STUDYING DIATOMS WITH THE HELP OF CIRCULAR OBLIQUE LIGHTING (COL)


By Yannis Tsamouris, ATHENS GREECE

 

                        In my February article I presented some results from using COL in diatom photography. From that time I received several questions about the method I am using. I feel obliged  to apologize for my  late response but the truth is that my method is the result of extensive trials and errors and  I have only a vague theoretical knowledge of the issues involved. In the following paragraphs I will try to describe the equipment I am using and the processing of the data.

                       The basic hardware consists of a Zeiss Photomicroscope III which I aligned according to the instruction given by Paul James in his Micscape article "Aligning the Zeiss Photomic's "Headrig"" on which I adapted a Leitz Heine condenser. The method is shown in the following photographs. The first photograph shows the base were I fitted the Heine condenser which I took from a spare Zeiss condenser after removing the lens and the iris diaphragm.

heine a

                                          After removing the central part of the Heine condenser from its base I inserted the cylindrical part into the above dovetail base as it is shown in the next photo.

heine 1

                                      I screwed the lower cap on the bottom part of the Heine condenser body and centered it with the screws.

heine 3

                                        Last I inserted the whole set on a regular Zeiss condenser holder. Under the Heine condenser you can see a Risley prism of my own construction which diverges the light for oblique lighting which I seldom use.

heine 4

                                        After fixing the condenser holder on the microscope body, it is of paramount importance to align the Heine condenser with the optical axis of the microscope. To achieve this one has to observe the concentric light rings on the back  focal plane of the objective lens to disappear symmetrically around the edges of the objective. It is also very important to use a green filter (for example I use the Zeiss Jena v232g) or a combined green and a blue filter to reduce the unnecessary glare. It is very helpful also to oil the condenser under the slide even if a dry objective is used. The lighting facility is a Schott fiber optic 250W illuminator.

                                         The camera I am using  is an Omax CMOS microscope dedicated camera with a 14 Mbits sensor which is very easy to use and gives very nice results. Recently I started using the image stacking of the Helicon focus software which gives the possibility to process the final image by retouching and choosing the best focused detail of each photograph of the stack. Finally I process the photographs with the help of the Adobe Photoshop to remove the unwanted elements of the background and at the same time to improve the contrast of the subject. The steps are the following:

            1. use the magic lasso to select the subject
            2.save the selection with the save selection function in the selection menu
            3.in the edit menu use the copy and  the paste function
            4. activate the background layer and use the load selection function and then the invert button to select the background of the image
            5.go to the edit menu and copy then paste the selected background
            6.use the Gaussian blur filter at 120 radius or more to blur the background
            7.go to image-adjustments and use the invert function or press ctrl and I. The background becomes dark.
            8. then if you like you can choose from image adjustments the photo filter function to give a particular tone to the background. Finally activate the layer with the darkened background             and using the opacity button reduce it to an acceptable level.
            9. activate the subject layer and use the curves or the brightness/contrast function from the image adjustment menu to improve the quality of the subject picture.
          10. use the merge function in the layer menu and get your final picture
          11. I am using extensively the healing brush tool to remove unwanted elements from the background.

                                        One can appreciate the results in the following photographs. It is an Actinoptychus splendens specimen and the final image was composed from 13 photographs.

actinoptychus

                       And its three dimensional representation:

actinoptychus 3D

                          Or again with a difficult subject (Campylodiscus adornatus) composed from 127 photographs:

campylodisc

                                      And its three dimensional representation:

campylodiscus 3D

                              The above results is another indication that in the light gathering capabilities of the light microscope there is a lot of information that it is usually lost because we do not know how to extract them or we do not have the proper means to do it.

All comments to the author are welcomed.

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