I have downloaded and installed a piece of freeware called “CombineZM” which rivals Helicon Focus in its ability to stack multiple images and produce 2D combined image at different focus planes. It also is not limited to the same extent in terms of the type of image it imports. It works up to Windows 10.
However, it does not produce 3D images.
The following pictures are re-processed from previously obtained stacks – see previous posts for comparable stacked images using Helicon Focus. The images below were stacked in CombineZM.
I love this image – vacuoles and other organelles are seen in the diatom, together with the crenulations of the chiton shell on the creature. Outside the main diatom, hundreds of bacteria can be seen, amongst various debris. See the end of this post for information on how to interpret what you see in this picture and the next one of the empty skeletal remains of a dead diatom.
Interpreting the pictures above:
The following diagram comes from Wikipedia and shows the different parts of the diatom. Which of these can you see in the photos above? https://en.wikipedia.org/wiki/Diatom
Here are some diatom photos I processed today using Helicon Focus 6 – the pictures are either combined across 20-26 sub-frames or 3D models produced from that data.
All on Zeiss IM microscope with Bresser MikrOkular camera.
Preparation for photography of these samples included 20ml pipette in jar – 5ml formalin 10% added to kill specimens so that they don’t move during photography – important when taking pictures at different focus distances. Helicon does not really work on live specimens!
Diatom as in life:
Combined frame – great views of organelles. Note also the large number of bacteria in the slide (small dark rods):
Empty Diatom Skeleton:
Combined frame – shows segmentation of diatom skeleton well:
Diatom skeletons and Volvox (green circles in middle):
Following are variations on above using different settings for smoothing and radius in Helicon Focus – variously brings out more or less the foreground diatom skeleton that goes from top to bottom just to right of Volvox:
A sample of moss from St Michael’s graveyard in Lichfield collected today 20/1/2018 was soaked in water and a small drop pipetted onto a slide and coverslip added. The pictures below were taken using my Bresser MikrOkular camera Zeiss IM microscope with 32x long working distance objective and Phase Contrast (Ph1 annulus). This is an amazing activity to do when it is snowing outside and solar observing is definitely not possible!
The following is the combined image of 15 subs at different levels – combined to produce single in-focus image using Helicon Focus:
The cell walls can be clearly seen in the image above.
The following picture is the depth map from the above slide – something not obvious in the flat image above:
Its relevance is that this ability to pick up depth from multiple photographs taken at different levels in the sample allows the software to model the 3D structure of the sample.
Frames captured from 3D model of the field of view above generated by Helicon Focus – cell walls are made of collagen and last after death of the cell (which is why you can sit on a wooden chair). The images below show the 3D structure of the cell walls in the sample from different angles:
I am very excited!
These images are amazing. My first attempts to produce 3D images from microscopic images on my Zeiss IM microscope. This software takes multiple images taken at different focus levels and combines them into a 3D model.
Tonight, I took a commercial stained slide of Volvox (pond water organism – ball of cells) and took a series of 7 photos and produced the 3D model frames of which can be seen below that. Following that, are photos and models of 4x objective view of stained flatworm.
Still photo – if you look carefully you can actually see that the central Volvox has collapsed centrally from its original ball in life during the fixing process:
The following is an image created from the seven images combined together to use the best focused parts of each image – this removes as far as possible the effect of depth of parts of the image on the slide to bring out as much detail as possible:
Frames from 3D model – this shows the central collapse and the different heights of different parts of the sample – down a microscope it looks like it is flat but the model shows that this is not really the case:
Using zoom function on 3D model – this blew my mind – the ability to zoom in to model in Helicon – however viewers must understand that the programme an combine detail from the images but not create detail where it is not present – so this image shows contours well but there is no individual cellular detail in this image as it was missing in the original photos:
3D depth map of the model:
Helicon combined image of one end of a flatworm (12 original images at different focus levels):