My Zeiss IM microscope came set up for phase contrast. Although phase contrast is an amazing technique, the condenser was limited in its ability to be used to set up Kohler illumination.
Kohler illumination is a method of illumination of microscopic objects in which the image of the light source is focused on the substage condenser diaphragm and the diaphragm of the light source is focused in the same plane with the object to be observed; maximizes both the brightness and uniformity of the illuminated field (http://medical-dictionary.thefreedictionary.com/Kohler+illumination)
To achieve Kohler illumination with the Zeiss IM or IM35 microscopes, I needed to obtain one of Zeiss’ bright light condensers. The manual showed the microscope in use with a flip top condenser so I purchased one of these from ebay together with the extension tube and condenser diaphragm shown in the manual – had to wait a bit until one was available.
Success! I can now focus the diaphragm edge in the same plane as the image of the slide, improving illumination and contrast to the maximum available for the microscope…..at least in theory – and seems to work today when I tried it.
Carl Zeiss 0.9 NA Swing Flip Top Condenser (below). With this arrangement, I am able to open the diaphragm up to the full field of view:
Condenser mounted on microscope – both with flip top lens in and out (below):
Image of condenser diaphragm edge shown against the background slide – I have closed down the diaphragm somewhat to show the edge (below):
I also tried mounting one of my other Zeiss condensers. This one has bright field, phase and dark field options. It achieved focus although the condenser had to be much closer to the slider, but, even when fully open, the diaphragm could not illuminate the whole field evenly (below):
The following photos show a piece of plant stem I cut up from a pot in our kitchen this evening. I don’t know what the plant was. It shows may chloroplasts and cell walls.
x20 bright field:
x20 objective Phase Contrast I annulus:
x32 objective Phase Contrast I annulus:
The following two photos are both taken using the x32 objective and phase contrast I annulus. The long thin features look like bacteria but they did not move so I wonder if they come from the plant? I wonder if they might be raphides. Raphides are oxalate of calcium needles secreted by some cells. Here, the edges of my blade may have cut an epithelial cell full of raphides throwing them on the cut surface, in a similar way to that experienced by Walter Dioni in his post on http://microscopy-uk.org.uk/mag/indexmag.html?http://microscopy-uk.org.uk/mag/artfeb04/wdstem.html
This sample was collected from our garden 7 days ago and kept in an open jar of water.
The contents of the jar had separated into a top layer of moss floating on the top, an intermediate layer of very cloudy water and a bottom layer of debris on the bottom of the jar. I have tried to sample all three layers in the pictures below.
x20 objective bright field sample from bottom of jar – debris layer. This shows large numbers of bacteria.
x32 objective bright field bottom debris layer:
Moss 7 day culture bottom jar layer video x32 objective Phase I annulus:
x20 phase contrast I debris layer bottom jar:
x32 objective phase contrast I debris layer jar:
x20 objective phase contrast I cloudy liquid layer between debris on bottom and floating moss – I am not convinced that this is phase contrast even though I labelled it as such – looks like bright field to me now:
x20 bright field liquid layer between debris and moss – video:
x20 bright field one single moss plant from the floating moss on top of the jar. If you look carefully you can see hundreds of bacteria surrounding this plant:
Tardigrades are water-dwelling, eight-legged, segmented micro-animals. They were first discovered by the German zoologist Johann August Ephraim Goeze in 1773. The name Tardigrada was given three years later by the Italian biologist Lazzaro Spallanzani. They have been found everywhere: from mountain tops to the deep sea and mud volcanoes (Wikipedia).
Tardigrades, often called water bears or moss piglets, are near-microscopic animals with long, plump bodies and scrunched-up heads. They have eight legs, and hands with four to eight claws on each. While strangely cute, these tiny animals are almost indestructible and can even survive in outer space. Tardigrade is a phylum, a high-level scientific category of animal. (Humans belong in the Chordate phylum — animals with spinal cords.) There are over 1,000 known species within Tardigrade. Water bears can live just about anywhere. They prefer to live in sediment at the bottom of a lake, on moist pieces of moss or other wet environments. They can survive a wide range of temperatures and situations (https://www.livescience.com/57985-tardigrade-facts.html)
I went looking for tardigrades today in St Michael’s church graveyard in Lichfield, Staffordshire, UK. No success – sadly – so you won’t see tardigrades in the photo and video below. However, the samples I obtained from moss on gravestones, some lichen off trees and a sample from a wood chipping pile, revealed a range of life shown in the video below.
Vorticella on a commercial stained slide, viewed using Zeiss IM microscope at different magnifications. Damian and I have previously seen Vorticella live in local pond water samples – see previous posts.
I used the Bresser MikOkular camera in “new” trinocular head (second hand from ebay) – this differs from previous trinocular head in that this is the one that is recommended in the Zeiss IM microscope handbook. I noted that the previous head, although it works, has small black ring around outside of field of view that I assume means field stop is too small for scope. This new one does not have this. This new one also provides 23mm ocular attachment on trinocular port, into which the Diagnostic Instruments adapter fits directly without needing a clamp.
I also tried out a dark field condenser on microscope today – did not work well – not sure why – so photos below are back to the phase condenser that came with the microscope, used without phase annulus (i.e. in bright field mode).
x63 objective (slide upside down so light only has to go through coverslip in this inverted microscope – 63x objective has only limited working distance). This is a panorama of 17 panes, joined using Microsoft’s Image Composite Editor:
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:
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: