Andrew Thornett

What grew on a slide left in the aquarium overnight

Over this last week I have created a temporary aquarium from pond water plus the Elodea pond weed I have been using in my previous posts this weekend. On various microscope blogs, it is recommended that slides are left in aquaria to see what grows on them….so I left a few in this one last night and the photos and video below are from one of those slides. Note that the organisms on this slide are directly growing and living on the slide so are unaltered. This is a live sample without any staining or centrifuge etc. Therefore the Helicon Focus 3D model below is probably the first as in vivo (because it is in vivo) model of a sphere of cells that I have obtained.

Today was also another chance to have a go at dark field microscopy and for the first time I used COL (circular oblique lighting). Still having problems with the dark field which I suspect is due to relative numerical apertures of condenser and objectives but COL was quite successful as you can see below – this uses an off axis one-sided aperture mask for the condenser.

Andy

Temporary aquarium – notice the frog’s eggs!

x10 Objective Bright Field

x10 Objective Dark field:

x20 Objective Bright Field:

x32 Objective Bright Field:

x63 Objective Bright Field:

Helicon Focus 3D model of above organism (ball of cells):

x40 Objective Circular Oblique Lighting (COL) – note in COL the apparent differences in 3D height are contrast differences and do NOT reflect actual contour changes in elevation on the slide (below):

 

Elodea movement in cells x63 obj 24/03/2018@1832

Another attempt at this – some fantastic images and video – I wonder what all those very small moving objects in the cells are? Organelles or another organism?

For photosynthesis plants use wavelengths that chlorophyll molecules can absorb, and these are blue (410-460 nm) and red (630-670 nm) light. There are 2 types of chlorophyll: a (max absorption at 430 and 662 nm) and b (max absorption at 453 and 642 nm) (http://answers.yahoo.com/question/index?qid=20060906053251AArv13c)

Andy

 

Disrupted early frog egg – unknown whether fertilised

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.

Andy

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:

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):

Lilly pollen grains & stigma

Following are photos from a lilly flower.

Andy

x32 objective, bright field, pollen grain group, Zeiss IM microscope, Bresser 5MP microscope camera:

 

On another slide of similar pollen mounted with stigma I found these – I think the background yellow fluid is probably nectar from the stigma. x32 objective bright field:

The following Helicon Focus 3D model from 4 images is of the above slide shows that the pollen grain sits above the fluid on the slide, x32 objective, bright field:

lilly stigma x10 objective bright field – I have followed a strand coming out (or is it going in?) of the stigma – not sure what it is but there is a bulbous end to it:

Horizontal section Tulip stem with and without polarisation, bright field

To interpret these pictures see 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

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

Compare these photos today with those taken through a vertical section through this same plant stem http://roslistonastronomy.uk/vertical-section-tulip-stem-180318

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

Andy

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):

Comparing effect with and without Optivar

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.

Andy

Without Optivar:

With Optivar 1x lens (Optivar provides 1x, 1.25x, 1.6x 2x lens):