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.
Cheers,
Aaron
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.
I had two issues when moving from the low cost imaging module to the high resolution module using a 40x objective.
The first was as mentioned by @WilliamW that you can’t focus through a standard slide only through a cover lens.
The second was that my optics module wasn’t straight when affixing it to the microscope via the fixing screw which meant I could only see items in focus in the bottom 10% of the screen.
After fixing both of these I got a clear image, and the resolution is crisssssp! Here is a comparison using the raspberry PI camera lens compared with the 40x objective on red blood cells fresh from my finger.
Can someone post some images where they have overcome the focusing issue with 40x objectives? I just finished my v7 and I’m having the same issue. I’ve only tried with some basic pre-made slides and a plastic petridish. To see whatever is on the pre-made slide (one of those that come with microscope kits) will only be in focus if I flip the slide over so it is facing the objective. I also had to lower the Object by loosening the fastening screw (maybe this can be made easier in future versions!) and moving it down a bit because it was pressing against the slide from below when I was trying to get it to focus…
I’m a bit lost now as to how this setup will work with live culture observing i.e. in a T25 flask? Any help would be greatly appreciated!
@Eiki it is usual to turn the slide over to image on an inverted microscope with high magnification objectives. The working distance of a 40x biological objective, like the one in the first post on this thread, is less than 1mm. They usually are designed to focus through a 0.17mm thick glass coverslip. This glass thickness is given in the marking 160/0.17 on the lens barrel.
I think that this may be a follow on from where you had got to with trying to focus with the slide the right way up. With the slide removed and the z-actuator centred (so that the nut hole in the actuator is lined up with the hole in the casing that you put the nut in through), the objective should sit just a little (maybe ~1mm) below the surface of the stage when it is pushed to the top of the optics mounting slot.
The first is to keep the same lens and find a flask with a thinner glass base. There are petri dishes with a coverslip thickness in the centre of the base, I don’t know about flasks with caps.
The second option is to consider whether you actually need 40x magnification. At 20x lenses usually do have enough working distance to image through 1mm of glass.
The third option is the easiest, but much the most expensive. Objectives do exist that are 40x with a long working distance. Mostly they are for metallurgical applications, so designed to view a surface, not through glass (marked 160/0 for zero thickness glass). They will work, but the image aberations will not be fully corrected. Lenses specifically designed for viewing through 1mm of glass do exist. I have now actually tried the one from Motic that I linked in post 2 of this thread. It does seem to work very well.
Thanks for the quick reply William. I did read about 2x well slides here somewhere with those super thin bottoms but the maker of those don’t sell direct. Any links to Petri dishes with those thin bases or other types of useful live culture holders that would work that you know of? I’m fairly good at finding stuff but unclear what to search for.
As for a 20x objective. Would that work without changes to the 3d print? Any suggestion on Objective?
Btw once the microscope managed to focus it actually looks great. I love being able to use my keyboard to move around.
Cell culture is a little far from my work. maybe @quigly might be able to point you to the name to search for.
A 20x objective would just screw in to replace the 40x that you have. Actually the ‘low cost optics’ version using the Pi camera lens gives very similar performance to a normal 20x objective Comparison of a pi-camera and a 20x optical lens. It will focus through a slide thickness (just)
The low cost optics version looks interesting. Am I right in understanding it also does not require the extra lens?
Btw would the “low cost optics” work on the v6 and is it worth it vs making a new v7?
I do have an extra pi-camera and I accidentally printed the whole v6 microscope before realising I had the older STLs I might be able to build another microscope from that if I can get the motor drivers that came with the stepper motors to work since I don’t have an extra Sangaboard. Worth the effort or just print another v7? I’m not sure what differences there are with v6 vs v7…
The ‘low cost optics’ only uses the lens from the Pi camera (if you have saved it ).
All of the optics modules interface to the microscope in the same way on V6 and V7, although the V7 effectively has the 10mm sample riser built in. So V7 modules should fit on a V6 with the sample riser installed. The slot in the optics mount dovetail is also longer in the V6, so you don’t push the optics module up to the top of the slot, you have to see the right place yourself.
If you build the V6 for this, I would leave off the motors and turn the gears manually to start with. That will be quick, and will let you see whether the working distance is enough to see into a culture flask and whether the resolution is enough to see the features that you are trying to study in there.
I might be able to build another microscope from that if I can get the motor drivers that came with the stepper motors to work since I don’t have an extra Sangaboard. Worth the effort or just print another v7? I’m not sure what differences there are with v6 vs v7…