#13
Post
by SunshineLW » Fri Apr 06, 2018 9:55 am
I know all microscopes and digital microscope softwares are different, but here are several things I fiddle with when assessing my color options for brightfieqld images (I know NOTHING about fluorescence! or DIC!). You probably know all of this, but I always use the same systematic process so I'll list it all for completeness sake. Also, I am a noob at this so this could be completely wrong in terms of how and what you are imaging. Besides, the photographer is the ultimate judge of exposure/ contrast/ color/ etc., and if you like what you are seeing, don't change it to please somebody else! I offer this list as a gesture of kindness and not to insult your technique because your images are stunningly beautiful. At this point, I would begin nitpicking every tiny detail to perfect every shot!
1. Place specimen on stage and focus image
2. SOFTWARE ADJUSTMENTS:
(A) If using "Auto-exposure", define the area of autoexposure to encompass the entire image.
(B) Look at the Red, Green, and Blue (RGB) histogram to ensure that the histogram is relatively evenly distributed from left to right. In general, if the RGB histogram is too far to the left, the image is underexposed. If the RGB histogram is too far to the right, the image is over exposed. Start with a histogram that has a nice spread (from left to right) and superimposition of Red, Green, and Blue humps. Ultimately, you are the judge of exposure, but the histogram can guide you to a starting point.
(C) Maneuver to an area that is clear and white and set white balance.
(D) IF NECESSARY, weak the above steps for each new objective and area of observation. I fight with my white balance the most. White balance and I have a love-hate relationship because we fight a lot, but in the end, we always compromise for a "whiteness" that makes us both equally satisfied.
3. MICROSCOPE ADJUSTMENTS:
(E) Find a spot to image, focus specimen, and begin preparations for imaging (do not change objectives or area of observation after this point)
(F) Set the aperture iris diaphragm of the condenser to 60-90% of the objective numerical aperture (may not be possible with every microscope) (do not touch the aperture iris diaphragm after this point).
(G) Use condenser focus knob and field diaphragm to put the image into Kohler illumination (may not be possible with every microscope) (do not touch the condenser focus knob or field diaphragm after this point).
(H) READY TO IMAGE!
Maybe this helps and maybe I am vomiting out a complete noob post, but these are some of the things that I found out on my own long after taking hundreds of images that could have benefitted from minor tweaks. I hope this was helpful and/or gave you ideas for improving upon that which you are already extremely proficient. Like I said, this is purely a gesture of kindness, and not to insult your technique. I hope you would do the same for myself should I ever post images that you would adjust differently.
Please, keep sharing with us your beautiful work!
EDIT: Hobbyst46, "IMHO, it would be better to set the condenser iris last, (reverse steps F and G) since the correct condenser aperture (whatever value you decide for it, I e.g. prefer towards the 60% not 90%) depends on the brightness of the illumination, which in turn depends on the centration of the image of the field aperture. I would start with centration and do it with the condenser iris fully open, or nearly so."
Last edited by
SunshineLW on Tue Apr 10, 2018 1:50 am, edited 2 times in total.
