Teaching Plan Information

Presentation

The subject of Diagnostic Imaging in Biomedicine is a basic training in the Degree in Human Biology. It has 4 ECTS credits. It is taught in the first term of the fourth year of the degree in the form of 20 theoretical hours, 8 hours of seminars and 16 practical hours. The languages used will be English.

Teaching will be by the lecturers Oriol Gallego (coordinator); Xavier Sanjuan, Anna Oddone and Pablo Guerra (Associate Professors), and Marta Puig (Professor UPF).

Associated skills

During the subject students should obtain the competences required by the educational authorities and stipulated in the degree syllabus, which are as follows:

• Fundamental concepts in optical microscopy
• Introduction to the main methods of fluorescence microscopy
• Latest developments in fluorescence microscopy
• Sample preparation and imaging
• Computational image analysis
• Recognize the morphology and structure of tissues, organs and systems using imaging techniques.
• Learn the basics of electron microscopy (EM).
• Atomic protein structures by EM
• Introduction to EM image processing

Learning outcomes

Teaching within the subject has the following aims:

• To provide students the basic theoretical basic fundamentals, competences and practical skills that allow them to approach to all the different techniques of imaging, understood as a tool for the study of the cell’s biology, development, biomedicine, both in humans and model organisms, from the visualization of protein structures to tissues.
• To inspire curiosity among students regarding bioimaging and encourage appropriate use of this technology.
• To involve students in their own learning and to provide them with an optimal methodology for the employment of imaging tools in biomedical research.
• To help students to achieve basic transversal competences.
• To acknowledge the importance of citizen science, the impact that frugal microscopy might have in preserving ecosystems (such as plankton in Barcelona’s beach).

Sustainable Development Goals

ODS3: Education of quality#ODS10: Reduce inequality#ODS14: Life below water

Prerequisites

The course has three parts: optical microscopy, electron microscopy and image-based ionizing radiation. Prior theoretical knowledge required: cellular biology, cellular cycle, and physics of the first year.

Contents

Lectures. Duration: 20 hours. All sessions are programmed to have duration equivalent to 50 minutes and will be in-person when possible, otherwise it will be online.

Theme 1. The principle of magnification. (Oriol)

Intro to the subject. Refraction and magnification.

Theme 2. The microscope. (Oriol)

History of the microscopy. Main parts of the microscope and types. Optical aberrations. Limits of light microscopy and biological relevance.

Theme 3. Light microscopy. (Oriol)

Optical lenses, physical principles and functioning. Focal distance and numerical aperture. Contrast: chemical contrast, dyes, and optical contrast.

Theme 4. Fluorescence microscopy I. (Oriol)

Physical principles of the fluorescence. Properties of fluorochromes and fluorescent proteins. Bleaching. Fluorescence microscope and filters. 

Theme 5. Resolution. (Oriol)

Resolution: waves diffraction, PSF, Abbe’s formula and numerical aperture.

Theme 6: Integration of concepts I. (Oriol)

Scientific discussion to consolidate theoretical concepts and Kahoot (or similar) test.

Theme 7: From qualitative imaging to quantitative measurements. (Oriol)

The concept of bioimage. ImageJ and Fiji. Bit depth. Brightness and contrast. Look-up tables. Object segmentation.

Theme 8: Image analysis (Oriol)

Pre-processing. Filters. Background subtraction. Automatization.

Theme 9. Optical sectioning I. (Xavi)

Focal plane and confocal microscopy.

Theme 10. Optical sectioning II. (Xavi)

Other techniques for optical sectioning: spinning disk and TIRF/HILO.

Theme 11. Fluorescence microscopy II. (Xavi)

Inverted microscope. Comparative analysis of live-cell imaging and imaging of fixed cells. Time (time-lapse and time scale). Particle tracking.

Theme 12. Fluorescence microscopy III. (Xavi)

X + Y (Automated microscopy/screening microscopy). Depth (Z-stacks, projections, 3D rendering). Colocalization.

Theme 13. Fluorescence microscopy in tissues and animals. (Xavi)

2-photon microscopy. Optical clearing and light-sheet microscopy.

Theme 14. Super-resolution I. (Xavi)

Diffraction limit. PSF engineering and STED microscopy.

Theme 15. Super-resolution II. (Anna)

Photoactivation Light Microscopy and Single Molecule Localization Microscopy (PALM and STORM).

Theme 16. Integration of concepts. (Anna)

Scientific discussion to consolidate theoretical concepts Scientific discussion to consolidate theoretical concepts and Kahoot (or similar) test.

