Imaging Flashcards
What determines colour of light
Wavelength
How to make invisible objects visible
Stain with dyes
Use phase altering properties of object to create contrast
Manipulating optical pathway of microscope
What determines brightness of light
Amplitude
What has caused a revival in microscopy
Better optics Computer interfacing User friendly Multi apps Molecular probes Genetic engineering
Purpose of objective lens
Critical for resolution
Focusing on image
What does PLAN on objective mean
Flat field - whole field is flat and in focus (no blurry edges)
What does apochromat mean
Lens is colour corrected so colours are transmitted accurately. (No chromatic aberration)
Higher NA equals
Better resolution
Why is thin cover glass better
Less refraction
Purpose of immersion lenses in microscopy
Reduce refraction improving resolution
What is the purpose of the condenser
Focuses light onto specimen
Fills objective lens with light (except in dark field)
What is Abbe’s Law
Minimum resolving distance (d) is related to wavelength of light divided by the numerical aperture, which is proportional to the angle of the light cone formed by a point on the object, to the objective
What is kohler illumination
Provides an evenly illuminated field of view whilst illuminating the specimen with a wide cone of light
How does kohler illumination work
Forms two conjugate planes of light:
One contains specimen image
Other the filament from the light
Why NB to have kohler illumination
Best for recording data
Best illumination, resolution
Standardized viewing and imaging conditions
Why don’t we visualize fresh tissue
Soft
Decays quickly
Can’t get thin enough samples
Living cells too thin to see when unstained
Methods of fixing tissue
Chemical - aldehydes, oxidizing agents, protein denaturation
Freezing (physical) - liquid nitrogen, propane
What molecule forms backbone of dyes
Benzene
How do dyes stain
Acid-base interactions
Permeability and displacement
Deposition, impregnation, precipitation
Chemical reaction with colourless dye to form colorful compound
Disadvantages of acid-base (h&e)
Different tissue components with similar charge stain the same.
Cytoplasm and ECM show similar staining
Fine extracellular fibres not visible
Lipids are unstained
How to overcome acid-base shortfalls
Trichromatic stains (all acid dyes. Permeability and displacement. Shows densities of tissue rather than charge) Metal/salt deposition (enhance size and contrast of fine structures) PAS (histochemical reaction. Stains mucopolysaccharides)
How does enzyme histochemical staining work
Active enzymes in tissue react with substrate to form a primary reaction product (invisible) . Combines with dye salt to form colorful insoluble precipitate.
How does one view unstained living cells?
Phase contrast microscopy
Differential interference microscopy
What is phase contrast microscopy
light background, uses inherent phase altering properties (modify optics) image is formed by diffraction and wave recombination increases contrast
Apps- living cells/tissues, thin unstained sections
What is differential interference microscopy
Similar to PCM but uses dif mods to optics
Reveals 3D effect on tissue.
Apps- see good structural detail on thicker samples
What is darkfield microscopy
Viewed on dark bkground (DF condenser)
Principle of deflection
Only dense objects detected
Higher resolution than BF
Apps- in situ hybridization, metal staining, live cell imaging.
Apps of fluorescence imaging
Detect autofluorescence Cytoskeleton Organelle tracking Non-living and living cells Ion channels, receptors Signal transduction etc etc
What is dif between colour pixel and black and white pixel
Black and white - each pixel has one binary unit eg. On or off
Colour - has one binary unit for each primary colour therefore, three times larger.
What must you ensure when editing images
Keep editing to bare minimum
Must not change ‘message’ of image
Must be same between control and sample
Always save original
Basic outline of immunohistochemical staining procedure
Fix, embed, section Wash with PBS Incubate with BSA Incubate w primary Ab (NB positive and negative controls) Wash with PBS Apply detection system Mount and analyze
Immunohistochemical detection methods
Fluorescent substances/fluorophores
Enzymatic conversion eg. HRP
Direct vs indirect immunofluorescence vs enzymatic
Direct - easy, not v sensitive, primary Ab need to be labelled.
Indirect - more steps, sensitive, more versatile as 2ndry Ab labelled (combos)
Enzymatic - BF microscope fine, resolution not as good but can use EM, long shelf life, toxic substrates used
What is ABC system
Avidin Biotin Complex
Biotin high affinity for avidin thus good linker
High high sensitivity
Endogenous biotin = background noise
Common problems w immunohistochemistry
Non specific binding of Abs (ionic/hydrophobic interactions) = background staining
Cross reactivity of Abs to unrelated Ags
Effect of exposure time
Increase chance of pixel saturation
Less exposure = more noise
What is binning
Combination of adjacent pixels. Faster. Less resolution. Better signal:noise
Role of field diaphragm
Diameter of light path on sample
Decrease in pinhole diameter causes
Reduced light detected
Better Resolution
Decrease signal intensity
More frequent z sections
Defn gain
Increase gain =
Digital photon detection amplification
More brightness
More noise
Ways to increase intensity of fast scan
Increase laser
Increase pinhole
Problems with fluorescent microscopy
Bleedthrough Blur Bleaching Phototoxicity Background/ autofluorescence
What is bleedthrough
With broad peaks and broad collection parameters fluorescence from one channel can appear in another
What is photbleaching
Irreversible destruction of an excited fluorophore
How to counter photobleaching
Reduce scan time Use high magnification Use wide emission filters Reduce excitation intensity Use 'antifade' reagents (only for dead cells)
Dif between fluorescent and confocal microscopes
F- arc lamp, emission filter, ocular lens
C- laser, emission pinhole, PMT
What is two photon excitation
Two photons of half the energy (double wavelength) arrive simultaneously
Will only occur at point of focus therefore, no out of focus emission
Adv of two photon confocal
Deeper tissue penetration
Less phototoxicity
2nd/3rd harmonic or CARS imaging of unlabeled specimen possible
What is 2nd harmonic imaging
Irradiation with femtosecond laser results in forward scatter of light which is double frequency.
In vivo and in situ wout staining.
High res: collagen, micro tubules, skeletal muscle.
What is PSF
Point spread function
Describes how light of point object is distorted by optical system. Can be fixed using de convolution software
