A comparison between different illumination methods.

harisA · Post by **harisA** » Thu Oct 19, 2017 4:28 am

I took these images with the purpose of making a brief comparison between different epi illumination methods. These were taken during servicing an old Zeiss Axioplan microscope which was used for wafer inspection back in the late eighties.I have difficulty to achieve uniform DIC illumination.

1)EPI Brightfield

2)Epi Darkfield

3)EPI DIC

carlos.uruguay · Post by **carlos.uruguay** » Thu Oct 19, 2017 5:55 am

Woooww!

JohnyM · Post by **JohnyM** » Thu Oct 19, 2017 7:12 am

Your images are very nice quality, but i dont see that much difference between those techniques. Im in a process of buying upright epi-microscope for photography of seeds and upper surface of in-vitro breed plants. Have you tried those with this kind of subject?

harisA · Post by **harisA** » Thu Oct 19, 2017 8:43 am

JohnyM wrote:Your images are very nice quality, but i dont see that much difference between those techniques. Im in a process of buying upright epi-microscope for photography of seeds and upper surface of in-vitro breed plants. Have you tried those with this kind of subject?

These may not the best examples to understand the differences between the various methods. Hope the following example will be more representative.(microchip surface)

Dark field

Bright field

Epi illumination microscopes are not suitable for organic materials.Brightfield illumination is not working at all giving ugly fuzzy images.Darkfield is ok but a)you need special bd objectives,b)it is a method that consumes a huge amount of light giving exposure times of several seconds at low iso.
Most people in this forum use common microscopes (transmitted light) with external light source (flash or leds) and some kind of diffuser around the specimen.This is the ideal method to photograph organic stuff like insects seeds plants etc.

JohnyM · Post by **JohnyM** » Thu Oct 19, 2017 11:36 am

harisA wrote: Epi illumination microscopes are not suitable for organic materials.Brightfield illumination is not working at all giving ugly fuzzy images.Darkfield is ok but a)you need special bd objectives,b)it is a method that consumes a huge amount of light giving exposure times of several seconds at low iso.
Most people in this forum use common microscopes (transmitted light) with external light source (flash or leds) and some kind of diffuser around the specimen.This is the ideal method to photograph organic stuff like insects seeds plants etc.

Thanks for those.
I dont see any reason why shouldnt i use oblique illumination while enjoying long working distance from metalurgical lenses? I've been using TTL epi illumination with 10x lens on seeds are good enough for scientific paper, but i want to use this technique for polished wood and ~1mm explants with 20x and 40x lenses - you say that EPI-DF would work while EPI-BF would be useless?

Pau · Post by **Pau** » Fri Oct 20, 2017 12:40 am

Excellent work!
Do your BF use crossed polarized light? if so, does it has a 1/4 wave plate?
Some time ago I tried epiillumination with very old Zeiss stuff and BF if used without cross pol gave horrible reflections and glare. Cross pol worked well for biological subjects but rendered metallic objects almost black, the 1/4 plate was useful to avoid it.

Reflected DIC was of no utility for bio. For metals it produced color contrast, very different of your image that looks about the same as BF

harisA · Post by **harisA** » Fri Oct 20, 2017 2:57 am

Pau:Thank you for your kind words.Althought the microscope is equipped with polarizers I haven't use them for these BF images.Also no wave plate.

JohnyM:If you have long working distance objectives then the best method to illuminate your specimens is to use external light source with a diffuser like a ping pong ball cut in half with the whole at the top .
I will try to post some seeds images so you can see the differences.Yes BF is not working for organic material,at least for me.

Marek Mis · Post by **Marek Mis** » Wed Oct 25, 2017 12:09 am

Very informative and high technical quality images !

Marek

carlos.uruguay · Post by **carlos.uruguay** » Fri Oct 27, 2017 5:59 am

harisA · Post by **harisA** » Fri Oct 27, 2017 6:00 am

Thank you Marek.

JohnyM asked my opinion about epi-microscopy with organic woody speciments. Here are a series of images of insects eggs. These are single images (no stacking) without any post processing except resizing.

1)Brightfield

2)Brightfield with cross polarizers*

3)Darkfield

It seems that bright field with cross polarizers works quite well. However it is an extremely light consuming method. In my case using a 100W halogen light source at maximum setting came out with an exposure of 1/2 sec at 100iso

Pau · Post by **Pau** » Fri Oct 27, 2017 10:56 am

Thanks for the test. The BF and cross pol concur with mines (I don't have epi DF). What stuns me is the lack of glare at your clock image.

My dirty tests: http://www.photomacrography.net/forum/v ... p?p=154251