Theme 17. Electron Microscopy. (Pablo)

Structural biology. Comparison of structural biology techniques. Transmission electron microscopy: basic principles. Processing of samples for electron microscopy. Applications of transmission electron microscopy in biomedical sciences.

Theme 18. Cryo-Electron Microscopy. (Pablo)

Basic principles. Single Particles Approximation (SPA):sample preparation and data processing. Cryo-Electron Tomography (cryoET): sample preparation and data processing. Sub-tomogram averaging. Outstanding examples.

Theme 19. Cryo-Electron Microscopy (Pablo). Limitations to high-resolution structure determination. The resolution revolution: instrumental and software improvements. In-cell cryo-electron microscopy. Outstanding examples.

Theme 20. Correlative Microscopy. (Oriol)

Electron microscopy Vs optical microscopy: Correlative Light-Electron microscopy. Main methods of correlative microscopy and examples of applications.

Seminars.

Duration: 8 hours. All seminars will be carried in two sessions of 50 minutes, will be online (synchronic) if necessary and they will be performed in groups of 30 students.

Seminar 1. Pre-processing and Automatization.

Introduction to the project. Group presentation of filters and other pre-processing tools. Automatization of measurements. Discussion of strategies to tackle the project.

Seminar 2. Macro design.

Answering questions and discussion of strategies. Problems solving using computational image analysis.

Seminar 3. Macro troubleshooting

Discussion about problems encountered during the coding of the groups software for automate image analysis.

Seminar 4. Macro presentation

Presentation of the projects developed.

Practices.

Duration: 16 hours. Practices will be in the campus, in groups of about 12 students that will work individually.

Practice 1.1 Microscope manipulation and sample observation.

Duration: 2 hours. Location: Microscopy room.

Goal: Introduction to the employment of optical microscopes.

Development: Each student will have a light microscope to observe histological samples. We will use laser pointers of different wavelength to discuss the functioning of fluorescence filters. At the end of the practice, the student will acquire the skills to design an optimal imaging.

Practice 1.2. Frugal microscopy to study Barcelona’s plankton

Duration: 4 hours. Location: teaching room. 

Goal: Learn the basic principles of frugal microscopy and plankton biology

Development: Students, in 2-3 people group, will construct their own microscope Curiosity, a frugal microscope designed for citizen science in the study of plankton. They will collect and analyze plankton samples from Barcelona’s beach.

Practice 2.1. Advanced Imaging.

Duration: 4 hours. Location: Microscopy room. The group of 15 students will be divided into groups of 3 people and visit the Laboratory of Live-cell structural biology.

Goal: Learn the specificities of an advanced fluorescence microscope and to design an imaging experiment. 

Development: Students will visit the live-cell structural biology lab, equipped with one of an advanced fluorescence microscope capable of doing live-cell imaging and super resolution microscopy.

Practice 2.2. Computational image analysis.

Duration: 4 hours. Location: Informatics room.

Goal: Quantitative analysis of bioimages using Fiji and iLastik.

Development: Students will use different computational tools to analyze the images obtained in the practices, including Fiji macro language and machine learning.

Practice 3. Electronic microscopy.

Duration: 2 hours.   Location: Informatics room 

Goal: Cryo-electron microscopy data processing using Relion

Development: Students will carry out an introduction to the use of Relion for cryo-EM structure determination, covering the first steps of single-particle analysis workflow.

Teaching Methods

As a requirement for passing the subject, students must undertake the activities scheduled during the academic year. Attendance at lectures will not be monitored. There will be a thorough check in the other activities. The following activities are planned for the teaching process:

• Lectures

Although the lecturers will discuss the content (in synchronic and asynchronic online lectures if needed), students' participation will be encouraged through online evaluation tools such as Kahoot.

• Seminars

The seminars will take place in groups of 20-30 students, and the aim is to apply the knowledge of the concepts explained in the lectures and to deepen in the field of bioimage analysis.

This activity will be performed online in a synchronic manner if needed. The content of seminars is subject to assessment through the development of a image analysis macro in groups.

• Practical sessions

Five practical sessions will take place at the campus classrooms, and in the laboratory of Live-cell structural biology. All sessions are considered mandatory and attendance will be checked.

Evaluation

The activity is assessed solely on the basis of specific objectives.

Assessment will be by means of multiple choice tests, essay tests, mostly of short answers with objective correction criteria.

