Flashcards in Darkfield Deck (35):
what is dark field used for?
special technique for viewing unstained specimens.
what does specimen look like with dark ground microscopy?
specimens appear bright on dark background.
describe the image with bright field.
->specimen is darker because some of the light is absorbed:
->bright background+ light from specimen participate in the final image
describe the image with dark field.
->specimen scatter/ diffract the light which does enter the objective
->no background light enters objective., thus not involved in final image formation
what are the general requirements for dark field image?
1. oblique illumination of specimen.
2. objective only collect light from specimen not background light.
3. high intensity light source
4. small structure specimen + medium with different refractive index.
what is oblique illumination of specimen ?
condenser set up illuminates specimen with a hollow cone of background light ( sides of cone are oblique)
How to make objective only collect light form specimen not background light?
when the NA of the objective is less than that of the condenser
what is example of specimen consisting of small structure?
what is principle of cark field microscope?
->direct rays from the light source eliminated by"
1. a patch stop
2. special condenser
->background light create hollow cone of direct light passing outside acceptance angle of objective
->specimen scatter or diffract some background light, this diffracted light enter objective
how is the image of specimen created?
the diffracted light enter the objective so specimen appears bright on a dark background
How is direct rays eliminated by special condenser?
by internal reflecting surfaces
What is diffraction maxima in diffraction by a single object point?
Diffraction maxima are bright points formed between zero order and second order diffraction. 3rd order is lost during scatter.
What is special features of dark field microscope ?
2.special oil immersion objective
What is special in dark field microscope in terms of condenser?
1. Standard condensers adapted with low power objective.
2. Use special dark field condenser called: cardioid or paraboloid.
3. No condenser iris
What is NA of low power objective?
How can special dark field condenser be purchased?
1. Purchase separately
2. purchase as part of a universal condenser
What is cardioid condenser like?
Direct light diffracted to enter the objective
What is parabioid condenser like?
Reflecting surface scatter light to enter the objectives
Why should use special oil immersion objective?
1. Dark field condensers produce very high NA cone of background light
2. objective with NA greater than 1.2 (short focal length)will still collect some of this background light.
What is the solution if objectives collect wine background light?
1. Use an oil immersion lens
2. Use a lens (NA=1.3or 1.4) with a build in iris diaphragm.
What is the NA of oil immersion lens?
Most standard oil immersion lenses are 1.25
What is function of this built in diaphragm?
1. allow reduction of NA to suit dark field setting
2. allow objectives to be used for ordinary purpose
When to use the oil immersion objectives?
To maximise NA
To oil condenser
How to abject the oil immersion setting?
1. Objective iris fully open
2. Adjust so that background becomes dark
What does field of view look like when condenser focused too high?
Black circle with black centre
What does field of view look like when condenser focused too low?
Black circle with black centre
What does field of view look like when condenser is focused?
Bright spot of minimum size but maximum brightness
What to do if bright spot/illumination part is off centre
Results in small field of view on high power not including bright spot-->
Centre the condenser
What does knurled ring control?
Internal iris diaphragm
->close to eliminate bright background
How to recognize iris
look into isolated lens from above
How to set up dark field?
1. Start on low power (return to low power if trouble shooting)
2. Oil condenser to bottom of slide (condenser NA> 0.95)
3. Focus on specimen
3. Focus condenser
4. Centre condenser
How to prepare specimen for dark field?
1. Small well separate particle
2. Mounting medium: different refractive index
3. Minimise dirt, bubble, scratch
4. Specimen not dry out
5. thin slides (
Why use different refractive index mounting medium?
Dark field show large difference in refractive index
Why minimise dirt, bauble, scratches
These will all add to the dark field image