Human and larger animal microscopy

Photographs of microscopy of ectopic pregnancy in Fallopian tube Zeiss IM microscope: comparison of images taken using Phil Dyer and Watec video cameras

Now that I have managed to get the video camera working with the microscope, I wanted to compare the colour Phil Dyer video camera images with the black and white (but more sensitive to light) Watec-120N images. Both use same LogiLink USB video frame grabber device and Yawcam software to capture the images.

To get good images I had to remove the blue filter again – this does not seem to work with H&E stained slides like these.

Andy

PHIL DYER CAMERA IMAGES:

x4 objective:

x20 objective:

x32 objective:

WATEC-120N VIDEO CAMERA:

The immediate effect of changing the camera was that the image became a bright white-out – too sensitive – I had to turn the power down on the LED illuminator from full to about 20-25% to be able to see the slide again!!

x20 objective:

x32 objective:

Photography of placental tissue (H&E staining) under Zeiss IM microscope using Phil Dyer video camera and Yawcam software

I ordered a second hand C-mount microscope video camera microscope adapter from ebay and it arrived earlier this week. Now I can attach video cameras to the microscope – this is great news! The Bresser MikrOkular camera produces great HD pictures but has low light sensitivity – not a problem with every day microscopy but a serious issue for dim subjects – as I found out when I tried to photograph fluorescein stained samples recently – the camera just showed a black screen even though I could see some fluorescence visually.

The Phil Dyer (PD) camera is a stalwart of astronomy and (with the proper adapter) now I can use it for microscopy. This is also not the most sensitive camera and you can see the brightness of the image falling off as the magnification increases. Admittedly this dim image is probably because I have not set the condenser up properly but it makes the point about brightness anyway!

These first pictures are of placental tissue – I am not sure if it is human or animal.

You can see masses of red blood cells clumping together (blood clots) – these do not have nuclei and are round/oval in shape. There are also blue formed pockets of tissue with nuclei which will be the placental stroma.

Andy

Following pictures captured using PowerDirector 15 trial version.

x10 objective:

x20 objective:

x32 objective:

The point about setting up the illumination correctly is proven in the image below – I discovered an inbuilt blue filter which I have rotated out of the way of the light and immediately the image is much brighter!

Now using the same setup, I tried a 100x objective – a step too far may be for this slide?

I had a go at processing the above image in GIMP – what do you think? Perhaps a little easier to make out detail……..

Following pictures x32 objective – this time I closed down PowerDirector software and instead used neat little free programme called Yawcam. I am impressed! Really easy to use and beautiful pictures.

Microscopy of petri-dish cultures of finger print and swab from around teeth in mouth 8/7/2017

Rhys, Hannah and I have just spent a couple of interesting hours preparing and viewing slides from petri-dish cultures that have been developing (much to Ean Ean’s disguist) on the window ledge in the spare bedroom for the last 2 weeks. One was from a finger print Hannah made in the petri dish and the other from a swab of Rhys’s mouth from around the teeth (in an attempt to get some plaque).

…..The children prepared the slides under my supervision using aseptic technique (very important – human samples – need to know what you are doing as serious risk of pathogenic bacteria and serious illness – don’t do it unless you are properly trained).

We looked at these slides using the Zeiss IM microscope and 3 objectives:

  • 4x objective – brightfield and with Phase 2 annulus (combination works really well to give excellent dark background so not sure whether this is really phase of simply darkfield but either way the view is great)
  • 20x objective – brightfield and phase contrast
  • 32x objective – brightfield and phase contrast

These latter 2 objectives were designed for this microscope and work incredibly well – when viewing samples their views have excellent depth of focus , long distance working, effective work with existing phase annuli and give contrasty bright views especially with my www.retrodiode.com 20W illuminator.

Andy

We prepared 7 different petri dish cultures initially and chose two today from which to prepare slides (below):

The following two photos show that, after two weeks, Hannah’s finger mark had led to growth of milky white bacterial culture, 2 white fungal areas, a yellow organism at around 2 o’clock and number of areas with pale haloes suggesting the production of antibiotics (below):

Hannah is taking a sample of one of the white fungal areas to put on a slide using homemade loop (below):

Rhys is looking at his mouth swab sample, as he gets ready to prepare his slides (below):

Hannah-fingerprint-in-petri-dish-white-fungus-culture-x4-objective-080717.bmp (below). This shows thousands of tiny blue/black dots. The round circle at top right is an air bubble:

 

Hannah-fingerprint-in-petri-dish-white-fungus-culture-x40-objective-080717.bmp (below). The extra magnification shows strings of spores in strings. No bacteria were present:

Hannah-fingerprint-in-petri-dish-bacterial-culture-x4-objective-080717.bmp (below). Again, at this low magnification, thousands of tiny dots seen but these turned out to be quite different from those seen above – this was on a different part of the slide – the milky white stuff that occupied most of the Petri dish:

Hannah-fingerprint-in-petri-dish-bacterial-culture-x20-objective-080717.bmp (below). With 20x objective (200-400x magnification), the dots resolve into strings of 1-3 cells:

Hannah-fingerprint-in-petri-dish-bacterial-culture-x32-objective-brightfield-080717.bmp (below). With 32x objective, green and blue/black organisms can be distinguished from each other in brightfield illumination:

Hannah-fingerprint-in-petri-dish-bacterial-culture-x32-objective-phase-I-080717.bmp (below). Phase contrast brings out the cells more effectively and demonstrates thousands of organisms, many in strings of cells but other isolated round organisms also present. Some are green indicating photosynthesis is occurring within them but others are not:

Hannah-fingerprint-in-petri-dish-bacterial-culture-x40-objective-080717II.bmp (brightfield, below). The following two images demonstrate that the 40x objective does not add much to the 32x objective data. In fact, the 40x objective here has smaller depth of field and this is a problem in terms of focusing on the organims in this unfixed specimen:

Hannah-fingerprint-in-petri-dish-bacterial-culture-x40-objective-080717.bmp (bright field, below):

Rhys-swab-around-teeth-petri-dish-culture-x4-objective-brightfield-080717.bmp (below). Again, a debris field with thousands of blue/black dots:

Rhys-swab-around-teeth-petri-dish-culture-x20-objective-brightfield-080717I.bmp (below). With magnification, the dots resolve into bacteria:

 

Rhys-swab-around-teeth-petri-dish-culture-x32-objective-brightfield-080717I.bmp (below). Now it is clear that there are some long organisms which might be actinomyces, believed to be involved in dental decay and uggesting Rhys needs to make mlre dffort to clean his teeth, and other small round blue/black organisms present, possibly Streptococcus mutans, a common commensal in the mouth:

Rhys-swab-around-teeth-petri-dish-culture-x32-objective-phase-I-080717I.bmp (below). Phase contrast brings out the fact that many of these organisms are in strings:

Rhys-swab-around-teeth-petri-dish-culture-x32-objective-phase-I-080717II.bmp (below):

Rhys-swab-around-teeth-petri-dish-culture-pale-white-area-inside-clear-halo-x4-objective-phase-II-080717I.bmp (below). Rhys was interested in what was growing inside one of the areas with a clear halo around it. The clear halo implies that a bacteriocidal substance is being produced. The image below shows that there are large numbers of organisms of different types in the centre of these halos with many interesting structures with a boundary and lots of organisms of one particular type within, as in the bottom left of the following image:

Rhys-swab-around-teeth-petri-dish-culture-pale-white-area-inside-clear-halo-x4-objective-phase-II-080717II.bmp (below). This gives a view of one of these structures (left of image):

Rhys-swab-around-teeth-petri-dish-culture-pale-white-area-inside-clear-halo-x20-objective-brightfield-080717I.bmp (below). Outside of the boundaried structures mentioned above, there are bacteria but, unlike the initial views within the bacterial area that Rhys first sampled, these bacteria are largely not moving – occasional small round fast bacteria moved through the field of view but otherwise these bacteria appeared dead:

Rhys-swab-around-teeth-petri-dish-culture-pale-white-area-inside-clear-halo-x32-objective-Phase-I-080717I.bmp (below). Within one of the boundaried structures, the cells form lined structures several cells long and sometimes branching. They appear to all be the same. The boundary is not composed of cells but its two colours suggest it is the boundary of a matrix of one refractive index with another. To me this suggests that the cells in the middle modify the fluid in which they live.

Rhys-swab-around-teeth-petri-dish-culture-pale-white-area-inside-clear-halo-x32-objective-Phase-I-080717II.bmp (below). This clearly shows the boundary between the boundaried area (left) and bacterial area (right). Note both of these are within the clear halo (milky white bit in the middle of the halo on the Petri dish) – I suspect that the organisms on the left within the boundary are fungi and are producing an antibiotic that has killed the bacteria both within and outside of the boundaried area. The limit of its effectiveness is up to the outer edge of the halo after which we see bacterial growth again:

Rhys-swab-around-teeth-petri-dish-culture-pale-white-area-inside-clear-halo-x32-objective-Phase-I-080717III.bmp (below):

Rhys-swab-around-teeth-petri-dish-culture-pale-white-area-inside-clear-halo-x32-objective-Phase-I-080717IV.bmp (below):

Rhys-swab-around-teeth-petri-dish-culture-pale-white-area-inside-clear-halo-x32-objective-Phase-I-080717V.bmp (below):

Videos:

Zeiss IM microscopy of Brunel Microscopes Ltd blood smears 9/6/2017

I obtained this small collection of six slides new from Brunel Microscopes Ltd – looking at the slides via my Zeiss IM microscope today I could see the following (all pictures below are with the same Bresser MikrOkular camera and 40x objective so at same scale):

(i) Mammalian red cells all are missing their nucleus – the process of production of these cells involves loss of the nucleus before the cells are released from the bone marrow – the cell hence has a doughnut shape and only lasts three months as it is already effectively “dead” without its nucleus.

Human-red-blood-cells-showing-lack-nuclei-Zeiss-IM-microscope-40x-LD-objective-090617.bmp (below):

Rat-red-blood-cells-showing-lack-nuclei-but-thicker-central-regions-Zeiss-IM-microscope-40x-LD-objective-090617.bmp (below):

Rabbit-red-blood-cells-showing-lack-nuclei-Zeiss-IM-microscope-40x-LD-objective-090617.bmp (below):

(ii) Sometimes nucleated forms of red cells are released from the marrow and a couple of these can be seen in my slides – alternatively I might be wrong and these might be white cells – I am not sure!

Rabbit-blood-showing-granulocytes-with-nuclei-Zeiss-IM-microscope-40x-Zeiss-LD-objective-090617.bmp (below):

Close up of nucleated cells in above image (below):

(iii) Bird, fish and amphibian red cells are nucleated – keep their nucleus after production. Does this mean that they are  closer together on the evolutionary scale or does it mean that nucleation of the red cells better suits aspects of the environments in which all three live as opposed to the environment in which mammals live?

Bird-red-blood-cells-showing-presence-nuclei-Zeiss-IM-microscope-40x-objective-090617.bmp (below):

Fish-red-blood-cells-showing-presence-nuclei-Zeiss-IM-microscope-40x-objective-090617.bmp (below):

(iv) There are differences in size and colour between red cells of different types of animal – for example, compare the views I obtained today of cells above in fish and bird blood with that I found from frog blood below.

Frog-red-blood-cells-showing-presence-nuclei-Zeiss-IM-microscope-40x-objective-090617.bmp (below):

Andy