Aeskulap-Stab
Introduction
Luminance Contrast
Relief 
Phase contrast
Aperture Reduction 
Phase Contrast
Aperture Reduction 
Darkfield
Digital
 Phase Contrast
 Photomicrography
and Analysis
Cytometry in
Reflection Contrast
Capillaroscopy
Video-Endoscopy
Calculation of
Cardiovascular Risk
Behavioral 
Risk Management
Efficiency in
Rehabilitation
Diagnostics in 
Rehabilitation
Complementary 
Medicine
Publications
Curriculum vitae
University 
of Oradea
U.N.E. Brussels
Journals of optics
and microscopy
Optical Society 
of America
Diagital Photomicrography
and Analysis

Digital documentation of microscopic images is very important in routine examinations as well as in fields of research. In comparison with conventional analogues images, digital techniques have various specific advantages.

Images are immediately seen on the camera display and poor images can be retaken immediately. The ISO speed level can be changed for each image according to the brightness of the specimen, in most cases up to ISO 400 when compact or bridge cameras are used and up to ISO 1600 or 3200 when a digital mirror reflex camera is available. In high ISO speed levels effects of noise in digital images are lower than effects of visible grain in analogue images. Some high end digital cameras can be used with a flash and off-camera shoe for high speed synchronisation up to 1/2000 or 1/ 4000 s in automatic TTL mode.

Digital images can be optimized by post-processing with the help of standard image optimizing software. Thus, fundamental parameters which determine the image quality can be influenced (gradation, brightness, contrast, colour saturation, white balance, sharpness). Moreover, impurities such as dust particles, artificial bubbles etc. can be removed by software based  retouching.

Digital images can be directly printed or integrated into other presentations or documents (e.g. scientific or medical letters). Moreover, they can be directly transferred to other addressees via the web. In special fields, e.g. histopathology, cytopathology, hematology, oncology and other medical areas, interesting findings can be directly discussed by long distance conferences based on telemedicine when digital images are available.

Decades ago, manufacturers of light microscopes had already developed connection modules that allow their microscopes to be used with analog cameras for photomicrography. For digital cameras, similar modules have not yet been widely developed. For digital microphotographic documentation specialised microscopy cameras are predominantly available which are usable just for photomicrography.

From an economic standpoint, consumer digital cameras are an attractive alternative solution for photomicrography as they are much cheaper and produced in high numbers. Moreover, high-end consumer digital cameras can produce high resolution images, similar to analogue or film images.

However, not all digital cameras are suitable for photomicrography because of potential artifacts, vignetting effects, deficiencies of coverage or sharpness and several other reasons.

Therefore, some technical solutions for digital photomicrography were elaborated and rigorously and successfully tested in practice, using several models of current digital cameras with various designs (compact, bridge or prosumer, and single-lens or mirror reflex). All aspects of mechanical and optical adaptation, exposure, manual focus, image control, potential artifacts, automatic and manual flash light control, monochrome techniques and other modes of light filtering based on various astronomic interference filters were evaluated using several suitable digital cameras. The quality of the respective images was visually compared using high resolution monitor images and colour prints. In addition to this, several software-based methods in postprocessing were evaluated, including stacking and sandwich techniques, HDR rendering and conversions in digital darkfield-like images.

These evaluations led to the result,  that several digital cameras (compact, bridge, mirror reflex) show good qualities when used for photomicrography. Thus, such cameras can be regarded as practicable and attractive technical solutions, cheaper than specialized microscopy cameras and useful for other photographic purposes, too. The image quality is comparable with the quality of conventional analogue images taken on a 24 x 36 mm film when the CCD- or CMOS-sensor is at least 6 or 8 megapixels.

When monochromatic green filters are used, the quality of monochrome digital images can be optimized even more. Contrast, sharpness and resolution can be especially improved because any chromatic aberration is eliminated. Several astronomic filters, especially contrast-booster filters and apochromatic filters work as RGB-intensifiers; by these filters, the primary colours (red, green, blue) are enhanced selectively. UV- and IR-cut filters and fringe killer filters can block ultraviolet and infrared or violet and short-wave blue radiation. Thus, the quality of colour images can be  fundamentally improved; colour saturation, colour purity, contour sharpness and resolving power can be enhanced in life microscopy as well as in photomicrography.

The depth of field can be significantly enhanced by use of stacking software, and ultra high ranges of brightness and contrast can be equalized with HDR software.

Moreover, some modern digital compact cameras are suitable for video documentations of dynamic processes in microscopy.

Figure 1: Native epithelial cell, objective oil 100x, phase contrast,
double-colour sandwich technique, superposition (stack) of 11 single images,
bulb and flash light illumination
 

Figure 2: Native epithelial cell, objective oil 100x, phase contrast,
monochromatic green light, superposition (stack) of 11 single images,
detail image with subcellular structures, horizontal field width: ca. 0,01 mm
 

Figure 3 and 4: Asteroid-arm, stereo-microscope, epi-illumination, objective 4x,
conventional still image (left), superposition (stack) of 7 single images (right),
visible improvement of the focal depth
 

Figure 5: Comparison of several digital cameras, suitable for photomicrography
a: Skeleton of a sea urchin, compact camera, 5 megapixels (Canon Powershot A 95)
b: Human retina, ultra compact camera, 6 megapixels (Casio Exilim EX-Z 110)
c: Gnat, mirror reflex camera, 8 megapixels, flash (Canon EOS 350D)
d: Living black gnat larva, bridge camera, 7,1 megapixels, flash (Olympus Camedia C 7070)
(Photographs taken by Timm Piper)

Figure 6: Bacillus megatherium, phase contrast, objective oil 100x, several green filter techniques
a: No filter
b: Yellow-green filter
c: Monochromatic green filter
d: Monochromatic green filter, black and white image
 

Figure 7: Software-based modification of the gradation histogram, small scratch on a polymer layer,
improved detection of the surface texture by colour contrast
a: Conventional brightfield (photograph taken by Timm Piper)
b-d: Multicolour images, achieved by variing the gradation histogram in multiple steps
 


Figure 8: Cartilage, flash, objective 40x, no filter (left), astronomic contrast booster filter (right)
visible improvement of the colour resolution by RGB-enhancement




Figure 9: Butterfly wing, polarized light, lambda compensator, range of exposure: 9EV,
quartet of single images at different exposures (left), HDR reconstruction (right),
complete equalization in brightness and contrast achieved by high dynamic range rendering




Figure 10: Radiolarian (length: 0.26 mm), photomicrograph based on interference contrast,
digital conversion in a darkfield-like appearance

 

Publications:

Piper, J.: Technical realisation of digital photomicrography using digital cameras for consumers
Part 1: Technical basics, compact and bridge cameras (in German)
(submitted: 07 August 2006, accepted: 10 August 2006)
Mikrokosmos 96, Heft 2, 111-124, 2007

Piper, J.: Technical realisation of digital photomicrography using digital cameras for consumers
Part 2: Digital mirror-reflex cameras, flashlight-imaging, future developements (in German)
(submitted: 07 August 2006, accepted: 10 August 2006)
Mikrokosmos 96, Heft 3, 173-182, 2007

Piper, J.: Adapting consumer digital cameras for photomicrography: 
Technical aspects
(submitted: 18 September 2006, accepted: 04 October 2006)
European Microscopy and Analysis106, S5-8, 2007

Piper, J.: Advanced techniques of software-based three-dimensional reconstructions and optimizations in sharpness of microscopic images (in German)
(submitted: 21 October 2006, accepted: 26 October 2006)
Mikrokosmos 97 / 4, 221-229, 2008

Piper, J.: Benefit of astronomic filters in microscopy and photomicrography (in German)
(submitted: 22 January 2007, accepted: 23 January 2007)
Mikrokosmos 97 / 1, 43-49, 2008

Piper, J.: Annular artifacts in mikroscopy and digital photomicrography
classifikation, causality, physical characteristics (in German)
(submitted: 05 February 2007, accepted: 07 February 2007)
Mikrokosmos 97 / 3, 169-176, 2008

Piper, J.: Use of astronomic filters in light microscopy and photomicrography
(submitted: 20 February 2007, accepted: 21 April 2007)
Microskopy Today 15 / 3, 30-35, 2007
(magazine owned of the Microscopy Society of America / MSA)

Piper, J.: Multicolor contrast effects by monochromatic astro filters - utilization in light microscopy and photomicrography.
(submitted: 12.08.2008, accepted: 06.12.2008)
Microscopy Today 16 / 5, 20-26, 2008
(magazine owned of the Microscopy Society of America / MSA)

Piper, J.: RGB-Splitting and multi-shot techniques in digital photomicrography - utilization of astronomic RGB-filters in true color imaging.
(submitted: 17.10.2008, accepted: 13.11.2008)
Microscopy Today 17 / 1, 48-51, 2009
(magazine owned of the Microscopy Society of America / MSA)

Piper, J.: Use of Baader astronomy filters in microscopy and photomicrography
- a propaedeutic intrstruction (in German)
Monograph for the Baader-Planetarium Company, 43 pages, July 2008

Piper, J.: Use of Baader RGB-filters (Contrast-Booster- und Semi-Apo-filter) in microscopy und photomicrography
- a propaedeutic instruction (in German)
Monograph für Fa.for the Baader-Planetarium Company, 8 pages, Jan. 2009

Piper, J.: Use of monochromatic astronomy filters in microscopy und photomicrography
- an introduction for practice (in German)
Monograph for the Baader-Planetarium Company, 8 pages, Jan. 2009

Piper, J.: Use of Baader RGB-color filters for multi-shot-techniques in microscopy  and photomicrography
- an introduction for practice (in German)
Monograph for the Baader-Planetarium Company, 7 pages, Jan. 2009

Piper, J.: Software-based three dimensional reconstructions and enhancements of focal depth in microphotographic images
(submitted: 27. March 2007, accepted: 15. Mai 2007)
In: Méndez-Vilas, A., Diaz, J. (Eds): Modern Research and Educational Topics in Microscopy, Vol. 2, 900-905
Formatex Research Center, Badajoz, SPAIN, 2007
ISBN-13: 978-84-611-9420-9
http://www.formatex.org/microscopy3/index.htm
http://www.formatex.org/microscopy3/pdf/pp900-905.pdf

Piper, J.: Wide field photomicrography in large specimens - comparison of various techniques (in German).
(submitted: 18. Oct. 2007, accepted: 22. Oct. 2007)
Mikrokosmos 97 / 5, 304-307, 2008

Piper, J.: Wide field and deep focus imaging in photomicrography - optical and software-based techniques.
(submitted: 17.12.2007, accepted: 30.12.2007)
Microscopy Today 16 / 2, 32-37, 2008
(magazine owned of the Microscopy Society of America / MSA)

Piper. J.: Use of software to enhance depth of field and improve focus in photomicrography.
(submitted: 02.01.2008, accepted: 06.03.2008)
European Microscopy and Analysis 113, 15-19, May 2008

Piper, J.: Improved techniques for imaging of high ranges in brightness and contrast in photomicrography (HDR- und DRI-techniques) (in German).
(submitted: 09.04.2008, accepted: 11.04.2008)
Mikrokosmos 97 / 6, 375-382, 2008

Piper, J.: Improved imaging of three-dimensional transparent specimens in modified darkfield techniques and digitized interference contrast (in German)
(submitted: 02.06.2008, accepted: 03.06.3008)
Mikrokosmos 98 / 2, 117-123, 2009

Piper, J.: Image processing for the optimization of dynamic range in photomicrography.
(submitted: 27.06.2008, accepted: 05.09.2008)
European Microscopy and Analysis 117, 23 (1), S5-S9 (EU), 2009

Piper, J.: Software-based amplification of low differences in brightness and density in bright field imagery (in German)
(submitted: 20.01.09, accepted: 02.02.2009)
Mikrokosmos 98 / 5, 315-318, 2009

Piper, J.: Improved techniques for imaging of three-dimensional transparent specimens in advanced darkfield and interference contrast modes.
(submitted: 15.02.2009, accepted: 06.04.2009)
Microscopy Today, 17 / 3, 20-28, 2009
(magazine owned of the Microscopy Society of America / MSA)

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 Universtiy Press
doi: 10.1017/S1551929510000222

Piper, J.: Software-based stacking techniques to enhance depth of field and dynamic range in digital photomicrography. In: Hewitson, T.D., Darby, I.A. (Hrsg.): Springer Protocols, Methods in molecular biology 611, Histology protocols, Chapter 16, 193-210, Humana Press, 2010

Piper, J., Chmela, G.: Optimized observation and photomicrography of small structures in diatom shells near the resolution limit in normal light microscopy (in German)
(submitted: 12.08.2009, accepted: 14.08.2009)
Mikrokosmos 99 / 2, 115-122, 2010

Piper, J., Chmela, G.: Advanced techniques in observation and photomicrography of subcellular structures in diatom shells
(submitted: 11.09.2009, accepted: 12.09.2009 )
Microscopy Today x / y, zz-zz, 2009
(magazine of the Microscopy Society of America / MSA)
Cambridge University Press

Piper, J.: Image processing for the optimization of dynamic range and ultra-high contrast amplification in photomicrography
(submitted: 06.03.2010, accepted: 12.03.2010)
In: Méndez-Vilas, A., Diaz, J. (Eds): Modern Research and Educational Topics in Microscopy, Vol. 3, xxx-xxx
Formatex Research Center, Badajoz, SPAIN, 2010
ISBN
http://www.formatex.org/microscopy3/index.htm
http://www.formatex.org/microscopy3/pdf/pp900-905.pdf

Piper J.: High-grade imaging of diatoms and radiolarians in light microscopy - optical and software-based techniques
(submitted: 05.05.2010, accepted: )
Diatom Research


Copyright: Joerg Piper, Bad Bertrich, Germany, 2010

 

     

[Introduction]
[Luminance Contrast]
[Relief Phase Contrast]
[Aperture Reduction Phase Contrast]
[Aperture Reduction Darkfield]
[Digital Phase Contrast]
[Digital Photomicrography and Analysis]
[Cytometry in Reflexion Contrast]
[Capillaroscopy]
[Video-Endoscopy]
[Calculation of Cardiovascular Risk]
[Behavioral Risk Management]
[Efficiency in Rehabilitation]
[Diagnostics in Rehabilitation]
[Complementary Medicine]
[Publications]
[Curriculum vitae]
[University of Oradea]
[U.N.E. Brussels]
[Journals of optics and microscopy]
[Optical Society of America]