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]