This is where the epi-illumination technique comes into its own. The following pictures are of a UK 1£ coin – showing up surface relief differences and tiny scratches that are not otherwise visible to naked eye.
I have used the white balance adjust function in the Bresser MikroCamLabII software (camera control software) to remove the effect of the colour tinge imparted by the mirror in the filter cube on the microscope.
Following photos x4 objective:
Helicon Focus stack – interestingly this does not appear to have improved a great deal on above best focus image:
Helicon Focus 3D model showing relief (below):
x2.7 objective – single image:
x2.7 objective, Helicon Focus stack (below):
Helicon Focus 3D model of above image:
The following images are taken on the Zeiss IM microscope using epi-illumination with the Zeiss 46 63 01 – 9901 filter cube from which I removed the filters. They show the effect of the coloured semi-silvered mirrors that remain in situ – these are required to direct the light and without them the microscope would not provide epi-illumination. Perhaps I can exchange them for non-pigmented mirrors in future?
Human-skin-commercial-slide-x20-obj-Kholer-illum-red-mirror-190218I.png – comparison image to above – same field of view – shows that epi-illumination is low contrast compared to transmitted light, particularly on this type of specimen. I have read similar in an article on Micscape website:
Comparison image of Aspergillus via transmitted light:
The following picture is a photo composite created from a video that goes through focus using Helicon Focus stacking software – this allows the best bits of focus at different levels to be combined:
I have purchased a spare second hand polarising filter cube. The Zeiss IM microscope (similar to IM35) uses an epi-illumination system for epi-fluorescence. However it was not designed with simple bright-light epi-illumination in mind. I am hoping that I can adapt this filter cube to allow me to introduce epi-illumination in bright-field on this scope.
My first step is to remove the fluorescence filters from the cube. These are extremely expensive filters so I want to ensure that I keep them carefully and know where to put them back if I wish to put the cube back to normal. Removal of the filters is very easy – a plastic ring holds them in and is very simple to remove.
I have 3 of these filter cubes. I am not sure if they have same filters or not so I have taken photos below during removal of the filters from this cube in order that I know how to replace them in the future.
List of filters included on filter cube:
The following pictures show the cube with the filters in situ, before removal:
Removal of filters from the cube:
The two filters at front (one each side) have labels on each filter. The following photos show the labels on those filters:
After filters are removed, it is possible to see the semi-silvered mirrors within the filter cube. The following pictures show that these are them selves coloured. I do not know how this will affect epi-illumination. They may removal too much light for effective epi-illumination and therefore require replacement or allow enough light that I can leave them in situ. I can easily see through them by eye allow they do give colour tinge to view:
The following comes from the manual for this scope and shows the originally used trinocular head with this microscope.
I was very excited to obtain a second hand trinocular head for my Zeiss IM microscope. However, when I took the phototube off the head, I wasxshocked to find it rusted inside- this was on both the phototube and head where they attached to each other.
The picture below shows the rust on the head. The good news is that this is the only place with rust. I took the head apart and there is no rust inside. So, I guess moisture has just got into this bit which was made of unprotected steel. The sellers are unlikely to have known it was there as I doubt they ever had reason to take the phototube off the head.
My wife suggested I do some research on the internet to find out the best way to remove this rust. I came up with italic acid as a particularly powerful rust remover, although with warnings to open windows due to the fumes. This sounded like the beasty I needed so I ordered some from the all-encomposing ebay. It arrived last week so I had a go this weekend at using it to remove the rust.
I started with phototube. This is just a simple metal tube so there are no optics to ruin. I prepared the italic acid solution – a few teaspoons in hot water stirred with an ice lolly stick (I did not want acid to ruin our household spoons!) I then prepared a shallow glass container of the acid (I did not know whether the acid would burn through plastic although I suspect it would be OK as the italic acid crystals were sent in a plastic container).
The following picture shows the container plus the amazing result of sitting the phototube is the acid for only a couple of minutes. The rust just disappeared as if by magic! Immrsion in this acid is very effective. A good wash under the tap followed to wash off the acid then 30 minutes in the oven at very low temperature to dry it out thoroughly so it did not immediately rust again. A light covering oil finished the job.
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: