MSc Neurosciences, VU University Amsterdam, Live Cell Imaging
Summary, Live Cell Imaging
Table of Contents
Background Movies Related to Lectures ............................................................................. 4
Characteris*cs of light ................................................................................................................. 4
Fluorescence ............................................................................................................................... 5
Point spread func*on .................................................................................................................. 5
Resolu*on ................................................................................................................................... 5
Fluorescent proteins.................................................................................................................... 5
Lecture 1, Introduc:on to fluorescence microscopy - Ruud Toonen ...................................... 6
Why use light?............................................................................................................................. 6
Op*cs........................................................................................................................................ 10
Fluorescence ............................................................................................................................. 14
Principle of fluorescence microscopy ......................................................................................... 16
Fluorescent molecules and proteins .......................................................................................... 19
Lecture 2, Building a LaCce lightsheet – Maxime Malivert ............................................... 25
Knowledge and technology gap ................................................................................................. 26
How to op*mally acquire an imaging volume? .......................................................................... 28
Light-sheet microscopy .............................................................................................................. 29
Endogenous protein labeling tools ............................................................................................ 33
Thick *ssue imaging from qm to nm resolu*on ......................................................................... 35
Superresolu*on imaging ........................................................................................................... 43
Lecture 3, Superresolu:on microscopy – Harold MacGillavry ............................................ 47
Photoac*vatable or photoconver*ble fluorophores (PALM) ...................................................... 54
Stochas*c switching of small organic fluorescent dyes (STORM) ................................................ 57
Points accumula*on for imaging in nanoscale topography (PAINT) ............................................ 61
Lecture 4, In vivo imaging – Angela Getz .......................................................................... 65
1. Biosensors (to observe neuronal ac*vity) .............................................................................. 66
Biosensor imaging – Calcium ....................................................................................................................... 66
Biosensor imaging – Glutamate ................................................................................................................... 72
Biosensor imaging – membrane poten9al / voltage .................................................................................... 77
2. Op*cal tools (to control neuronal ac*vity)............................................................................. 78
Op9cal tools – Rhodopsin family ................................................................................................................. 79
Op9cal tools – photoac9vatable kinases ..................................................................................................... 79
Op9cal tools – photoac9vatable kinase inhibitors ....................................................................................... 80
Op9cal tools - Dimerizers ............................................................................................................................. 81
3. Advanced protein labeling techniques (to observe molecules) ............................................... 82
Direct protein labeling – self-reac9ng enzymes ........................................................................................... 87
, MSc Neurosciences, VU University Amsterdam, Live Cell Imaging
Direct protein labeling – gene9c code expansion & click chemistry............................................................ 88
Lecture 5, Superresolu:on Live cell imaging – Max Koppers ............................................. 89
S*mulated emission deple*on (STED) ....................................................................................... 90
MINFLUX: nanometer resolu*on imaging .................................................................................. 91
Structured illumina*on microscopy (SIM).................................................................................. 92
Total internal reflec*on fluorescence (TIRF)-SIM ....................................................................... 93
Grazin incidence (GI)-SIM .......................................................................................................... 93
Airyscane -confocal microscope ................................................................................................. 94
Instant SIM (iSIM) ..................................................................................................................... 94
SoRa spinning disc ..................................................................................................................... 95
Lecture 6, Single Vesicle Imaging – Ruud Toonen .............................................................. 97
Imaging vesicle dynamics at the membrane à TIRF .................................................................. 98
Visualisa*on of synap*c vesicle cycling ................................................................................... 108
Imaging of synap9c vesicle cycle with FM dyes ......................................................................................... 109
pHluorin: a pH sensi9ve variant of GFP ..................................................................................................... 113
Lecture 8, FRAP – Jurjen Broeke ...................................................................................... 119
Lecture 9, Image J – Jurjen Broeke (not part of exam) ..................................................... 129
Lecture 10, In vivo 2 photon imaging – Chris:an Lohmann ............................................. 133
Labelling neurons with fluorescent dyes .................................................................................. 134
2-photon microscopy .............................................................................................................. 136
Choice of indicators ................................................................................................................. 138
Quan*fica*on of op*cal signals .............................................................................................. 142
Lecture 11, Optogene:cs – Wilco Nijenhuis .................................................................... 146
Neuronal optogene*cs: A tale of algal phototaxis, structural biology and fiber op*c cables..... 147
Non-neuronal optogene*cs, the only limit is our crea*vity...................................................... 158
Exam
- Wri@en exam
- Open quesDons on topics addressed during lectures
- One quesDon (a-d) per lecture
- SomeDmes you need to draw!!
- 10 mulDple choice quesDons (most are open quesDons!)
- Level of difficulty is fine
PracDcal informaDon:
- The day before the lecture, you have the slides
- You don’t need to be present every day during the pracDcal weeks
Summary, Live Cell Imaging
Table of Contents
Background Movies Related to Lectures ............................................................................. 4
Characteris*cs of light ................................................................................................................. 4
Fluorescence ............................................................................................................................... 5
Point spread func*on .................................................................................................................. 5
Resolu*on ................................................................................................................................... 5
Fluorescent proteins.................................................................................................................... 5
Lecture 1, Introduc:on to fluorescence microscopy - Ruud Toonen ...................................... 6
Why use light?............................................................................................................................. 6
Op*cs........................................................................................................................................ 10
Fluorescence ............................................................................................................................. 14
Principle of fluorescence microscopy ......................................................................................... 16
Fluorescent molecules and proteins .......................................................................................... 19
Lecture 2, Building a LaCce lightsheet – Maxime Malivert ............................................... 25
Knowledge and technology gap ................................................................................................. 26
How to op*mally acquire an imaging volume? .......................................................................... 28
Light-sheet microscopy .............................................................................................................. 29
Endogenous protein labeling tools ............................................................................................ 33
Thick *ssue imaging from qm to nm resolu*on ......................................................................... 35
Superresolu*on imaging ........................................................................................................... 43
Lecture 3, Superresolu:on microscopy – Harold MacGillavry ............................................ 47
Photoac*vatable or photoconver*ble fluorophores (PALM) ...................................................... 54
Stochas*c switching of small organic fluorescent dyes (STORM) ................................................ 57
Points accumula*on for imaging in nanoscale topography (PAINT) ............................................ 61
Lecture 4, In vivo imaging – Angela Getz .......................................................................... 65
1. Biosensors (to observe neuronal ac*vity) .............................................................................. 66
Biosensor imaging – Calcium ....................................................................................................................... 66
Biosensor imaging – Glutamate ................................................................................................................... 72
Biosensor imaging – membrane poten9al / voltage .................................................................................... 77
2. Op*cal tools (to control neuronal ac*vity)............................................................................. 78
Op9cal tools – Rhodopsin family ................................................................................................................. 79
Op9cal tools – photoac9vatable kinases ..................................................................................................... 79
Op9cal tools – photoac9vatable kinase inhibitors ....................................................................................... 80
Op9cal tools - Dimerizers ............................................................................................................................. 81
3. Advanced protein labeling techniques (to observe molecules) ............................................... 82
Direct protein labeling – self-reac9ng enzymes ........................................................................................... 87
, MSc Neurosciences, VU University Amsterdam, Live Cell Imaging
Direct protein labeling – gene9c code expansion & click chemistry............................................................ 88
Lecture 5, Superresolu:on Live cell imaging – Max Koppers ............................................. 89
S*mulated emission deple*on (STED) ....................................................................................... 90
MINFLUX: nanometer resolu*on imaging .................................................................................. 91
Structured illumina*on microscopy (SIM).................................................................................. 92
Total internal reflec*on fluorescence (TIRF)-SIM ....................................................................... 93
Grazin incidence (GI)-SIM .......................................................................................................... 93
Airyscane -confocal microscope ................................................................................................. 94
Instant SIM (iSIM) ..................................................................................................................... 94
SoRa spinning disc ..................................................................................................................... 95
Lecture 6, Single Vesicle Imaging – Ruud Toonen .............................................................. 97
Imaging vesicle dynamics at the membrane à TIRF .................................................................. 98
Visualisa*on of synap*c vesicle cycling ................................................................................... 108
Imaging of synap9c vesicle cycle with FM dyes ......................................................................................... 109
pHluorin: a pH sensi9ve variant of GFP ..................................................................................................... 113
Lecture 8, FRAP – Jurjen Broeke ...................................................................................... 119
Lecture 9, Image J – Jurjen Broeke (not part of exam) ..................................................... 129
Lecture 10, In vivo 2 photon imaging – Chris:an Lohmann ............................................. 133
Labelling neurons with fluorescent dyes .................................................................................. 134
2-photon microscopy .............................................................................................................. 136
Choice of indicators ................................................................................................................. 138
Quan*fica*on of op*cal signals .............................................................................................. 142
Lecture 11, Optogene:cs – Wilco Nijenhuis .................................................................... 146
Neuronal optogene*cs: A tale of algal phototaxis, structural biology and fiber op*c cables..... 147
Non-neuronal optogene*cs, the only limit is our crea*vity...................................................... 158
Exam
- Wri@en exam
- Open quesDons on topics addressed during lectures
- One quesDon (a-d) per lecture
- SomeDmes you need to draw!!
- 10 mulDple choice quesDons (most are open quesDons!)
- Level of difficulty is fine
PracDcal informaDon:
- The day before the lecture, you have the slides
- You don’t need to be present every day during the pracDcal weeks
