Microscopy Flashcards
The resolving power of the light microscope is the …
… the smallest distance between two objects
Abbe’s Law
d = (lambda)/2* N.A
where d is the maximum resolution, with values lower not being resolvable
Whether you can see an image depends on …
… the brightness and not the size
Fluorescence Diagram (3)
- Excitation to higher energy level
- Decay back to lower energy level
- Energy given out as fluorescent light
Fluorescent Microscope (3)
- First barrier filter - lets trough only blue ligh with a wavelegth between 450 and 490 nm
- Beam spliting mirror - reflects light below 510nm but transmits light above 510nm
- Second Barrier Filter - cuts out unwanted fluorescent signals, passing the specific green fluorescein emission between 520 and 560nm
GFP facilitates …
… facilitates live cell imaging
What does GFP stand for?
Green Fluorescent Protein from the pacific ocean jelly fish
autofluorescent allows direct visualisation
Why use wide-field microscopy?
Used fixed filter blocks e.g. for specimens but motorised filter wheels and shutter enable more complex time-lapse experiments
What does CLSM stand for?
Confocal Laser Scanning Microscopy
Confocal Microscopy Technique (3)
- Fluorescent specimen is illuminated with a focused point of light from a pinhole
- Emitted fluorescent light from in-focus point is focused at pinhole and reaches detector
- Emitted light from out-of-focus point is out os focus at pinhole and is largely excluded from detector
Pinhole confocal imcroscopy limits …
… detection to in-focus fluorescence known as “optical Sectioning”
Using sectioning of the cell, the …
… 3D reconstruction can be built up
What does WFM stand for?
Wide Field Microscope
Deconvolution can signigicantly enhance the …
… clarity of WFM images depending on the sample properties
How to have a higher resolution? (3)
- Selective Illumination (TIRF)
- Beyond the diffraction limit
- Electron microscopy
What does TIRF Microscopy stand for?
Total Internal Reflection Fluorescence Microscopy
What is a method of Super Resoluton Light Microscopy?
PhotoActivation Localization Microscopy (PALM)
STochastic Optical Reconstruction Microscopy (STORM)
Using fluorophor, we can detect nanometres by …
… switching on and off fluorophors
Another type of Super Resolution Light Microscopy
STimulated Emission Depletion (STED)
STED Method (2)
Excitation spot created with doughnut spot to create..
A spontaneous emission, but gives off very little light and signal.
Confocal can now detect up to …
… 1 micrometre
3D Light Microscopy (2)
- Imaging in 3D with less damge
- Sheet Plane Imaging Microscopy (SPIM) uses a sheet of light rather than a point
How is contrast achieved in the EM?
Specimen is stained with an electron dense material
- Electrons passing through the specimen are scattered by the structures
- Others pass through parts of the cell not stained to form an image on a phosphorescent screen.
Scanning EM is analgous to the …
… Stereo Binocular light microscope because it looks at surfaces rather than through the specimen.
