Aeskulap-Stab
Introduction
Principles of 
 Phase Contrast
Principles of 
 Relief Phase Contrast
Material and  
 Methods
Results
Further technical 
 Developements
Discussion
Summary
Links
Contact
References
Results

When phase-contrast objectives that were not highly corrected were used, the quality of the images could be dramatically improved. Relief phase contrast achieved a higher contrast with enlarged focal depth and often improved sharpness. The relief of the specimen mostly appeared more three dimensional, similar to interference contrast images. The planarity of the microscopical image was improved, because effects of spherical aberration were lower. Halo artifacts were often reduced.

Even when highly corrected phase-contrast objectives were used (e.g. planachromatic or planaopchromatic lenses), the images resulting from relief phase contrast had more contrast, enlarged focal depth and more apparent three-dimensional aspects.

Compared with interference contrast, relief phase contrast oftenly produced images with higher or complementary information of specimen details.

The brightness of the microscopical image was lower than using conventional phase contrast, because the area of the illuminating light beams was more reduced (about – 2,0 or – 3,0 EV). Therefore, higher light intensities have been necessary.

As specimens were illuminated from one direction by oblique light beams, the background of relief phase-contrast images sometimes appeared with variable brightness, especially when objectives with low magnifications were used. Similar effects are also known from interference contrast and reflection contrast (6, 7, 8).

All relevant findings are presented in the table which compares the major similarities and differences of conventional phase contrast with relief phase contrast.
 

 

Conventional
Phase Contrast

Relief
Phase Contrast

use objectives from all manufacturers

no

yes

illuminating light beams

concentric

eccentric

condenser aperture diaphragm

open

Open or smaller (various modifications)

3D images / relief-effects

no

yes

contrast

good

higher

sharpness

good

potentially higher (depending on the specimen)

depth of focus

narrow

higher

resolving power

high

sometimes lower (when aperture diaphragm is smaller)

halo artifacts

possibly higher

sometimes lower (depending on the specimen)

influence of spherical aberration

high

lower

influence of chromatic aberration

high

sometimes lower

brightness of the microscopical image

high

lower

homogenity of background

high

sometimes lower (when low-magnifying objectives are used)


The following images demonstrate the effects of relief phase contrast in comparison with conventional phase contrast and interference contrast.


Fig. 6:
Small drops of water (condensation on a micro slide), surrounded by oil, thin-layer preparation, cover glass.
Objective 40x, conventional phase contrast (left), relief phase contrast (right).
Horizontal field width (HFW) = 0.1 mm


Fig. 7:
Bucal epithelial cells, basic corrected 40x objective (Olympus A 40x 0.65NA), HFW = 0.1 mm.
Conventional phase contrast (left), relief phase contrast (right)


Fig. 8:
Buccal epithelial cells, highly corrected 40x objctive (Leica Phaco Plan Apo 40x 0.75NA), HFW = 0.1 mm.
Conventional phase contrast (left), relief phase contrast (right)


Fig. 9:
Buccal epithelial cells in a very thin layer of saliva with Newton´s rings. 40x objectives, HFW = 0, 07 mm.
Relief phase contrast using Leica Plan 40x 0.65NA objective (left),
Interference contrast using Leica NPL Fluotar 40x 0.65NA ICT objective (right).
Exposure by electronic flash.
 

Fig. 10:
Thin-layer crystallisation of a water solubile pigment. Coverslip preparation with Newton´s rings.
Conventional phase contrast using Leica Plan 40x 0.65NA objective (left)
Relief phase contrast using Leica Plan 40x 0.65NA objective (center)
Interference contrast using Leica NPL Fluotar 40x 0.65NA ICT objective (right)
HFW = 0.1 mm


Copyright: Joerg Piper, Bad Bertrich, Germany, 2007
 

 

[Introduction]
[Principles of Phase Contrast]
[Principles of Relief Phase Contrast]
[Material and Methods]
[Results]
[Further technical developements]
[Discussion]
[Summary]
[Links]
[Contact]
[References]