Zeiss IM/IM35 microscope

Algae growing on Pot Plant Slide 1/12/2018 – Zeiss IM microscope; QHY6 mono camera vs. Bresser Mikrocam SP5.1 colour camera

About a month ago, I placed a slide in my pot pond outside and the algae on the photos below are those that have grown in situ. The slide was in the top ten centimetres of the pot pond – where sunlight would hit the slide every day.

This has also been an opportunity to try out my QHY6 mono astronomical camera on my Zeiss IM microscope & compare photos taken with that camera with those taken with my trusty Bresser Mikrocam SP 5.1 camera – the main issue is the small sensor on the QHY6 gives highly magnified image & loss of detail as in monochrome.

Andy

x4 objective, QHY6 mono camera:

x4 objective, Bresser Mikrocam SP5.1 colour camera:

x32 objective, QHY6 mono camera:

x32 objective, Bresser Mikrocam SP5.1 colour camera:

Crossed-polarised images of set of five NWA meteorite thin section slides from Sahara (Morocco)

Set of five thin sections of NWA meteorites from the Sahara in Morocco.

These are from un-numbered meteorites and were sold as set of ebay by SDFossils in the UK. I do not know whether they are from different meteorites or five sections from the same meteorite. They are all show chrondules.

A meteorite is debris from space that survives impact with the ground.

NWA Meteorites: Northwest Africa (from https://en.wikipedia.org/wiki/Meteorite): Meteorite markets came into existence in the late 1990s, especially in Morocco. This trade was driven by Western commercialization and an increasing number of collectors. The meteorites were supplied by nomads and local people who combed the deserts looking for specimens to sell. Many thousands of meteorites have been distributed in this way, most of which lack any information about how, when, or where they were discovered. These are the so-called “Northwest Africa” meteorites. When they get classified, they are named “Northwest Africa” (abbreviated NWA) followed by a number. It is generally accepted that NWA meteorites originate in Morocco, Algeria, Western Sahara, Mali, and possibly even further afield. Nearly all of these meteorites leave Africa through Morocco. Scores of important meteorites, including Lunar and Martian ones, have been discovered and made available to science via this route. A few of the more notable meteorites recovered include Tissint and Northwest Africa 7034. Tissint was the first witnessed Martian meteorite fall in over fifty years; NWA 7034 is the oldest meteorite known to come from Mars, and is a unique water-bearing regolith breccia.

I don’t think my meteorite thin sections are not from Mars or the Moon! They are probably from a range of different unclassified meteorites – and this explains differences seen in the photos below.

It is quite possible that one or more of these thin sections come from “NWA meteorite 869”. The meteorite bulletin describes NWA 869 as follows at https://www.lpi.usra.edu/meteor/metbull.php?code=31890:

Northwest Africa 869

Northwest Africa

Find: 2000 or 2001

Ordinary chondrite (L4–6)

History: It is quite clear that meteorite collectors in Northwest Africa have discovered a large L chondrite strewn field at an undisclosed location. At least 2 metric tons of material comprising thousands of individuals has been sold under the name NWA 869 in the market places of Morocco and around the world. Individual masses are known to range from <1 g to >20 kg. It is certain that NWA 869 is paired with other NWA meteorites, although no systematic survey has been done. It is also possible that some stones sold as NWA 869 are not part of the same fall, although dealers are confident that most of the known masses are sufficiently distinctive from other NWA meteorites in terms of surface and internal appearance that the error rate should be fairly low. Scientists are advised to confirm the classification of any specimens they obtain before publishing results under this name.

Petrography and Geochemistry: (A. Rubin, UCLA) A fragmental breccia of type 4–6 material; one thin section dominated by an L5 lithology gave olivine (Fa24.2).

Classification: Ordinary chondrite (L4–6); W 1, S3.

Specimens: A 189.3 g type specimen is on deposit at UCLA.

Andy

Below with x25 Leitz objective, crossed polarised filters, Zeiss IM microscope, Optovar x1.0 setting:

Below with x32 Zeiss LWD objective, crossed polarised filters, Zeiss IM microscope, Optovar x1.0 (1st) and x2.0 (2nd photo) setting:

Successful polarised microscopy of NWA Sahara (Morocco) meteorite slide using Zeiss IM microscope & Optovar

It has taken me some time to get there but I am here now – success with polarised microscopy through the Zeiss IM microscope!

The following images are of a Chrondrite-rich NWA meteorite from the Sahara desert, Morocco, purchased from SDFossils on ebay.co.uk May 2018. There is no date on the slide nor meteorite designation number.

It wasn’t obvious exactly which bits were needed but eventually I found that a Zeiss 47 36 68 polarised filter fits into the filter slot below the objective turret – this gives plane polarisation alone. However the purchase of another polarised filter (this time a modern one) designed to fit in the filter slot above the condenser (if you are used to normal upright microscopes remember this is an inverted microscope so turn everything upside down in your imagination and it then makes sense) allows for crossed polarisation. The latter filter can be turned through 90 degrees.

