In standard bright-field illumination, colorless specimens such as unstained cells and micro-organisms (so-called phase objects) are just barely visible because of their very low
optical density. These specimens do not absorb light in a relevant manner; they only modify the phase of transmitted light by about one-quarter
wavelength (λ/4). Such low differences in phase are associated with minimal differences in density; they cannot be directly perceived by the eye or a photographic film. Thus,
phase and interference contrast are the most widely used methods for examinations of phase specimens,
Depending on the configuration and properties of the phase ring in the objective, the natural phase shift within phase preparations (circa λ/4) is amplified, so that the resulting final difference in phase of the specimen and the background is around one-half wavelength (λ/2, positive phase contrast) or one wavelength (λ, negative phase contrast). In positive phase contrast, the specimen is visible with medium or dark grey features, surrounded by a bright halo, and the background is of higher intensity than the specimen. In negative phase contrast, the contrast of these features
is inverted. In both methods, the intensity of contrast is solely determined by the design of the objective lens phase ring and the optical density of the specimen and its surrounding medium. Since phase contrast is
usually optimized for observations of native cells in their natural environment and calculated for an amplification of λ/4-phase-shifts, the quality of traditional phase contrast images will be degraded the
more the natural phase difference deviates from λ/4.
Standard phase contrast is affected by several specific limitations: (a) Halo artifacts are prevalent, especially in specimens, which induce large phase shifts. (b) The condenser iris
diaphragms should always be wide open so that the contour sharpness and the depth and planarity of field cannot be influenced by the condenser aperture. (c) The intensity of contrast cannot be adjusted with
regard to the optical characteristics of the specimen, and all existing phase shifts remain constant and cannot be trimmed. (d) The phase ring within phase contrast lenses may reduce the image quality in
general when compared with corresponding lenses designed for bright-field. (e) Phase contrast images may be
negatively affected in certain circumstances, e. g. in living cells when cultured and examined in small
Thus, a new technique for improved
visualization of unstained transparent specimens has been developed based on bright-field images promising an extraordinary high resolution and
contrast. When compared with traditional phase contrast images, the new method can lead to superior results in many specimens.
Principles of ultra-high contrast amplification
In unstained phase objects, the natural dynamic range is much lower than in any other kind of specimen because there are only minimal differences in regional brightness, contrast and
optical density. Thus, ultra-high contrast amplification is necessary for visualizing these low natural differences
with extended clarity. This procedure can be carried out by use of particular software, so-called
HDR-software (HDR = high dynamic range rendering). HDR-rendering is the first step in this specific postprocessing procedure.
In the first line, HDR-software has been developed for contrast equalizations in digital photographs showing high ranges in brightness and contrast so that some parts in single shot
photographs appear over- and underexposed.
When specimens or illumination modes are affected with a very low variation in brightness and contrast (e.g. bright-field images of unstained phase specimens), the same software can
act as ultra-high contrast amplifier. For this task, two or more single images have to be superimposed; otherwise, HDR-rendering cannot work. When the respective low contrast images are superimposed
on each other
and HDR-rendering is appropriately carried out, all pre-existing very low contrasted details which may just barely be visible in the original images can be transformed into a high contrast
picture. It is
important for the authenticity of the visual information within the finally reconstructed images that only pre-existing details which are situated in at least one of the original images and associated with
pre-existing low differences in brightness can be amplified and high contrasted by HDR-rendering.
In HDR-imagges, the number of tonal values can be enlarged from 256 (typical of 8-bit images) up to around 4.3 billion graduations per channel. Such high graduations can neither be
printed nor observed on a standard screen in a satisfying manner. Therefore, these images have to be transformed into new images characterized by a normal number of tonal values (so-called low dynamic range / LDR
LDR images can be visualized on a normal screen or printed out by a normal printer. This separate step in image processing (tranformation of a HDR image into a new LDR image) is called tone mapping. When the tone mapping is adequately carried out, the resulting final image shows more tonal nuances, sharpness, and detail in fine structures, and it is free from any visible over- or under-exposed zones. Also very low natural differences in brightness, corresponding to minimal differences in density and very discrete local phase shifts, are transformed into high contrast.
When the procedure of tone mapping is finished, the resulting reconstructed phase images can be optimized further in additional steps with the help of normal digital image processing.
In particular, the gradation, histogram, brightness and contrast level can be re-adjusted in tiny steps, so that the contrast can be optimized with regard to the existing real density and phase shift (digital
contrast trimming). Moreover, the color balance can be re-adjusted in color images. Finally, each unstained phase structure is documented in optimum contrast, and the regional contrast will no longer be determined
by the optical density within the specimen and the surrounding medium. Facultatively, digitally reconstructed color images taken from colorless specimens can be transformed into black and white.
According to our own practical evaluations, Photomatix Pro can be successfully used for HDR rendering in low contrast bright-field images. The software needs at least two separate
single images which have to be superimposed. Thus, either two bright-field images can be taken from a specimen in identical views, or only a single image has to be duplicated on the computer by re-naming and
re-saving. Photomatix Pro offers several different tools and presets for image rendering. For ultra-high contrast amplifying of bright-field images of unstained phase objects, the tone mapping should be carried out
using the so-called “Details Enhancer”. To obtain a suitable quality in this special task, the strength of contrast has to be set to the maximum level (“100”) when low regional differences in brightness have to be
amplified. Moreover, the light smoothing has to be regulated to a high level. In unstained phase objects, the color saturation should be reduced to a low level to achieve a good reproduction of all tonal values.
When color saturation is set to “zero”, phase structures are directly amplified in black and white.
FDR Tools advanced can also be used for high contrast amplification. By this software, also a single one-shot photograph can be transformed into a high dynamic range image.. In
HDR-rendering, the so-colled “Creative mode” leads to the best HDR images in most cases when low contrast bright-field material has to be processed. For the tone mapping procedure, the so-called “Compressor
mode” should be preferred promising most complex results. W
The Figures 1-3 demonstrate that digitally reconstructed bright-field-based images from unstained specimens (phase objects) can lead to a very high level of contrast and detail,
optimized sharpness, and enhanced depth and planarity of field. Figure 1 shows a thin-layer crystallization in normal bright-field (a), common phase contrast (b), reconstructed by HDR-rendering (c), and
converted in B & W (d). The clarity of all structures is improved when HDR-rendering is carried out, whereas the phase contrast image contains haloing and blooming. In the original bright-field image, the
colorless thin crystals appear with the lowest clarity and contrast.
Fig. 1: Unstained alum crystallization, thin-layer preparation, horizontal field width: 1.10 mm
a: conventional bright-field, b: normal phase contrast, c and d: contrast amplified bright-field
Figure 2 demonstrates the usefulness of the new method with regard to observations of native and unstained cells. The epithelial
cell of the oral mucosa is just barely visible in bright-field (a) because of its low contrast. In normal phase contrast (b) the cellular
structures appear in higher contrast, as usual for phase contrast images. In the HDR-color reconstruction (c) and in the B & W
conversion (d) fine details within the cell and the nucleus appear with improved distinctness and resolution.
Fig. 2: Native epithelial cell from the oral mucosa, horizontal field width: 0.07 mm
a: conventional bright-field, b: normal phase contrast, c and d: contrast amplified bright-field
Figure 3 shows a stained cross section of a pine leaf in normal brigh-field and rendered by HDR software. Such specimens cannot
be examined well in normal phase contrast illumination because of their high optical density. Nevertheless, in digital phase contrast
based on high contrast amplification and adequate contrast equalization, fine details within the specimen appear in superior clarity
when compared with conventional bright-field imaging. More detail and structural nuances can be visualized eith the new technique, especially in regions with a high local density.
Fig. 3: Leaf of a pine, stained section, horizontal field width: 1,5 mm
a: normal bright-field, filtered in ideal white, b: contrast amplified bright-field,
ideal equalization of brightness in specimen and background (image b)
All reconstructed images are either completely free of haloing and blooming, or the halo and blooming effects are significantly
reduced. Thus, very- fine and low-contrasted details remain visible in enhanced contrast and clarity. The reserve for post
-magnification seems to be higher than in usual phase contrast images so that a higher level of sharpness and resolution will be
visible in HDR-reconstructions when a small region of interest is clipped from the total image.
Further technical develepements
A new generation of improved digital live observation microscopes could be created if the new techniques were integrated into a
software-based workflow for image processing in real-time microscopy. For this purpose, bright-field images could be directly
detected by a suitable CCD-camera equipped with a high-resolution sensor. When this camera generates 30 single frames per
second for instance, each pair of two consecutive images could be superimposed and processed to an optimized HDR-image
within the refresh rate period. In this case, 15 HDR-images could be created per second so that native living cells and other
suitable specimens could be directly observed by this new technique. When the rendering algoriothm should also be able to work
with single images instead of pairs, even 30 images per second could be created in he given example. All technical and optical
advantages presented above could be used for digital live-microscopy of native motile specimens. Further technical developments
could aim toward a real-time trimming of several quality determining parameters in the so-created HDR-based image sequences
when the contrast enhancement and other parameters (e.g. gradation and brightness) might be manually adjusted during live
-examination. In this respect, the new techniques described here should be of interest to manufacturers engaged in the development of new technical solutions for digital live microscopy.
Piper, J.: Software-based contrast amplification in bright-field imagery (in German)
(submitted: 20.01.09, accepted: 02.02.2009)
Mikrokosmos 98, Heft 5, 315-318, 2009
Piper, J.: Ultra-high contrast amplifying in bright-field images
(submitted 11.07.2009, accepted 16.07.2009)
Microscopy Today x / y, zz-zz, 2010
(magazine of the Microscopy Society of America / MSA)
Cambridge University Press
Copyright: Joerg Piper, Bad Bertrich, Germany, 2010