Type and number of assessments

During the course assessment will be followed with the Kahoot tool in the theoretical lessons.

Seminars on bioimage analysis will be evaluated as the result of a macro for image analysis to quantify biological features of the sample such as protein expression levels, colocalization, etc. The macro will be developed in small groups. During the last seminar session, each group will present their macro and defend the strategy used. Evaluation will take into account performance of the macro (group mark), the consolidation of concepts by each student (individual defense) and the contribution according to members of the group (individual contribution). “Seminars mark” = Average (“group mark”, “individual defense”, “individual contribution”).

At the end of the teaching process, a final evaluation of the theory will take place, consisting of a multiple choice test with questions on all the subjects covered and an essay test with short questions.

Impact of the various types of assessment on the final accreditive mark

Final assessment: MCT                                              35%

                           Test                                              30%

Assessment during course:

Kahoot                                                                   10%

Seminars                                                               25%

Pass criteria and qualitative grades

Student must participate in the programmed activities and obtain a mark of 4 or higher in final assessment (MCT + Test) and 5 or higher in global mark to pass the subject.

Recovery process criteria

Students that after the evaluation process have not passed the subject, have the option of a recovery test of the final evaluation in July. Under no circumstances, the continuous evaluation can be recovered. The student undergoing recovery will keep the mark of the continuous evaluation and this will be computed with the new mark of July with the contingencies described before.

Bibliography and information resources

Reference books and articles

1. Douglas E. Chandler and Robert W. Roberson
   Bioimaging: Current Techniques in Light and Electron Microscopy.  Jones and Bartlett Publishing, Sudbury, Massachusetts (1st Edition). (2008).  
2. H. S. M. Bradbury and Brian Bracegirdle. Introduction to Light Microscopy.  Garland Science, Taylor and Francis, Florence, Kentucky (2nd Edition). (1998).
3. David L. Spector and Robert D. Goldman
   Basic Methods in Microscopy: Protocols and Concepts from Cells: A Laboratory Manual.  Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1st Edition). (2005). 
4. M. J. Dykstra. Biological Electron Microscopy. 1992. 
5. Pawley, James. Handbook of Biological Confocal Microscopy. 3a. ed.. Springer, 2006.
6. Miura K. Bioimage Data Analysis. Wiley-VCH, 2016. 
7. Wheeler, A. and Henriques, R. Standard and super-resolution bioimaging data analysis. Wiley- RMS, 2017.

8. Nature Milestones in Light Microscopy: https://www.nature.com/collections/ptjfzfbkwv
9. Alison J. North. Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. J Cell Biol (2006) 172 (1): 9–18. https://doi.org/10.1083/jcb.200507103  
10. Jennifer C. Waters. Accuracy and precision in quantitative fluorescence microscopy. J Cell Biol 185 (7): 1135–1148 (2009). https://doi.org/10.1083/jcb.200903097
11. Jonkman, J., Brown, C.M., Wright, G.D. et al. Tutorial: guidance for quantitative confocal microscopy. Nat Protoc 15, 1585–1611 (2020). https://doi.org/10.1038/s41596-020-0313-9 . (this revisión is accompanied by a poster: https://www.nature.com/articles/s41596-020-0307-7) 
12. Sahl, S., Hell, S. & Jakobs, S. Fluorescence nanoscopy in cell biology. Nat Rev Mol Cell Biol 18, 685–701 (2017). https://doi.org/10.1038/nrm.2017.71

Web resources

1. Full collection of videos with the basic principles of Optical microscopy and Advanced fluorescence microscopy: https://www.ibiology.org/online-biology-courses/microscopy-series/microscopy-series-table-contents/

2. Molecular Expresions Optical Microscopy Primer: http://www.micro.magnet.fsu.edu/primer/

3. Web focused on GFP: https://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm

4. Fluorescencetutorials: basic principles of fluorescence, edited by the company comercial Molecular Probes: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html

5. Confocal microscopy: http://www10.uniovi.es/tutoriales/confocal/presentacion.swf

6. Introduction to Bioimage analysis (by Dr. Robert Haase): https://www.youtube.com/channel/UC-hlwQ9Q4GS3rtv2EwSStAQ

7. NEUBIAS (Network of European Bioimage analysts) academy: Webminars about different computational tools for bioimage analysis. Advanced level. https://www.youtube.com/channel/UC-oy7UpEhRfHQ-5ePCviKFg

8. UCLA course about molecular imaging: http://laxmi.nuc.ucla.edu:8248/index.html

Teaching Plan Information

Presentation

The subject of Diagnostic Imaging in Biomedicine is a basic training in the Degree in Human Biology. It has 4 ECTS credits. It is taught in the first term of the fourth year of the degree in the form of 20 theoretical hours, 8 hours of seminars and 16 practical hours. The languages used will be English.

