Microscopy staining techniques

Some limited success with fluorescence microscopy

Yesterday I tried staining a pond water slide with fluorescein without success. Today I repeated this process using the H&E stained slide. I added fluorescein dye and left it for 30 mins to maximise uptake. I then viewed it on the microscope and discovered that there is evidence of uptake BUT that the Bresser MikrOkular camera is not sensitive enough to pick it up. I could see it by eye through the oculars and resorted to using my Samsung S7 phone afocally to take photos of the phenomenon. Unfortunately, these are not good enough to allow me to work out where in the cell the fluorescein dye is sitting. However, it has proved that the concept can work – I need to look at however to attach a more sensitive camera to the microscope. 1.25 inch adapters are too wide for the ocular holder. Perhaps I can buy a smaller one for my Watec or PD cameras?

Andy

Applying-fluorescein-dye-to-slide-260817.jpg

UV-ring-light-flourescing-flurescein-dye-on-slide-260817.jpg (below):

Ring-UV-light-on-microscope-280817 showing that the UV is directed onto slide (below):

The green light on the slide below is fluorescent light from the dye on the sample as result of the purple/blue UV light – note the fluorescent light is at different (lower energy) wavelength than the light used to fluoresce the dye – the process of exciting the electrons within the dye is not 100% efficient so energy must be lost and the resultant wavelength of light given off by the dye changed to longer one.

Volvulus on pond water slide – this is brightfield image – H&E stained – fluorescein dye has also been added but will not be visible with microscope visible light illuminator switched on as here:

Samsung S7 phone camera hand-held afocal image x10 objective (below – some areas of green fluorescence indicate that fluorescein dye has been taken up on the sample):

Following images are also afocal hand-held Samsung S7 photos – with 40x objective (below – again they show uptake of dye in specific areas of sample but are not adequate to indicate where that is occurring):

    

H&E staining of pond water sample

This is my first attempt to do H&E staining – I used the pond water sample from John Brockley’s garden yesterday which showed so much Volvulus. Heat fixed it first then applied the Haematoxylin for around 20 mins, washed it off, and then eosin for 5 mins. The former should bring out nuclear material in purple and the latter cytoplasmic material – I will leave you to decide whether I was successful from the photos below!

Haematoxylin and eosin stain is one of the principal stains in histology. It is the most widely used stain in medical diagnosis and is often the gold standard; for example when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E and termed “H&E section”, “H+E section”, or “HE section”. A combination of hematoxylin and eosin, it produces blues, violets, and reds (Wikipedia).

I used this staining technique a lot in my anatomy degree and most pathology slides use H&E.

Andy

For comparison non-stained photos from yesterday from same water sample (below – these first two are NOT stained with H&E):

Volvulus-from-John-Brockleys-pond-270817-HE-stain-10x-Planapo-obj-Zeiss-Photomic-III (below – reds and purples are evident but not as pronounced as I expected. However this is only using the 10x objective so higher magnification would be more insightful into whether stain is effective):

 

16x objective H&E stain:

40x objective H&E – panorama of 7 photos (below – try downloading this image and zooming in – it is a much bigger image than previous ones as it is created from 7 photos – there is a great amount of detail within the cytoplasms of the cells other than chloroplasts (which were visible in unstained images)):