Zeiss IM/IM35 microscope

Diatom photos processed using Helicon focus 6 from Stowe Pool jar culture 20/1/2018

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!

Andy

Diatom as in life:

Combined frame – great views of organelles. Note also the large number of bacteria in the slide (small dark rods):

Depth map:

3D model:

Empty Diatom Skeleton:

Combined frame – shows segmentation of diatom skeleton well:

3D model:

Diatom skeletons and Volvox (green circles in middle):

Combined frame:

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:

3D Models:

Demonstrating 3D structure of cell walls in moss from St Michael’s Church graveyard 20/1/2018

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!

Andy

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:

Helicon Focus 6.8.0 – 3D modelling of microscopic images

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.

Andy

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):

 

3D models:

Changing the Zeiss 47 17 61 flourescence epi-illuminator condensor on the Zeiss IM microscope

When I purchased my Zeiss IM inverted microscopy just over one year ago, there was no epi-fluorescence condenser present. I purchased one off ebay (Zeiss part 47 17 61 – in this post I will call it the EFC). Unfortunately, the filter slider was stuck. Luckily another in full working condition came up very cheaply recently and I have now replaced the first one. However, the technique for taking one out and putting in another was not obvious so this post is to record the process for future reference, should I or anyone else want to make this change on the Zeiss IM or IM35 microscope (both use same component). I will probably wish to do this myself. After I find a way to fix the stuff filter slider, I also have a Zeiss IM35 and will probably put the spare one on that, although epi-fluorescence requires use of the fluorescence cube for the microscope and I only have one of those and they are quite expensive second hand, so I doubt I will be acquiring a second one soon!

It is worth noting that the EFC provides epi-illumination but was only designed to be used for fluorescence microscopy. I am hoping to adapt the fluorescence filter cube to remove one of the filters and replace with plain glass so that I can use it for more general epi-illumination. Today’s project was the first step in this process as it allowed me to slide out of the way a blue filter in the filter slider which was stuck in the light path. In fact this needed to be done prior to the removal of the old EFC and – given that the reason for Today’s project is that it was stuck – this required the use of a hammer to be achieved! I was gentle and put something between hammer and microscope with softening covering (spare piece metal coveted in kitchen absorbent roll) and managed to succeed without causing further damage, although my heart was in my mouth while I did it.

Andy

Zeiss 47 17 61 epi-flourescence condenser  (EFC). This is the one that I removed today. You will notice that there is one slight difference between this one and the new one I installed in its place, which is shown in the later photos on this post – the adjustment screws for centering the illuminator light are longer in the new version. These can be seen in the later views from the back of the microscope.

Back of microscope – epi-illuminator is central round bit. The illuminator will be attached here (not attached in this photo):

The following pictures show an illuminator attached to the back of the Zeiss IM35 microscope. It is the square box hanging off the back, opposite the eyepieces (oculars). It would be exactly the same with the IM microscope as the only difference between the two varieties of microscope is that the IM35 has an inbuilt camera port whereas the IM does not have one.

View of epi-fluorescence condenser (EFC) from next to the eyepiece turret. This view shows the filter slider within the epi-fluorescence condenser component that was stuck on the first one I installed on the microscope. The epi-fluorescence condenser comprises both the white and black round parts on left side of the picture. The black part is seen to contain a circular filter holder with small black handle. This can be removed. For installation of the whole component or taking it out again, both the filter slider and the round filter holder need to be removed. This allows the whole component to then be slid out through the hole in the casing on the left – that hole is currently obscured by the black circular component which sits over it.

The the following photo shows the EFC from the other side. The long thin silver metal handle controls the aperture control within the EFC. To install or remove the EFC from the microscope, this handle must be first removed so that the EFC can slide out through the hole, otherwise it will obstruct it. The handle is removed by twisting it anti-clockwise, which will unscrew it from the EFC.

PROCESS OF REMOVAL OF EFC:

1. Start at back of microscope.

2. Locate the four retaining screws on the back which hold the etc In place and remove them.

3. Unscrew and remove long thin handle.

4. Locate and remove tiny screw from end of filter slider. This screw prevents filter slider being removed from EFC. Once screw is remembered bed, slide filter slider completely out of the EFC.

5. Remove content recluse filter holder (left below) from EFC. Note in this picture, filter slider is also shown as present but of course by now you will have already removed that in previous steps.

 

6. By now the EFC should be quite loose. Note it only pushed again eyepiece turret and not attached to it so there are no screws etc that need to be loosened there. The EFC should now be easy to thread through the hole in the back of the microscope and removed.

7. Insert new EFC by reversing the above.