Teaching will be by the lecturers Oriol Gallego (coordinator); Xavier Sanjuan, Anna Oddone and Pablo Guerra (Associate Professors), and Marta Puig (Professor UPF).

Associated skills

During the subject students should obtain the competences required by the educational authorities and stipulated in the degree syllabus, which are as follows:

• Fundamental concepts in optical microscopy
• Introduction to the main methods of fluorescence microscopy
• Latest developments in fluorescence microscopy
• Sample preparation and imaging
• Computational image analysis
• Recognize the morphology and structure of tissues, organs and systems using imaging techniques.
• Learn the basics of electron microscopy (EM).
• Atomic protein structures by EM
• Introduction to EM image processing

Learning outcomes

Teaching within the subject has the following aims:

• To provide students the basic theoretical basic fundamentals, competences and practical skills that allow them to approach to all the different techniques of imaging, understood as a tool for the study of the cell’s biology, development, biomedicine, both in humans and model organisms, from the visualization of protein structures to tissues.
• To inspire curiosity among students regarding bioimaging and encourage appropriate use of this technology.
• To involve students in their own learning and to provide them with an optimal methodology for the employment of imaging tools in biomedical research.
• To help students to achieve basic transversal competences.
• To acknowledge the importance of citizen science, the impact that frugal microscopy might have in preserving ecosystems (such as plankton in Barcelona’s beach).

Sustainable Development Goals

ODS3: Education of quality#ODS10: Reduce inequality#ODS14: Life below water

Prerequisites

The course has three parts: optical microscopy, electron microscopy and image-based ionizing radiation. Prior theoretical knowledge required: cellular biology, cellular cycle, and physics of the first year.

Contents

Lectures. Duration: 20 hours. All sessions are programmed to have duration equivalent to 50 minutes and will be in-person when possible, otherwise it will be online.

Theme 1. The principle of magnification. (Oriol)

Intro to the subject. Refraction and magnification.

Theme 2. The microscope. (Oriol)

History of the microscopy. Main parts of the microscope and types. Optical aberrations. Limits of light microscopy and biological relevance.

Theme 3. Light microscopy. (Oriol)

Optical lenses, physical principles and functioning. Focal distance and numerical aperture. Contrast: chemical contrast, dyes, and optical contrast.

Theme 4. Fluorescence microscopy I. (Oriol)

Physical principles of the fluorescence. Properties of fluorochromes and fluorescent proteins. Bleaching. Fluorescence microscope and filters. 

Theme 5. Resolution. (Oriol)

Resolution: waves diffraction, PSF, Abbe’s formula and numerical aperture.

Theme 6: Integration of concepts I. (Oriol)

Scientific discussion to consolidate theoretical concepts and Kahoot (or similar) test.

Theme 7: From qualitative imaging to quantitative measurements. (Oriol)

The concept of bioimage. ImageJ and Fiji. Bit depth. Brightness and contrast. Look-up tables. Object segmentation.

Theme 8: Image analysis (Oriol)

Pre-processing. Filters. Background subtraction. Automatization.

Theme 9. Optical sectioning I. (Xavi)

Focal plane and confocal microscopy.

Theme 10. Optical sectioning II. (Xavi)

Other techniques for optical sectioning: spinning disk and TIRF/HILO.

Theme 11. Fluorescence microscopy II. (Xavi)

Inverted microscope. Comparative analysis of live-cell imaging and imaging of fixed cells. Time (time-lapse and time scale). Particle tracking.

Theme 12. Fluorescence microscopy III. (Xavi)

X + Y (Automated microscopy/screening microscopy). Depth (Z-stacks, projections, 3D rendering). Colocalization.

Theme 13. Fluorescence microscopy in tissues and animals. (Xavi)

2-photon microscopy. Optical clearing and light-sheet microscopy.

Theme 14. Super-resolution I. (Xavi)

Diffraction limit. PSF engineering and STED microscopy.

Theme 15. Super-resolution II. (Anna)

Photoactivation Light Microscopy and Single Molecule Localization Microscopy (PALM and STORM).

Theme 16. Integration of concepts. (Anna)

Scientific discussion to consolidate theoretical concepts Scientific discussion to consolidate theoretical concepts and Kahoot (or similar) test.

