Are there different disk options on the DSD for work with objectives other than the 60X?
Not at this time.The DSU from Olympus would offer different disks for different objectives but this instrument doesn’t.In our experience so far the instrument’s active background subtraction gives you very good confocality, even with lower magnification and moderate NA objectives.So our experience is that it is not necessary to switch disks. Do note however that the disk has two regions: one for high contrast with 1:3 transmission to reflection ratio; and the other for high throughput with a 1:1 transmission to reflection ratio.
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Are you planning to have a CSU with a 10,000 rpm spinning rate?
We do have a CSUX with a 10,000 rpm spinning rate.Where I mentioned up to 2000 frames per second, this is actually the scan rate of the 10,000 rpm CSUX disk.
Are you planning to work with CoolLED’s instead of metal halide lamps?
CoolLED’s are excellent sources but at this moment are not bright enough for this application. However we are keeping this under constant review and will certainly consider in the future.
Can you comment on the advantages and disadvantages of using lamp based excitation vs. laser based excitation?
Lamp based excitation is very cost effective and allows us to deliver the DSD confocal instrument at a mid price point. The DSD is designed for use with our metal halide source and will perform optimally and with great flexibility in this configuration. A metal halide light source provides a broad spectrum of excitation wavelengths and so can be adapted to many fluorophores by selecting suitable excitation and emission filters.
But a white light source is not the best source for a CSUX because a single mode fibre is required for illumination at the present time and it is not possible to couple the broad emission pattern of the lamp to such a tiny optical fibre. This can be appreciated when it is noted that a single mode optical fibre will typically have a core diameter of ~ 3.5 μm with NA as low as 0.1.
Laser illumination systems with multiple excitation lines or wavelengths require one solid state laser module for each wavelength and the beams need to be co-aligned. This requires a combiner like our ALC with wavelength selection and modulation. Since lasers are relatively expensive, this results in a much more expensive source. But lasers have their own advantages in terms of parallel emission beam, coherence and power density. They therefore lend themselves to complex illumination techniques such as TIRF and beam scanning applications. The ability to deliver the beam with high precision is widely used for laser scanned microscopy, fluorophore activation and switching, uncaging and ablation of intra-cellular organelles and laser tweezers.
However, there is evidence that incoherent light produced by a metal halide lamp results in a slightly lower bleach rate than an equivalent laser.
So it is really a matter of choosing appropriately for the relevant application and budget.
Can you confirm that you are using online processing of the DSD imaging?
I can confirm that we do both online and offline processing.There is a fast online algorithm and for the highest precision you can apply an offline method that uses a spline distortion correction algorithm.
Note: since the webinar, we have reduced the processing time of the matching and subtraction algorithms by a factor of about twelve. Hence it is unnecessary to differentiate between on-line and off-line processing. With the use of multi-threading we are able to perform the confocal and/or widefield calculations during exposure of the next frame and high quality confocal (or widefield or both) images are available in real time.
Do we have or can provide any data about confocality or PSF when using 40x-wi NA1.1 objectives?
We do not have data yet on confocality with a 40X WI objective, but I have expectation that it will deliver a strong confocal section because of the activity background rejection. I cannot give you a figure yet. This is something we are working to deliver more information about in the near future.
How looks the final confocal image? Is it half width of the CCD sensor, that means we loose 50% of field of view in one dimension? The pictures in your webinar show rectangular full height, half width images for "what the camera sees".
Yes the field of view is reduced by 50% in the long axis of the camera. We combine the two halves of the image to create the confocal and/or the widefield. So the field of view is 50% of the camera and the camera is the CLARA -10x 7 mm – hence our Field of View is 5x7 mm.
Is Firewire faster than USB 2?
Firewire A or IEE1394A is actually slower than USB2; 1394A is specified at 400 mega bits per second (Mbps), while USB2 is rated at 480 Mbps, but the latest version, Firewire B is rated at 800 Mbps.However as you have seen from our frame rate calculations the limiting step may not be the bit rate of the channel through which you are transmitting data but the readout speed which you are able to achieve with the camera.So, in summary Firewire B is faster and Firewire A is slower than USB 2 but channel is not the limiting factor to frame rate.
Is there any software algorithm applied to the raw images like in Apotome or Optigrid, if so, is that limiting the frame rate?
Yes there is processing involved – you could think of the DSD as combining the strengths of spinning disk with structured illumination while avoiding many of the artefacts. So the confocal image is created by alignment or registration followed by weighted subtraction of the two halves of the image. To minimize the impact of any processing delay we have made the processing happen in its own software thread so that the processing of the current frame is implemented while the next frame is being acquired. We can process full frames at about 20 fps on a mid-range dual core Pentium processor.
What is the status of third party driver for the Clara specifically Micromanager, MetaMorph and Nikon Elements software?
Nikon Elements software has been integrated with Clara.MetaMoprh has now provided support and we have written a Micromanager driver for those people who would like to use that particular open source solution.
Will you be integrating the new sCMOS camera with the DSD?
Currently the aperture of the DSD aperture is matched to the Clara – that is it is 10x7mm; however we have not ruled out the possibility that the aperture may be increased and therefore make it suitable for use with the sCMOS camera. But even with this aperture mismatch, the relative speed and sensitivity of the sCMOS as well as its cost-effectiveness may make the combination viable in the near future.
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