DaRk Field &PhAsE ConTRaST MiCRoScoPy

• Light microscopes–Bright field microscope–Dark field microscope–Phase contrast microscope–Fluorescent microscope

I. Dark field contrast microscopy takes advantage of objects that “scatter” light - this requires a special condenser that can “angle” the incident light

II. Phase contrast microscopy takes advantage of objects that alter the phase of incident light - This requires “phase rings” in the condenser and in the objective lens

III. Fluorescence microscopy take advantage of inherently fluorescent Material of biological objected that can be fluorescenlty labeled.

Single Cell Organism ( Tetrahymena) observed with:

“Advanced” Light Microscopic Methods

Bright Field

Dark Field

Phase Contrast

Fluorescence

(Dr. Gorovksy)

Recall that a specimen (e.g a live cell) with few structures that strongly absorb light provides littlecontrast with bright field microscopy

condenser lens

objective lens

e.g., the single celledprotist Tetrahymena

Dark Field Microscopy: Some objects can alter the light path by diffraction & or light scattering

condenser lens

objective lens

If the light pathway is angled without a specimen the field is dark...

cond

ense

r len

s

objective lens

…but any object that diffracts light will be detected!

Dark Field seen

cond

ense

r len

s

objective lens

• The most commonly employed light microscope allows

light to pass through the object -> bright field microscopy

• The limitation of such microscope is that transparent &

semitransparent objects are not readily visible (needs

staining)

• Visibility ->contrast between object & background

• Contrast can be greatly improved by creating a dark

background

Dark field microscopy

Principle• If the aperture of the condenser is opened

completely & darkfield stop is inserted below the condenser, the light rays reaching the object forms a hollow cone

• If a stop of suitable size is selected, all direct rays from the condenser can be made pass outside the object

• Any object within this beam of light will reflect some light in to the objective & become visible

• Method of illumination of object-> object become self illuminous against dark background -> dark-field illumination

Phase contrast microscopy

• The phase contrast microscopy is a special adaptation of the light microscopy & helps to obtain a clear picture of living or unstained cells.

• The adaptors convert minute difference in phase changes in transmitted light due to refractive indices of all cell organelles in to perceptible shades of grey

• This allow organelles of the living cell to become visible with fair contrast in them.

Principle of phase contrast microscopy (PCM)

• Different wavelength of our light rays detect differences in colours

• Different shade of grey are distinguished to our eyes due to differences in amplitude of light rays

• PCM convert invisible small phase changes caused by the cell component in to visible intensity changes

• What is phase change & how r they caused?• Phase changes are caused by the biological

material through which the light ray passes• If a material is absorbent, it causes the ray to

undergo a change in amplitude. This is distinguished by our eyes

• Eg---- light passing through window glass n without them

• This is because the type of light rays reaching our eyes in the two different cases are different

• Light rays goes change in amplitude when they pass through glass

• The change increases with the thickness of glass plates

• Similarly when light ray passes through a living cell they undergo an invisible phase changes due to different refractive indices & thickness of the cell organelles

• Consider three different material & its effect

on light ray

1)Transparent & non absorbent material of

higher refractive index

2)Transparent, non absorbent but thicker

3)Transparent & absorbent

Inference:1)A light ray undergoes a phase change

depending on refractive index of the transparent material through which it passes

2)The phase change is in direct proportion to thickness of the material

3)A light ray undergoes a change in amplitude when it passes through an absorbent material

• More refractive index & thickness, more change in phase

• If biological material absorbs light ray they show contrast but living cell generally do not absorb light ray.

• Cells & their component do not show phase change

• The value of phase change is ¼ the wave length of light

• But this phase change is not distinguished by our eyes

• The principle behind PCM is to convert the

undistinguishable phase change in to

distinguishable phase change in terms of

variation of contrast, with the help of two

adaptors called the annular diaphragm &

annular phase plate.

Phase contrast is obtained with the help of the annular diaphragm by separating the central & direct ray from the diffracted rays