Theme 17. Electron Microscopy. (Pablo)

Structural biology. Comparison of structural biology techniques. Transmission electron microscopy: basic principles. Processing of samples for electron microscopy. Applications of transmission electron microscopy in biomedical sciences.

Theme 18. Cryo-Electron Microscopy. (Pablo)

Basic principles. Single Particles Approximation (SPA):sample preparation and data processing. Cryo-Electron Tomography (cryoET): sample preparation and data processing. Sub-tomogram averaging. Outstanding examples.

Theme 19. Cryo-Electron Microscopy (Pablo). Limitations to high-resolution structure determination. The resolution revolution: instrumental and software improvements. In-cell cryo-electron microscopy. Outstanding examples.

Theme 20. Correlative Microscopy. (Oriol)

Electron microscopy Vs optical microscopy: Correlative Light-Electron microscopy. Main methods of correlative microscopy and examples of applications.

Seminars.

Duration: 8 hours. All seminars will be carried in two sessions of 50 minutes, will be online (synchronic) if necessary and they will be performed in groups of 30 students.

Seminar 1. Pre-processing and Automatization.

Introduction to the project. Group presentation of filters and other pre-processing tools. Automatization of measurements. Discussion of strategies to tackle the project.

Seminar 2. Macro design.

Answering questions and discussion of strategies. Problems solving using computational image analysis.

Seminar 3. Macro troubleshooting

Discussion about problems encountered during the coding of the groups software for automate image analysis.

Seminar 4. Macro presentation

Presentation of the projects developed.

Practices.

Duration: 16 hours. Practices will be in the campus, in groups of about 12 students that will work individually.

Practice 1.1 Microscope manipulation and sample observation.

Duration: 2 hours. Location: Microscopy room.

Goal: Introduction to the employment of optical microscopes.

Development: Each student will have a light microscope to observe histological samples. We will use laser pointers of different wavelength to discuss the functioning of fluorescence filters. At the end of the practice, the student will acquire the skills to design an optimal imaging.

Practice 1.2. Frugal microscopy to study Barcelona’s plankton

Duration: 4 hours. Location: teaching room. 

Goal: Learn the basic principles of frugal microscopy and plankton biology

Development: Students, in 2-3 people group, will construct their own microscope Curiosity, a frugal microscope designed for citizen science in the study of plankton. They will collect and analyze plankton samples from Barcelona’s beach.

Practice 2.1. Advanced Imaging.

Duration: 4 hours. Location: Microscopy room. The group of 15 students will be divided into groups of 3 people and visit the Laboratory of Live-cell structural biology.

Goal: Learn the specificities of an advanced fluorescence microscope and to design an imaging experiment. 

Development: Students will visit the live-cell structural biology lab, equipped with one of an advanced fluorescence microscope capable of doing live-cell imaging and super resolution microscopy.

Practice 2.2. Computational image analysis.

Duration: 4 hours. Location: Informatics room.

Goal: Quantitative analysis of bioimages using Fiji and iLastik.

Development: Students will use different computational tools to analyze the images obtained in the practices, including Fiji macro language and machine learning.

Practice 3. Electronic microscopy.

Duration: 2 hours.   Location: Informatics room 

Goal: Cryo-electron microscopy data processing using Relion

Development: Students will carry out an introduction to the use of Relion for cryo-EM structure determination, covering the first steps of single-particle analysis workflow.

Teaching Methods

As a requirement for passing the subject, students must undertake the activities scheduled during the academic year. Attendance at lectures will not be monitored. There will be a thorough check in the other activities. The following activities are planned for the teaching process:

• Lectures

Although the lecturers will discuss the content (in synchronic and asynchronic online lectures if needed), students' participation will be encouraged through online evaluation tools such as Kahoot.

• Seminars

The seminars will take place in groups of 20-30 students, and the aim is to apply the knowledge of the concepts explained in the lectures and to deepen in the field of bioimage analysis.

This activity will be performed online in a synchronic manner if needed. The content of seminars is subject to assessment through the development of a image analysis macro in groups.

• Practical sessions

Five practical sessions will take place at the campus classrooms, and in the laboratory of Live-cell structural biology. All sessions are considered mandatory and attendance will be checked.

Evaluation

The activity is assessed solely on the basis of specific objectives.

Assessment will be by means of multiple choice tests, essay tests, mostly of short answers with objective correction criteria.

