I just got my microscope camera working with the “High Resolution Optics”. I am trying to scan droplets of algae culture on slides, looking up from underneath with a 40x plan objective (link below). I have used many inverted scopes before for this without difficulty, but I am finding that when I try to get the objective close enough to focus on the algae in the droplet, even those sitting on the top surface of the slide, the objective bumps against the bottom of the slide before the image comes into full focus.
Here is the objective:
It costs $70 on Amazon and has a lot of good reviews, but it apparently has too short a working distance to look through a slide.
What 40x objectives do other folks employ, and do you all get good resolution looking through slides from underneath?
Most 40x objectives will have a working distance which is considerably less than 1mm. As you have found, that means that you cannot focus through the thickness of a microscope slide. Usually you would put a coverslip on a sample and then turn the slide over so that the coverslip is on the bottom, next to the lens.
It sounds as though turning the slide over is not possible in your case? Would the upright version of the microscope help - putting the objective above the slide instead of below? This is designed for version 7.0 of the microscope, but should also basically fit V6 or the Deltastage. It would be less stable as there is an extra mounting screw on V7.0
Alternatively you would need a long working distance objective. They are often called metallurgy objectives. Unfortunately they are usually much more expensive. I have used ones from the Motic range. Something like CCIS® Plan achromatic objective LWD PL 40X/0.5 (WD=3.0mm) (moticeurope.com) has a 3mm working distance and is not too expensive. It is infinity corrected, so would need the infinity optics version for your microscope. It actually appears to be designed to look through 1.1mm glass slide, rather than metallurgical objectives that are designed to look directly at the object with no cover.
Thanks, this is very helpful, I will look for a longer working distance. Inverted scopes are much more convenient for looking at liquid samples, we are planning on putting several droplets on a slide, scanning them automatically, then applying machine vision.
I also have a feeling that you can get well slides with a thin base, so that the thickness through the glass to the lens below is the normal 0.17mm coverslip thickness. I have never used one, I just have a vague feeling that they do exist for exactly your situation.
The lens that you have, used with those slides, will in principle give a higher resolution as the NA is quoted as 0.65 rather than 0.5 for the long working distance Motic objective. That of course depends on the lenses being designed and manufactured to the physical limit of their stated NA, and the illumination also being at least the same high NA. Transmission illumination at high NA from above those well slides may be difficult with the standard Openflexure illumination. There is about 5mm space below the condenser lens.
The LED grid illumination (thread here as well) is not particularly intended to address that problem, but does give you a bigger gap for 0.65NA I think, as it is physically wider than the standard condenser lens.