Fluorescence microscopy

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?



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):