527/11/8/011. C. Roider, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Axial super-localisation using rotating point spread functions shaped by polarisation-dependent phase modulation,” Opt. Express, OE, vol. 22, no. 4, pp. 4029–4037, Feb. 2014, doi: 10.1364/OE.22.004029. “Microspheres—Section 6.5 - CO.” Accessed: May 23, 2024. [Online]. Available: https://www.thermofisher.com/ht/en/home/references/molecular-probes-thehandbook/ultrasensitive-detection-technology/microspheres.html M. A. Perilla Rubio, “Óptca adaptativa en microscopios para la reducción de aberraciones en volúmenes,” Dec. 2022, Accessed: Mar. 26, 2024. [Online]. Available: http://hdl.handle.net/1992/69093 A. Klauss, F. Conrad, and C. Hille, “Binary phase masks for easy system alignment and basic aberration sensing with spatial light modulators in STED microscopy,” Sci Rep, vol. 7, no. 1, p. 15699, Nov. 2017, doi: 10.1038/s41598-01715967-5. “Physics of agarose fluid gels: Rheological properties and microstructure - PMC.” Accessed: May 23, 2024. [Online]. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8255179/ A. Castelblanco, “AriCastel/SPIM2-revisited.” Mar. 10, 2024. Accessed: May 02, 2024. [Online]. Available: https://github.com/AriCastel/SPIM2-revisited |
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Forero Shelton, Antonio Manuvirtual::20167-1Castelblanco Villalobos, Alex ArtemisNuñez Portela, Mayerlinvirtual::20166-1Facultad de Ciencias::Biofísica2024-12-02T18:29:21Z2024-12-02T18:29:21Z2024-12-02https://hdl.handle.net/1992/75217instname:Universidad de los Andesreponame:Repositorio Institucional Sénecarepourl:https://repositorio.uniandes.edu.co/This project implemented techniques to track single molecules in 3D space inside living cells using a specialized microscope called a light sheet fluorescence microscope. One major challenge was extending the depth range over which molecules could be accurately tracked. To address this, we used a liquid crystal spatial light modulator (SLM) to engineer the point spread function (PSF) of the microscope, which is the image produced by a point source of light. We employed a special "double helix" phase mask pattern to project onto the SLM. This modified the PSF in a way that encoded information about the depth position of the molecule into the shape of the PSF image. Careful alignment and sizing of the phase mask on the SLM was required. Additionally we developed software to automatically detect the positions of single fluorescent particles from the modified PSF images and reconstruct their 3D trajectories over time. This allowed us quantify the diffusion dynamics of the particles, which relates to how molecules move around inside cells. To validate our approach, we measured the diffusion coefficients of fluorescent microbeads suspended in a gel, confirming our method worked accurately. In summary, this phase mask technique coupled with the 3D tracking software enabled extending the depth range for precise single molecule tracking in microscopy of living cells. This could help provide insights into the intricate dynamics underlying biological processes at the molecular level.Pregrado39 páginasapplication/pdfengUniversidad de los AndesFísicaFacultad de CienciasDepartamento de FísicaAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Implementation of phase masking for single molecule tracking in 3D space.Trabajo de grado - Pregradoinfo:eu-repo/semantics/bachelorThesisinfo:eu-repo/semantics/acceptedVersionhttp://purl.org/coar/resource_type/c_7a1fTexthttp://purl.org/redcol/resource_type/TPPSFPhase maskMicroscopySLMDiffusionLSMSPIMTrackingFluorescenceMoleculeFourier opticsOpticsFísicaJ. J. H. Ackerman and J. J. Neil, “Biophysics of Diffusion in Cells,” in Diffusion MRI: Theory, Methods, and Applications, P. Jones Derek K., Ed., Oxford University Press, 2010, p. 0. doi: 10.1093/med/9780195369779.003.0008.“Adaptive optical microscopy: the ongoing quest for a perfect image | Light: Science & Applications.” Accessed: Apr. 02, 2024. [Online]. Available: https://www.nature.com/articles/lsa201446Y. Shechtman, S. J. Sahl, A. S. Backer, and W. E. Moerner, “Optimal Point Spread Function Design for 3D Imaging,” Phys Rev Lett, vol. 113, no. 13, p. 133902, Sep. 2014.K. Hampson et al., “Adaptive optics for high-resolution imaging,” Nature Reviews Methods Primers, vol. 1, p. 68, Oct. 2021, doi: 10.1038/s43586-021-00066-7.V. Lakshminarayanan and A. Fleck, “Zernike polynomials: a guide,” Journal of Modern Optics, vol. 58, no. 18, pp. 1678–1678, Oct. 2011, doi: 10.1080/09500340.2011.633763.R. J. Mathar, English: Zernike polynomials. 2010. Accessed: Feb. 22, 2024. [Online]. Available: https://commons.wikimedia.org/wiki/File:Zernike_polynomials3.pdf“Digital Imaging Processing.” Accessed: Mar. 31, 2024. [Online]. Available: https://www.pearson.com/en-us/subject-catalog/p/digital-imageprocessing/P200000003224/9780137848560J. W. Goodman, Introduction to Fourier Optics, 2nd ed. McGraw-Hill, 1996.C. Williams and O. A. Becklund, Introduction to the Optical Transfer Function, 1st ed. Bellingham: SPIEóThe International Society for Optical Engineering, 2002.R. Phillips, J. Kondev, and J. Theriot, Physical Biology of the Cell, 2nd, illustrated ed. New York: Garland Science, 2013.W. Hong et al., “Adaptive light-sheet fluorescence microscopy with a deformable mirror for video-rate volumetric imaging,” Applied Physics Letters, vol. 121, p. 193703, Nov. 2022, doi: 10.1063/5.0125946.M. Lelek et al., “Single-molecule localization microscopy,” Nat Rev Methods Primers, vol. 1, no. 1, Art. no. 1, Jun. 2021, doi: 10.1038/s43586-021-00038-x.A. Martins, “ngmsoftware/blender_wavefront_sensor.” /dev/.mind, Jun. 27, 2023. Accessed: Apr. 02, 2024. [Online]. Available: https://github.com/ngmsoftware/blender_wavefront_sensorC. Manzo and M. F. Garcia-Parajo, “A review of progress in single particle tracking: from methods to biophysical insights,” Rep Prog Phys, vol. 78, no. 12, p. 124601, Dec. 2015, doi: 10.1088/0034-4885/78/12/124601.T. Kuhn, J. Hettich, R. Davtyan, and J. C. M. Gebhardt, “Single molecule tracking and analysis framework including theory-predicted parameter settings,” Sci Rep, vol. 11, no. 1, Art. no. 1, May 2021, doi: 10.1038/s41598-021-88802-7.I. Wohl and E. Sherman, “ATP-Dependent Diffusion Entropy and Homogeneity in Living Cells,” Entropy (Basel), vol. 21, no. 10, p. 962, Oct. 2019, doi: 10.3390/e21100962.S. C. Weber, A. J. Spakowitz, and J. A. Theriot, “Nonthermal ATP-dependent fluctuations contribute to the in vivo motion of chromosomal loci,” Proceedings of the National Academy of Sciences, vol. 109, no. 19, pp. 7338–7343, May 2012, doi: 10.1073/pnas.1119505109.E. J. Gualda, H. Pereira, G. G. Martins, R. Gardner, and N. Moreno, “Threedimensional imaging flow cytometry through light-sheet fluorescence microscopy,” Cytometry Part A, vol. 91, no. 2, pp. 144–151, 2017, doi: 10.1002/cyto.a.23046.T. F. Holekamp, D. Turaga, and T. E. Holy, “Fast Three-Dimensional Fluorescence Imaging of Activity in Neural Populations by Objective-Coupled Planar Illumination Microscopy,” Neuron, vol. 57, no. 5, pp. 661–672, Mar. 2008, doi: 10.1016/j.neuron.2008.01.011.R. A. Abello Verbel, “Implementación de un microscopio de fluorescencia con una máscara de fase para ampliar su profundidad de campo,” Universidad de los Andes, Física, Facultad de Ciencias, Departamento de Física, 2022.“Shearing Interferometers.” Accessed: Dec. 13, 2023. [Online]. Available: https://www.thorlabs.comA. D. Chandra and A. Banerjee, “Rapid phase calibration of a spatial light modulator using novel phase masks and optimization of its efficiency using an iterative algorithm,” Journal of Modern Optics, vol. 67, no. 7, pp. 628–637, Apr. 2020, doi: 10.1080/09500340.2020.1760954.M. J. Townson, O. J. D. Farley, G. O. de Xivry, J. Osborn, and A. P. Reeves, “AOtools: a Python package for adaptive optics modelling and analysis,” Opt. Express, OE, vol. 27, no. 22, pp. 31316–31329, Oct. 2019, doi: 10.1364/OE.27.031316.B. Dong, D. Ren, and X. Zhang, “Stochastic parallel gradient descent based adaptive optics used for high contrast imaging coronagraph,” Res. Astron. Astrophys., vol. 11, no. 8, pp. 997–1002, Aug. 2011, doi: 10.1088/1674-4527/11/8/011.C. Roider, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Axial super-localisation using rotating point spread functions shaped by polarisation-dependent phase modulation,” Opt. Express, OE, vol. 22, no. 4, pp. 4029–4037, Feb. 2014, doi: 10.1364/OE.22.004029.“Microspheres—Section 6.5 - CO.” Accessed: May 23, 2024. [Online]. Available: https://www.thermofisher.com/ht/en/home/references/molecular-probes-thehandbook/ultrasensitive-detection-technology/microspheres.htmlM. A. Perilla Rubio, “Óptca adaptativa en microscopios para la reducción de aberraciones en volúmenes,” Dec. 2022, Accessed: Mar. 26, 2024. [Online]. Available: http://hdl.handle.net/1992/69093A. Klauss, F. Conrad, and C. Hille, “Binary phase masks for easy system alignment and basic aberration sensing with spatial light modulators in STED microscopy,” Sci Rep, vol. 7, no. 1, p. 15699, Nov. 2017, doi: 10.1038/s41598-01715967-5.“Physics of agarose fluid gels: Rheological properties and microstructure - PMC.” Accessed: May 23, 2024. [Online]. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8255179/A. Castelblanco, “AriCastel/SPIM2-revisited.” Mar. 10, 2024. Accessed: May 02, 2024. [Online]. 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