I obtained some frog’s eggs from a friend’s pond and we are growing them in my ad hoc “pond” (a bucket that also contains my pond weed – see previous post).
I sacrificed a couple of the eggs today to see what they looked like under a microscope.
The picture below shows the general structure of a frog egg.
My photos show a large amount of green-coloured but otherwise featureless oval objects which look to me similar to starch when I look at bread under the microscope – I suspect (but am not sure) that this is the contents of the yolk sac.
Apart from this, I also see multiple small motile cells that look like bacteria in the video. Seems strange to have bacteria in the egg but it in water so possibly is not sterile. Also possible that the bacteria come from outside egg and have contaminated sample from water in bucket when I prepared the slide.
All x32 objective, bright field, Zeiss IM microscope.
Large numbers of small dots in next photo which ? bacteria – especially see centrally
Even more small black dots here which are moving visually…..
The following videos show these moving black dots which I suspect are bacteria:
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.
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:
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 image comes from this resource and is labelled to show which structure is which (Walter Dioni, http://microscopy-uk.org.uk/mag/indexmag.html?http://microscopy-uk.org.uk/mag/artfeb04/wdstem.html):
ep = skin of the stem – col = colenchyma – par = cortical parenchyma (colenchyme + parenchyma form the cortex) –cb = cambium interfasciculaire – Paq.vasc. = package or vascular bundle formed by the phloem (ph) and the xylem (xy). Later it will be seen with more details – moelle, the central cylinder of parenchyma (the pith). Cambium plus pith form a central cylinder: the stele. The raphides are oxalate of calcium needles secreted by some cells. Here, the edges of the razor blades have cut an epithelial cell full of raphides throwing them on the cuted surface
I think the dark brown/black areas between cells in my photos below are vascular bundles seen end on, with the thick spiral supporting structure shown in the photos in my photos of the vertical section seen at the link above acting to block out light when seen end on. I can not identify raphides in the pictures below but I think I may have seen them in another post – see http://roslistonastronomy.uk/plant-stem-from-kitchen
My photos from today:
x10 objective, bright field:
x20 objective, bright field:
x32 objective, bright field:
x10 objective bright field crossed polarisation – varying rotation angle of one filter (below):
I tried adding in the Optivar into the optical train today. This is an additional magnifying lens that does the same job as a Barlow lens on a telescope. In a previous post, I noted that this particular Optivar suffers from extensive delamination so today was my first chance to find out if this is a significant issue and what difference an Optivar makes on my microscope. When I purchased it and it arrived and I found that it had the delamination I managed to get a massive discount so I am not concerned if it isn’t that good but it would be nice if it worked!
The following two pictures show an image using 32x objective bright field with and without Optivar of the cell boundary area on the tulip stem from the previous post.
The Optivar loses some of the sharpness of the image and with higher power lenses in the Optivar (it provides 1x, 1.25x, 1.6x and 2x lens – photo below with 1x lens to compare with photo without Optivar) the image is not as well focused – sadly the delamination does affect the image. Mind you, the image is not that bad and it should still be helpful for some projects.
I bought my wife some tulip cut flowers last week – an ideal place to obtain a short section of stem. The biggest problem was finding a fresh sharp blade to cut it. Ideally, a new razor blade would do the job but I did not have one to hand!
An excellent resource which discusses what can be found in such sections can be read at http://microscopy-uk.org.uk/mag/indexmag.html?http://microscopy-uk.org.uk/mag/artfeb04/wdstem.html
In particular, see the image that author has published on that page of the spiral reinforcing structures in the vascular bundles of the stem – you can see these spiral reinforcing structures, cell walls, chloroplasts in my images below – although there are also linear artifacts from my cuts/folding tissue as I cut (basically it goes crinkly – although I floated the section on a drop of water on the slide to try and smooth this out. It is for this reason that I have not published any images of the Helicon Focus 3D depth maps – as the crinkling dominates the depth maps).
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