A quartet of epi-illumination methods

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Discussion Overview

The discussion revolves around the use and characteristics of various epi-illumination microscopy methods, particularly focusing on epi-phase contrast. Participants share personal experiences with microscopy techniques, equipment acquisition, and image results, while exploring the nuances of different illumination methods.

Discussion Character

  • Exploratory
  • Technical explanation
  • Debate/contested

Main Points Raised

  • One participant describes their acquisition of epi-phase microscope objectives and compares the principles of epi-phase contrast to transillumination phase contrast, noting the differences in phase ring specifications.
  • Another participant humorously questions the term "cheap" in relation to the cost of the microscope objectives, suggesting a comparison to luxury items.
  • A participant shares that the total cost of the objectives was $120, indicating they were in new condition and likely unused.
  • Discussion includes the challenges of microscopy, such as the need for flat surfaces for optimal epi-phase imaging and the impact of optical cleanliness on image quality.
  • One participant explains the mechanism of epi-phase contrast, mentioning the use of filters for destructive interference and the resulting image characteristics.
  • Another participant requests help identifying a logo seen in the images shared by the original poster.

Areas of Agreement / Disagreement

Participants express enthusiasm for the microscopy techniques discussed, but there is no consensus on the desirability or utility of epi-phase objectives, as some view them as less sought after compared to other methods.

Contextual Notes

Participants note the specific requirements for successful epi-phase imaging, such as the need for clean optics and proper alignment, which may affect the results and usability of the technique.

Who May Find This Useful

This discussion may be of interest to microscopy enthusiasts, researchers in imaging techniques, and those exploring different methods of illumination in microscopy.

Andy Resnick
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TL;DR
I finally obtained a set of epi-phase microscope objectives (Zeiss)
Well, it took almost 20 years (!!!), but I finally obtained a set of epi-phase microscope objectives (Zeiss). The principles of epi-phase contrast is nearly identical to transillumination phase contrast, but the phase ring is a 1/8 wave retarder rather than a 1/4 wave retarder (because with epi-illumination, the light passes through the ring twice). This method was popular only for a very short period of time before epi-DIC (differential interference contrast) became widely available. So, not only are epi-Ph lenses impossible to find, but they are cheap because nobody wants them :)

Here are 4 images of a razorblade comparing the different epi illumination methods, all using 40x lenses. In order: brightfield, phase contrast, DIC, and darkfield:

DSC_7951 copy.webp



DSC_7952 copy.webp


DSC_7953 copy.webp


DSC_7954 copy.webp


While epi-phase looks a bit like epi-darkfield, the biggest difference is the light throughput- 1/30 s shutter (phase) instead of 5 s (darkfield).

Success! I feel like Captain Ahab, if he had finally caught Moby Dick. But I guess that would moot the point of the story....
 
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Andy Resnick said:
TL;DR Summary: I finally obtained a set of epi-phase microscope objectives (Zeiss)

but they are cheap because nobody wants them :)
II that 'cheap' like a second hand Maserati or cheap cheap? Captain Ahab went overboard , remember so be careful.

I guess they need all you existing gear to do their stuff. I'm pleased for you and the images are interesting. Could you do labels?
:thumbup:
 
sophiecentaur said:
II that 'cheap' like a second hand Maserati or cheap cheap? Captain Ahab went overboard , remember so be careful.

I guess they need all you existing gear to do their stuff. I'm pleased for you and the images are interesting. Could you do labels?
:thumbup:
cheap-cheap: Including shipping from Europe, the cost was $US 120 for all three, all in new condition, likely never used :)

Thanks, re: the images. More to come as I improve my technique!
 
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I'm starting to get the hang of it; in microscopy (unlike astronomy), the photographer must prepare their own objects to photograph. Epi-phase works best on extremely flat surfaces (polished metal, for example... or computer chips), and here's what I was able to produce: all images are JPG straight off the camera, no post-processing/photoshop/etc. The first image is epi-brightfield, the second epi-phase.

First, an overprocessed CCD array:

DSC_8567.webp


DSC_8568.webp


Epi-Phase contrast works by using 2 filters to cause specularly reflected light to destructively interfere with itself, so that the image intensity is proportional to the phase difference between "scattered" and "unscattered" reflected light. (I don't know better terminology). The colors here are most likely from thin-film interference created by multiple transparent layers.

And next, another IC but completely "blank" (obviously there's something going on underneath the opaque layer):

DSC_9063.webp


DSC_9065.webp


Here, epi-phase clearly shows the topography of the top layer. This image also shows how unforgiving the technique is for (ahem) dirty optics (see the dark rings along the right side) and misaligned components anywhere in the entire optical path (uneven illumination).

Also- if anyone can identify that cryptic logo (not the year or copyright symbol, duh....) on the second set of images, I'd appreciate it!
 
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