Summary midterm test: Visualizing health and disease: from molecule to man
Learning objectives:
1. Know the fundamentals of the major types f imaging modalities available.
2. Gain hands-on experience of several of the major types of imaging modalities available.
3. Understand the applications of different imaging techniques, both clinical and preclinical.
4. Able to oervorm laboratory experiments with various imaging modalities, from same
preparation to the imaging and analysis of the data.
5. Choose the most appropriate imaging modality for a particular application.
Table of contents:
1. Imaging at different scales
- Electron microscope
- Optical microscope
2. Microscopy techniques
- Fluorescence microscopy
- SPT
- FRAP
- FRET
- DIC
- Widefield
3. Resolution
- Spatial resolution
- Spectral resolution
- Radiometric resolution
- Temporal resolution
4. Super resolution
- Selective plane microscopy/light sheet microscopy
- TIRF
- PALM/STORM
- Confocal laser scanning microscopy
- STED
5. Cell culture the basics
6. Cell viability assay: MTT
7. Molecular labeling strategies
- Fluorescent probes/dyes
- Zymosan
8. Pathology
- Cancer pathology
- Computational pathology
- Tumor microscopy
- MSI and Lynch
- Breast cancer
9. Transfection methods for live imaging
- Plasmids
- Transformation and transfection
- Isolation of plasmid
10. Dynamic imaging of cancer: from cell to tissue
- Dynamic tumor growth invasion, metastasis and therapy
11. Intravital imaging using multiphoton microscopy to monitor cell dynamics
- Multiphoton microscopy
- Label free imaging of tissue structures
- Imaging windows
,1. Imaging at different scales
- UV and IR light is not used for light microscopy because it can damage the cells
- Visible wavelengths are 760nm (red) - 390nm (slightly in UV, which starts at 500nm)
The use of microscopy:
- Anatomical research
- Reconstruction of the systematics
- Viewing the specimen with a high magnification
Electron microscope
• The electron microscope can measure down to 0.2nm
• There is no use of light, but use of a filament which heats up
• Instead of lenses, it uses magnets which make sure the signal makes a certain path to the cell
Types of electron microscopy: transmission, scanning, immune-EM and 3D ET (further in end-term
test).
Optical microscope
• The optical microscope can measure down to 200nm
• Under this size, different objects cannot be seen separated anymore, the object then needs to
be labelled to tell if there are multiple objects. For example with GFP.
How do we track single cells in a living tissue?
- 3D in vitro cultures
- Intravital microscopy of tissues in living animals
, 2. Microscopy techniques
Fluorescence microscopy
Fluorescence microscopes are capable of revealing the presence of a single molecule.
Basic function: to irradiate the specimen with a desired and specific band of wavelengths, and
then to separate the much weaker emitted fluorescence from the excitation light.
Principle:
- The specimen is illuminated with light of specific wavelength(s) which is absorbed by the
fluorophores, causing them to emit light with longer wavelengths (different color).
- The illumination light is separated from the weaker emitted fluorescence through the use of a
spectral emission filter
- Components:
• Light source
• The excitation filter
= a bandpass filter that passes only the wavelengths absorbed by the
fluorophore
• The dichroic mirror
= a very accurate colour filter used to selectively pass light of a small range of
colors while reflecting other colors.
• Emission filter
= a bandpass filter that passes only the wavelengths emitted by the fluorophore
and blocks all undesired light outside this band – especially the excitation light.
- The filters and dichroic mirror are chosen to match the spectral excitation and emission
characteristics of the fluorophore used to label the specimen.
• Multi colour images of several types of fluorophores must be composed by combining
several single-colour images.
Explanation figure:
Light of the excitation wavelength is focused on the specimen though the objective lens. The
fluorescence emitted by the specimen is focused to the detector by the same objective that is
used for the excitation. Since most of the excitation light is transmitted through the specimen,
only the reflected excitatory light reaches the objective together with the emitted light and the
epifluorescence method therefore gives a high S/N ratio. The dichroic mirror acts as a wavelength
specific filter, transmitting fluoresced light through to the eyepiece or detector, but reflecting any
remaining excitation light back towards the source.
Learning objectives:
1. Know the fundamentals of the major types f imaging modalities available.
2. Gain hands-on experience of several of the major types of imaging modalities available.
3. Understand the applications of different imaging techniques, both clinical and preclinical.
4. Able to oervorm laboratory experiments with various imaging modalities, from same
preparation to the imaging and analysis of the data.
5. Choose the most appropriate imaging modality for a particular application.
Table of contents:
1. Imaging at different scales
- Electron microscope
- Optical microscope
2. Microscopy techniques
- Fluorescence microscopy
- SPT
- FRAP
- FRET
- DIC
- Widefield
3. Resolution
- Spatial resolution
- Spectral resolution
- Radiometric resolution
- Temporal resolution
4. Super resolution
- Selective plane microscopy/light sheet microscopy
- TIRF
- PALM/STORM
- Confocal laser scanning microscopy
- STED
5. Cell culture the basics
6. Cell viability assay: MTT
7. Molecular labeling strategies
- Fluorescent probes/dyes
- Zymosan
8. Pathology
- Cancer pathology
- Computational pathology
- Tumor microscopy
- MSI and Lynch
- Breast cancer
9. Transfection methods for live imaging
- Plasmids
- Transformation and transfection
- Isolation of plasmid
10. Dynamic imaging of cancer: from cell to tissue
- Dynamic tumor growth invasion, metastasis and therapy
11. Intravital imaging using multiphoton microscopy to monitor cell dynamics
- Multiphoton microscopy
- Label free imaging of tissue structures
- Imaging windows
,1. Imaging at different scales
- UV and IR light is not used for light microscopy because it can damage the cells
- Visible wavelengths are 760nm (red) - 390nm (slightly in UV, which starts at 500nm)
The use of microscopy:
- Anatomical research
- Reconstruction of the systematics
- Viewing the specimen with a high magnification
Electron microscope
• The electron microscope can measure down to 0.2nm
• There is no use of light, but use of a filament which heats up
• Instead of lenses, it uses magnets which make sure the signal makes a certain path to the cell
Types of electron microscopy: transmission, scanning, immune-EM and 3D ET (further in end-term
test).
Optical microscope
• The optical microscope can measure down to 200nm
• Under this size, different objects cannot be seen separated anymore, the object then needs to
be labelled to tell if there are multiple objects. For example with GFP.
How do we track single cells in a living tissue?
- 3D in vitro cultures
- Intravital microscopy of tissues in living animals
, 2. Microscopy techniques
Fluorescence microscopy
Fluorescence microscopes are capable of revealing the presence of a single molecule.
Basic function: to irradiate the specimen with a desired and specific band of wavelengths, and
then to separate the much weaker emitted fluorescence from the excitation light.
Principle:
- The specimen is illuminated with light of specific wavelength(s) which is absorbed by the
fluorophores, causing them to emit light with longer wavelengths (different color).
- The illumination light is separated from the weaker emitted fluorescence through the use of a
spectral emission filter
- Components:
• Light source
• The excitation filter
= a bandpass filter that passes only the wavelengths absorbed by the
fluorophore
• The dichroic mirror
= a very accurate colour filter used to selectively pass light of a small range of
colors while reflecting other colors.
• Emission filter
= a bandpass filter that passes only the wavelengths emitted by the fluorophore
and blocks all undesired light outside this band – especially the excitation light.
- The filters and dichroic mirror are chosen to match the spectral excitation and emission
characteristics of the fluorophore used to label the specimen.
• Multi colour images of several types of fluorophores must be composed by combining
several single-colour images.
Explanation figure:
Light of the excitation wavelength is focused on the specimen though the objective lens. The
fluorescence emitted by the specimen is focused to the detector by the same objective that is
used for the excitation. Since most of the excitation light is transmitted through the specimen,
only the reflected excitatory light reaches the objective together with the emitted light and the
epifluorescence method therefore gives a high S/N ratio. The dichroic mirror acts as a wavelength
specific filter, transmitting fluoresced light through to the eyepiece or detector, but reflecting any
remaining excitation light back towards the source.
