We got one (very) recently! It’s using laser illumination, rather than LED. The compromises are, essentially, flexibility; it has a 5-position filter wheel with a manual bypass of the spinning disk lightpath if you want to use it as an epifluorescence system as well. So we essentially have 4 emission filters because we need to leave a blank slot for using the microscope in Epi mode.
I assume that the disk (50µm holes, 250µm spacing) is the same as the equivalent disk in an X-light V2 or V3; if you have weak signals you need to do a lot of binning because the disk does throw away a lot of light. It’s feasible that CREST might consider opening the door to other disk designs in the Cicero if people asked for them. I think if you have medium levels of expression and a good camera, then the Cicero is actually quite good, it’s just that our samples (endogenously-tagged proteins) are low expression.
@CMCI, can I ask an approximate price of CICERO and the light source? I got a quote from a local distributor in korea and it is ~$100K for both the spinning disc unit and LDI light source (4channel), no camera. Thanks!
That sounds about right, to be honest. Make sure that you’re getting the right laser lines - I think 470nm lasers are now common on the smaller laser launches, and if you’re doing live imaging of GFP, then I think you would want to have a 488. You probably already know this, but I almost got caught out by it, so I thought I should mention it!
When measured in the wieldfield mode, lasers give >300mW at the objective at 100%, but when measured in the disk mode, all lasers are <10mW at the objective.
Final image is dimmer than what I would expect even for the 10mW illumination.
e.g. It takes 1s exposure with the 60x oil lens to image a cell stained with an actin antibody.
Could it be simply some mistakes in the setup? Can any alignment error throw it off like this?
@smson This sounds normal to me. I have the identical setup. The Kinetix is super sensitive and will enable you to have just a few milliseconds for exposure time when you are not using the spinning disc. The moment you add the disc light power will significantly drop and you will have to go up to 200-300 ms exposure time to get the same signal intensity.
In spinning disc mode 1s or higher exposure would indicate that you probably have quite a weak antibody staining. What fluorophore are you imaging?
Hi @Daniel_Furth , thanks for the quick reply and sharing your setup. I’m using Alexa 555, which should be bright and photostable.
I’ve used Yokogawa CSU-X1 + Andor ILE for years and cell imaging required ~100ms with <30% power for simple GFP or Alexa staining. Using oil lenses it became even more bright.
From what I read in this thread, I expected Crest V3 be comparable to CSU-X1 so I’m still confused about current brightness of our V3 demo system. I’ll check the pinhole alignment first, but I’d appreciate other suggestions regarding the device setup.
@smson I just measured the power with a Thorlabs PM100D using S120VC sensor. The Lumencor CELESTA laser “GREEN” widefield mode gives 2 mW out at 545 nm from the 100x Plan Apo 1.45NA objective when laser is set at 1%. Increasing to 10% gives 17 mW at 545 nm.
When spinning disc is active you’ll get a little bit above 35 µW at same 1% setting, increasing to 10% gives 270 µW. So not quite linear response.
A reasonable setting for most imaging situations in my experience is to start at 50% laser power and 500 ms exposure for the spinning disc and adjust from there. 50% at the CELESTA GREEN this gives you about 1.25 mW.
I also used a CSU-X1 before on a Nikon Ti-E with Andor camera but the Nikon lasers which are just Agilent lasers in a box. CSU-X1 has a microlens system that boosts excitation light unlike V3 where the microlens system dont quite add that functionality. In addition the lasers from Agilent are more standard wavelengths (e.g. 488 nm, 555 nm) and more power than what you have with a Celesta system.
@Daniel_Furth Thanks very much for sharing the detailed info. I brought this up to our distributor and they adjusted the setup (“body alignment” is what they said…) and everything looks more normal now. This is the first Crest demo in our country and the local distributor just started learning about this system.