The following images show the Zeiss 47 36 68 filter and where it fits onto the microscope:

The following images show the second polarised filter and where that fits onto the Zeiss IM microscope – both the above filter and this second one are needed to achieve crossed polarisation:

This is also the first time I have used my new Optovar – this is another ebay purchase with optics in excellent condition.

Optovar on Zeiss IM microscope:

The different positions of the Optovar’s ens wheel showing different magnifications offered – these are in addition to that provided by the eyepiece/camera and objective lens:

The Optovar was not originally designed for the Zeiss IM microscope nor for the dual head block I am using to allow me to place a camera below the binocular head. This extra round accessory with the black lever on it is necessary to act as a spacer between dual head block and Optovar so they can fit together, otherwise the lens on top of the Optovar projects too far out and so does that on the bottom of the dual head block and they won’t fit together. The extra accessory with the level is in fact an aperture diaphragm attachment for use with illuminators and I have another one under my illuminator (see diagram above):

The following information about the Optovar comes from http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artjun12/fs-optovar.html:

The Optovar magnification changer was originally introduced by Carl Zeiss Oberkochen / West Germany in 1954 as an accessory to the Stand W. This microscope was the first post-war microscope designed by Dr. Walter Kinder at Oberkochen and incorporated a number of major innovations. As such it deserves a separate essay. What I would like to discuss here is the Optovar magnification changer. Still today it is a much valued accessory to any Zeiss microscope of the Standard series and has, in principle, been incorporated in many newer stands since.

The Optovar is an intermediate tube with a magnification changer, it also features an Amici-Bertrand lens (=auxiliary microscope) and an analyser.

The multi-step magnification changer introduces factors of 1x, 1.6x, and 2.5x .Later versions have the factors 1x, 1.25x, 1.6x, and 2x. The Optovar integrated in the larger Universal, Photomicroscope, and Ultraphot offers the factors 1.25x, 1.6x. and 2x. This allows the microscopist to bridge over the magnification gaps between objectives in small steps and saves him from having to change the eyepieces frequently. The optical systems to achieve this are arranged on a rotatable disc and can be switched in as desired.

For instance, with a standard set of objectives 2.5x, 10x, 40x, and 100x and an eyepiece 8x the following magnifications can be obtained:

20 – (25) – 32 – (40) – 50 – (63) – 80 – (100) – 125 – (160)

200 – (250) –320- (400) – 500 – (630) – 800 – (1000) -1250 -(1600) and

2000x (= the DIN series in steps of 1.6x, steps of 1.25x in brackets – figures are rounded off).

The Amici-Bertrand lens as it is officially called, together with the eyepiece, forms what is commonly also known as a phase contrast centering telescope or an auxiliary microscope, and is well known to users of polarizing microscopes. It serves to view the rear focal plane (exit pupil) of the objectives. In other words: it shifts the filament plane into the image plane. In the Optovar, the A-B lens is set in a helical mount that can be controlled by a separate wheel so one can focus up and down to reach the rear focal plane of objectives from 16x to 100x. With it the microscopist can see, focus on, and center the phase rings of his phase-contrast system. He can also check the setting of the condenser aperture diaphragm and the correct centering of the filament of the light source.

It is a most useful tool to inspect the optical system for any misalignment, vignetting, dirt in the objective or air bubbles in the oil immersion. With it one can also detect any cracked or damaged lenses, fungus or delamination in the objective. The main convenience is the elimination of having to remove the eyepiece and to insert the auxiliary telescope each time one wants to check the objective’s aperture or the phase rings.

Lastly, the Optovar includes a swing-out analyser or slot to insert one. The biologist needs only to place a polarizer under the condenser to render his instrument into a simple polarizing microscope to examine crystals or other birefringent material.

All images below with Bresser 5MP Microcam through Zeiss IM microscope, bright field. The advantage of polarised light is that it brings out birefringence in the crystal structure of the rock which can be used to identify the type of mineral in the rock and gives pretty pictures! The advantage of doing this with a biological microscope is the ability to use a range of objectives and imaging accessories that might not normally be available on polarised microscopes – however the disadvantage is that the biological microscope is more likely to suffer from strain in the glass of the instrument and accessories and objectives, which itself will affect the images produced.

Medium resolution images of NWA meteorite:

25x Leitz objective:

Unpolarised light:

Plane polarised light:

Crossed polarised light:

Effect of Optovar on above meteorite thin section showing extra magnification this accessory provides:

x1.0 lens:

x1.25 lens:

x1.6 lens:

x2.0 lens:

High magnification images of NWA meteorite chondrite:

x63 objective + Optovar:

x63 objective, x1.0 Optovar lens = x63 (plus magnification from camera = approx. real magnification of 1758 times – see calibration slide below):

x63 objective, x1.6 Optovar lens = x101 (plus magnification from camera – see below for calibration to convert this into actual magnification). Individual crystals can be seen and it is also obvious that they have a preferred direction:

Calibrating above to give actual magnification – for this purpose I used a calibration slide where each division is 0.01mm.

Calibration slide 0-01mm per division x63 obj x1-6 Optovar 090618:

Therefore across the slide photo there are 7 divisions or 7 x 0.01mm = 0.07mm. On my laptop computer screen the photo appears as 197mm across, meaning that magnification of the on-screen image compared to the original structures on the slide’s actual size = 197/0.07 = 2814 times magnification.

What we found in tap water in Lichfield

This weekend we have had guests – and they asked me what tap water looked like under the microscope. We filled 18 Eppendorf 1.5ml centrifuge tubes with tap water and centrifuged them at 10000 revs/minute for 10 mins. We then pipetted off the top 95% of the water and moved the remaining water into a single tube and centrifuged it again at similar settings. Removing the top 90% of that tube and then putting the bottom few drops onto a slide and using x40 objective revealed that there is was a small amount of grit and organic debris only. We could not see bacteria. This suggests that our tap water is pretty clean and safe.

Andy

Grit:

Organic debris:

Chloroplast movement in Elodea (a form of pond weed)

Cellular Turbulence. Rhys and I went with the family to the Big Bang Show at the NEC in Birmingham. On the Zeiss stand were a number of microscopes – and on one of them some Elodea showing chloroplast movement around the cell. This pond weed has particularly mobile chloroplasts and the site is amazing. This movement is referred to as cyclosis or cytoplasmic streaming.

See the photo and video below – wow! I wonder what the small things are, much smaller than chloroplasts? Organelles or parasitic protozoa or bacteria? Magnifications here using x32 and x63 objectives so bacteria would show up at this magnification.

Andy

Some facts about chloroplasts:

Chloroplasts are organelles, specialized compartments, in plant and algal cells. The main role of chloroplasts is to conduct photosynthesis, where the photosynthetic pigment chlorophyll captures the energy from sunlight and converts it and stores it in the energy-storage molecules ATP and NADPH while freeing oxygen from water. They then use the ATP and NADPH to make organic molecules from carbon dioxide in a process known as the Calvin cycle. Chloroplasts carry out a number of other functions, including fatty acid synthesis, much amino acid synthesis, and the immune response in plants. The number of chloroplasts per cell varies from one, in unicellular algae, up to 100 in plants like Arabidopsis and wheat (from https://en.wikipedia.org/wiki/Chloroplast)

The average chloroplast is about 3 µm (micrometers) in diameter. In one square millimeter of the surface of a leaf, there are about half a million chloroplasts (from www.answers.com/Q/What_is_the_size_of_a_chloroplast)

Chloroplasts in vascular plants range from being football to lens shaped and as shown in Figure 1, have a characteristic diameter of ≈4-6 microns (BNID 104982, 107012), with a mean volume of ≈20 μm3 (for corn seedling, BNID 106536). In algae they can also be cup-shaped, tubular or even form elaborate networks, paralleling the morphological diversity found in mitochondria. Though chloroplasts are many times larger than most bacteria, in their composition they can be much more homogenous, as required by their functional role which centers on carbon fixation. The interior of a chloroplast is made up of stacks of membranes, in some ways analogous to the membranes seen in the rod cells found in the visual systems of mammals. The many membranes that make up a chloroplast are fully packed with the apparatus of light capture, photosystems and related complexes.  The rest of the organelle is packed almost fully with one dominant protein species, namely, Rubisco, the protein serving to fix CO2 in the carbon fixation cycle. The catalysis of this carbon-fixation reaction is relatively slow thus necessitating such high protein abundances (from http://book.bionumbers.org/how-large-are-chloroplasts/)

Components of cells seen in photos and video:

   

Today’s photos and video:

If you want the wow factor go straight to the videos at bottom of page using x63 objective! I am quite excited by the views with the x63 objective below as this is first time I have used it so successfully with this microscope. The slide is turned upside down and Kohler illumination has been achieved using my “new” (second hand off ebay) NA 0.9 bright field condenser with Zeiss 475638 illuminator collimation tube (at least I think that is what it is for!).

The slide was prepared using free hand sections of Elodea leaf using razor blade put on slide with drop water and covered with cover slip. Edges of cover slip help firmly to slide by electrical insulating tape strips and then slide turned upside down and put on microscope stage (upside down as Zeiss IM microscope is an inverted microscope).

Photo of Elodea leaf section (cut free hand with razor blade) x32 objective, bright field, Zeiss IM microscope, showing cell walls and chloroplasts:

Photos of Elodea x63 objective bright field, now also shows small inclusions much smaller than chloroplasts – in later videos these are shown to move as well as chloroplasts:

Videos – first two videos are with x32 objective, bright field:

Videos – next videos used x63 objective, bright field:

Kohler illumination on Zeiss IM microscope using Carl Zeiss 0.9 NA Swing Flip Top Condenser, Zeiss 475638 extension tube and Zeiss condenser diaphragm

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.

Andy

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):

Plant stem from kitchen

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.

Andy

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

Microscopy of culture of moss collected 7 days ago

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.

Andy

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: