Pond, lake, stream and river water (fresh water) microscopy

Four algae cells showing cilia around edge from Hopwas Canal

Ean Ean and I collected the sample of water from Hopwas canal 19/4/2018 and today I viewed it in the Zeiss IM microscope using 40x objective. The picture below shows multiple cilia (hair-like structures) around edge of the cells which the cells use to move around. I have used Curves function in GIMP2 to enhance the image to demonstrate these cilia more effectively.

Andy

Ean Ean collecting sample from canal:

Attempts at dark field microscopy using x50 infinity-corrected objective with diaphragm

I still can’t seem to get dark field to work well!

Photos below using infinity-corrected 50x objective with iris diaphragm, curves adjusted in GIMP2.

Andy

Algae from my pot pond 12/4/2018:

Volvox on commercial slide viewed by myself today using same setup as above:

 

Home pot pond algae showing cilia

I have created a pond at home using a very large plastic pot from Homebase. It even obtained a new resident yesterday (a fish) who has joined the frogspawn and Elodea and some other water plants obtained from a friend’s pond – whence the fish also originated. The friend in question insists that the fish should not be lonely so another will be joining it later in the week!

The photos below are of an algal sample from the pot pond. 18 x Eppendorf 1.5ml centrifuge tubes were centrifuged at 10000 revolutions/minute for 10 minutes. I pipetted off excess water above the centrifuge pellet. I then combined the pellets into a single Eppendorf tube and centrifuged again using the same settings. This concentrated the algae to make microscopic observation easier.

The main findings today are of cilia on most of the algal cells. I have annotated the photos below to show whether these can be seen. In some cells the green chloroplasts are bunched up at one end and I suspect this is due to the centrifuging process.

Finally bacteria abound – not sure why I used to have such difficulty seeing bacteria initially but now I see them all the time – I suspect it is a bit like training yourself to observe faint fuzzies in the night sky – once it clicks it becomes a lot easier – perhaps it is also just about knowing what to look for!

Andy

Live bacteria amongst the algae:

https://youtu.be/WsejNez8AwQ

 

Commercial slide of Volvox showing daughter colonies inside parent colony

The following photos show daughter colonies inside Volvox colonies – blue circles within the blue circles.

I used x10, x20 and x40 objectives for these photos in bright field. Unable to get dark field even with x10 objective for this slide for some reason. Best I obtained was a sort of partial oblique illumination which you can see on photo with the partially dark background.

Andy

Bright field and dark field images of commercial slide of Ulothrix Algae

My favourite view from this session – x10 objective, dark field, Zeiss IM microscope, image edited in GIMP2 (other photos below have not been edited, only this one):

My wife looked at the dark field images below and immediately suggested they looked like a star field – to me the cosmic web! The white blobs are I think mountant rather than sample.

These images are part of my ongoing project to get dark field microscopy working well on the Zeiss IM microscope. From my reading, it looks like my previous difficulties obtaining dark field images was due to having objectives that are simply too good – dark field requires a condenser with larger numeral aperture (NA) than the objectives but mine all have enormous NAs!

The following information is taken from “Microscopy Primer by Frithjof A. S. Sterrenburg: 8. SPECIAL MICROSCOPY TECHNIQUES” (http://www.microscopy-uk.org.uk/index.html?http://www.microscopy-uk.org.uk/primer/index.htm):

Darkfield [microscopy]:

If sunlight enters an otherwise dark room through a slit in the curtains, tiny dust motes that were first invisible are seen against the dark background because they scatter the light. The undeviated light is excluded, the deviated light produces the image. A simple way to obtain such darkfield illumination is to place a central stop in the filter ring of the condenser. This central stop can be a coin, cemented centrally on a plastic disc cut to fit the filter ring (CDROM cassettes are good material). This central stop blocks the direct light, only rays at an angle larger than the “angle of admittance” of the objective in use are allowed to strike the object. The background is dark, the light scattered (deviated) by the object – drawn bright blue in Fig. 43 – is imaged by the objective.

The size of the central stop will obviously depend on the “angle of admittance” (i.e. the NA) of the objective. A central stop of about 15 mm in diameter will generally yield darkfield with objectives up to about NA 0.5, depending on the properties of the microscope condenser. A central stop of 20 mm will generally permit darkfield with a 40x/NA 0.65 objective also. Adjust the focus of the substage condenser for maximum brightness of the central field of view. The field stop of the lamp can be wide open and there’s no major difference between critical or Köhler illumination. For darkfield at higher NA, special “cardioid” darkfield condensers using mirror surfaces are manufactured. These are oiled to the slide (instead of oil, water may be used for convenience) and require exact focusing and centering with their centering screws. Even these high-power darkfield condensers cannot be used with objectives of NA above 1.2 or higher, however. The reason is that darkfield condensers have two NA’s that should be considered: the inner NA (i.e. the portion of the light that is blocked) and the outer NA (which for technical reasons is limited to at most 1.4). For an objective of NA 1.25 one would have to block everything up to at least NA 1.3 and the cone of light that would remain would thus be limited to an NA between 1.3 and 1.4. That’s simply not enough to produce practical darkfield. The inner NA limit attainable with current darkfield condensers appears to lie near 1.0 , corresponding to the medium-power (40 – 60x) oil immersion objectives offered by several manufacturers.

Contrast in darkfield is extreme and resolution is as high as the objective can yield. Darkfield is spectacular for observations of live protozoa or bacteria (cilia or flagella are visible) and for diatoms. I always use darkfield to scan diatom slides at low to medium power because it’s easy on the eyes and even the smallest and faintest diatoms stand out clearly. The limitations of darkfield:

  • thick or extended objects and even dense strews are unsuitable (glare)
  • residual errors in objectives (notably spherical aberration) become maximally visible. This is especially the case with a dry 40x objective and forms the reason why a 40x immersion is highly advantageous here. For examination of live specimens in darkfield a 40x water immersion is ideal.
  • all specks of dirt or traces of grease also become maximally visible. You need a scrupulously clean optical train: top lens of condenser, both surfaces of slide, clean and “dilute” sample, grease-free object glass.

In my experiments with different objectives today, the x10 objective works well in dark field – but then its NA = 0.10.

x40 objective, NA=0.65 – not able to obtain dark field illumination.

Andy

Bright field, x10 objective. The two images differ in colour due to white balance settings in MikroCamLabII software for the Bresser Microcam 5.0 MP camera and show the difference in detail that can be seen with such a simple setting change:

Dark field, x10 objective:

 

Helicon Focus stack, dark field, x10 objective, and 3D model from Helicon Focus from same stack of frames (from AVI video of slide):

x40 objective, bright field:

x40 objective, Helicon Focus stack & 3D model showing difference heights of filamentous algae on slide:

The following picture shows the effect on resolution of closing the illuminator aperture somewhat and switching in the high NA lens on the condenser:

 

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

 

Second attempt at oblique dark field illumination of algae from neighbour’s pond using halogen swan-neck illuminator on Zeiss Standard microscope

This time I used my centrifuge to concentrate the sample – snow outside/cold means number algae per ml in the water low.

Bright field images of algae x40 objective – unfortunately they show that I have some work to do aligning the optics as lot of colour fringes…there is also quite a lot of dust on the optics of this microscope – I need to give it a good clean! However, not all out of focus rings are dust – much of it is algae in different planes on this live sample.

I particularly like the second and third pictures as they show long cilia from the spherical organism.

Video from this session showing motile organisms:

Dark field using oblique illumination – set up with Zeiss Standard microscope:

I have found that the best dark field is when the fibre-optic tips are placed on the stage pointing virtually horizontally at the end of the objective.

Dark field with x10 objective, using above equipment – looks like a star field in the telescope! Can you recognise the constellations?

The above two images came directly from the camera.

The following are the same two pictures but this time I have used curves in GIMP to remove part of curve below the data and hence blacken the background:

Dark field using x40 objective – this is where I am breaking new ground with success at dark field using x40 objective. So far, using my Zeiss IM microscope, I have been able to obtain excellent dark field images using the 10x non-phase objective and a phase annulus, but the higher power objectives don’t seem to work so well using that system. I think maybe the NA on the objectives is too high compared to the NA on the condenser, but am not sure of the reason.

I found that if I varied the position of the swan neck heads to direct the light more downwards (angled the lights to point down rather than horizontally) then this varied the lighting effect. The lighting is no longer true dark field but is still interesting! Example picture below: