Microscopy Flashcards
Light Microscopy
- Uses visible light and system of lenses to magnify images
- Commonly used to examine tissues
Super-Resolution Light Microscopy
- Encompasses fluorescence techniques with capabilities to resolve objects below the classical diffraction limit of optical resolution
Electron Microscopy
- Uses a beam of concentrated electron against the sample at a greater resolution
- Does not work on living cells because it will fry them
Atomic Force Microscopy
- Scanning probe microscope which primary roles include measuring properties such as magnetism, height, and friction
Fluorescent Microscopy
- Revolutionized the field of cell biology
- Provides an enormous contrast (black, white, etc.)
- Linked to specific probes, such as antibodies or small molecules to localize discrete cell types or cellular compartments
- Possible by virtue of the principle of fluorescence
Principle of Fluorescence
Increased absorption of photons -→ reaches an excitatory state
When released from the excitatory state, emission of photons at a longer wavelength
First Barrier Filter
Lets through only blue light with a wavelength between 450 and 490nm
Restrict; Allows light that excites
Beam Splitting Mirror
Reflects light below 510nm and transmits light above 510nm
Second Barrier Filter
Cuts out unwanted fluorescent signals, passing the specific green fluorescein emission between 520 and 560nm
Restricts; Allows you to view the range you’re expecting
Phase Contrast Microscopy
- If staining is not possible
- Takes advantage of the change in phase caused by regions of the sample have differential refractive index to introduce context into the image
- Look at occlusion within cells
Refractive Index
Measurement of a bending of light ray when passing through one medium and into another
Differential Interference Contrast Microscopy
- If staining is not possible
- Another method of generating contrast is based on the interference pattern generated by light passing through adjacent regions of a sample
Fluorescent Protein Technologies and in vivo Imaging
- The creation of fusion proteins between a protein like GFP and a cellular gene in yeast or any other organism permits us to light that cellular product in a living organism
- The presence of GFP does not interfere with the function of the cellular protein
- No antibody or fixation required
Jellyfish Aequorea Victoria - Green Fluorescent Protein (GFP)
- System to TURN ON GFP: Aequorin + Coelenterazine (Ca2+) Aequorin (Blue emission energy transfer to GFP) GFP
- Light around 480nm will image a longer wavelength
GFP and DsRed
- Two families of fluorescent proteins
- Both tinkered and engineered to emit most of the visible spectrum
- DsRed and GFP functions are similar but DsRed can emit different wavelengths
Photoactivable Fluorescent Proteins
- Useful for looking at the dynamics of specific molecules
- Can be turned on with a laser of specific wavelength and turned off by an intense laser, photobleached (bright light)
PhotoActivable Localization Microscopy (PALM)
- Only activates specific molecules at a time
- Current iterations of PALM technology enable 20nm resolution
Eukaryotic Cell
The size of an organelle is just at the limit of resolution for a conventional microscope
Factors that affect the Quality of Data
- Contrast
- Microscope Design
- Resolution
Contrast
- The use of stain to selectively mark cellular components
- To observe cellular structure stains or dyes were necessarily introduced into samples to increase contrast between different cellular constituent
Stains
Stains impart differential absorption of light as it passes thru samples, thus providing contrast to an otherwise uniformly translucent sample
- Different stains will bind to different regions
- “Differential absorption of light”
- Common stain: Hematoxylin/Eosin (H&E)
Hematoxylin (BLUE)
Binds arginine and lysine AA side chains enriched in nuclei (Basophillic)
Eosin (PINK)
- Stains protein the cytoplasm
- Staining is done on fixed tissue (dead)
Benefits of Fixed Tissues
- Fixation procedures allow for stains to enter the sample (more stable)
- Helps in catalog and archiving samples
Antibody Probes
Antibodies can be linked to fluorescent molecules and become stains themselves
- Antibodies to many cellular antigens exists to recognize many cellular constituents
Covalently Coupled Markers
Enhance contrast in the cell or sub-cellular region in which the antigen was contained
Cell Impermanent
Opens hole using alcohol to allow the stain to enter
Digitization
- Of image data using digital cameras extend the ability to accurately detect very weak signals and increase contrast
- Digitization permits processing to enhance image quality or presentation
Digitization Examples
- In Calcium imaging, where the sperm enters. Color coding identifies different concentrations of calcium
- 3D structures of organelles can be determined through digitization and processing
Microscope Design - 3D Reconstruction
- Removes out of focus light
- Amending the microscope design
Standard Fluorescence Microscope
- Out of focus light
- As you image a particular focal plane (optical section) light from above and below that plane is illuminated transmitted to the detector
- Fuzzy image that is a composite of the plane of focus and the out of plane light
Cortical Microscope
Producers an image of all the light coming off the sample both above and below the point at which you are focused
- “ Just look where you are focused”
Deconvolution
Algorithm is used to remove out of focus based on the behavior of light in the system
Laser Scanning Confocal Microscope
- Removal of out of focus light through fine engineering
- Confocal pinholes are controlled precisely by and relative to one another where light illuminates to the point of focus and what emitted light makes it to the detector
- Only fluorescence emitted from the point of focus makes it through the second pinhole to the detector
- Coupled with digitization of data permits us to view the cell in 3D
Resolution
Development in super-resolution technology
Diffraction Limited Resolution
The Raleigh criterion defines the lower limit of resolution
The Raleigh Criterion
two images are just resolvable when the center of the diffraction pattern of one is directly over the first minimum of the diffraction pattern of the other
- The size of the disc, its interference rings, and the overall resolution of the optical system is a function of the following: (Resolution eq: wavelength/numerical aperture)
- Lower value for resolution is desirable: Decreased Resolution = Increased Light