Type and number of assessments

During the course assessment will be followed with the Kahoot tool in the theoretical lessons.

Seminars on bioimage analysis will be evaluated as the result of a macro for image analysis to quantify biological features of the sample such as protein expression levels, colocalization, etc. The macro will be developed in small groups. During the last seminar session, each group will present their macro and defend the strategy used. Evaluation will take into account performance of the macro (group mark), the consolidation of concepts by each student (individual defense) and the contribution according to members of the group (individual contribution). “Seminars mark” = Average (“group mark”, “individual defense”, “individual contribution”).

At the end of the teaching process, a final evaluation of the theory will take place, consisting of a multiple choice test with questions on all the subjects covered and an essay test with short questions.

Impact of the various types of assessment on the final accreditive mark

Final assessment: MCT                                              35%

                           Test                                              30%

Assessment during course:

Kahoot                                                                   10%

Seminars                                                               25%

Pass criteria and qualitative grades

Student must participate in the programmed activities and obtain a mark of 4 or higher in final assessment (MCT + Test) and 5 or higher in global mark to pass the subject.

Recovery process criteria

Students that after the evaluation process have not passed the subject, have the option of a recovery test of the final evaluation in July. Under no circumstances, the continuous evaluation can be recovered. The student undergoing recovery will keep the mark of the continuous evaluation and this will be computed with the new mark of July with the contingencies described before.

Bibliography and information resources

Reference books and articles

1. Douglas E. Chandler and Robert W. Roberson
   Bioimaging: Current Techniques in Light and Electron Microscopy.  Jones and Bartlett Publishing, Sudbury, Massachusetts (1st Edition). (2008).  
2. H. S. M. Bradbury and Brian Bracegirdle. Introduction to Light Microscopy.  Garland Science, Taylor and Francis, Florence, Kentucky (2nd Edition). (1998).
3. David L. Spector and Robert D. Goldman
   Basic Methods in Microscopy: Protocols and Concepts from Cells: A Laboratory Manual.  Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1st Edition). (2005). 
4. M. J. Dykstra. Biological Electron Microscopy. 1992. 
5. Pawley, James. Handbook of Biological Confocal Microscopy. 3a. ed.. Springer, 2006.
6. Miura K. Bioimage Data Analysis. Wiley-VCH, 2016. 
7. Wheeler, A. and Henriques, R. Standard and super-resolution bioimaging data analysis. Wiley- RMS, 2017.

8. Nature Milestones in Light Microscopy: https://www.nature.com/collections/ptjfzfbkwv
9. Alison J. North. Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. J Cell Biol (2006) 172 (1): 9–18. https://doi.org/10.1083/jcb.200507103  
10. Jennifer C. Waters. Accuracy and precision in quantitative fluorescence microscopy. J Cell Biol 185 (7): 1135–1148 (2009). https://doi.org/10.1083/jcb.200903097
11. Jonkman, J., Brown, C.M., Wright, G.D. et al. Tutorial: guidance for quantitative confocal microscopy. Nat Protoc 15, 1585–1611 (2020). https://doi.org/10.1038/s41596-020-0313-9 . (this revisión is accompanied by a poster: https://www.nature.com/articles/s41596-020-0307-7) 
12. Sahl, S., Hell, S. & Jakobs, S. Fluorescence nanoscopy in cell biology. Nat Rev Mol Cell Biol 18, 685–701 (2017). https://doi.org/10.1038/nrm.2017.71

Web resources

1. Full collection of videos with the basic principles of Optical microscopy and Advanced fluorescence microscopy: https://www.ibiology.org/online-biology-courses/microscopy-series/microscopy-series-table-contents/

2. Molecular Expresions Optical Microscopy Primer: http://www.micro.magnet.fsu.edu/primer/

3. Web focused on GFP: https://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm

4. Fluorescencetutorials: basic principles of fluorescence, edited by the company comercial Molecular Probes: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html

5. Confocal microscopy: http://www10.uniovi.es/tutoriales/confocal/presentacion.swf

6. Introduction to Bioimage analysis (by Dr. Robert Haase): https://www.youtube.com/channel/UC-hlwQ9Q4GS3rtv2EwSStAQ

7. NEUBIAS (Network of European Bioimage analysts) academy: Webminars about different computational tools for bioimage analysis. Advanced level. https://www.youtube.com/channel/UC-oy7UpEhRfHQ-5ePCviKFg

8. UCLA course about molecular imaging: http://laxmi.nuc.ucla.edu:8248/index.html