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For once I pretty much totally agree with Jim! It is just impossible to compare something as peaky as the Hg spectrum with a continuum source. If one of the Hg peaks matches the excitation you need - Bingo! Hg is fantastic in the near UV at 360, and even better in the violet at ~405 and ~470. It is pathetic in the blue (480-500) but brilliant at 550. Obviously it isn't as specific as a laser source, but you still have to think hard about where your peaks are. LED sources are much broader, and therefore more versatile.
As for critical illumination - can you actually do it with a commercial epi illuminator?
Guy
Guy Cox, Honorary Associate Professor
School of Medical Sciences
Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of James Pawley
Sent: Thursday, 7 November 2013 1:06 AM
To: [log in to unmask]
Subject: Re: Brightness difference Hg vs LED
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Hi all,
Lots of good answers already but I think that it is important to remember that Hg has a lot of 10x peaks in the visible part of its spectrum. So an answer with one bandpass filter may not indicate a general trend.
One also needs to know a bit about the optics used. Critical illumination (source focused onto the image plane) will usually be brighter than Kohler, but whereas critical illumination that focuses the brightest "plasma ball" of the arc into the centre of the imaged area can be relatively uniform over a small field-of-view, it is not so clear what will happen when one images an LED into the image plane (It will depend on the construction of the LED).
(Note: The actual ball of a 100 w Hg source is usually about 150µm in diam. just near the electrode. Assuming both the collector lens and the objective are of about the same, high NA, the most efficient optics to convey this to the focus plane will do so at a magnification of 1:1. Of course the field of view of the objective (in µm) will vary inversely with its magnification, but the area that can be properly sampled with a given CCD/sCMOS will not vary: a 1000x1000 array of 0.1µm pixels will be about 140µm across its diagonal). However each manufacturer has made different compromises in terms of the magnification of their epi-illumination system (at very least to also accommodate low-magnification use) and some may utilize more of the light leaving the original light source (arc or fibre) than others.
The idea of measuring the output at the microscope end of a fibre-optic seems sensible as long as this is how you will illuminate your sample on your exact setup. However, such fibers have an NA (angle at which the light leaves them) and so not all the light leaving them necessarily makes it to the image plane. For instance, a given system may under- or overfill the entrance pupil of a given objective. As one can never make the light brighter (in photons/second/µm*2) simply by focusing it, if the end of the fibre is much larger than 150 µm (in the example above) then some of the light leaving it must inevitably be lost somewhere before it reaches that part of the focus plane covered by the
1000x1000 image sensor.
My preference would be to measure the light leaving the objective once the field diaphragm has been set to repeatable diameter (say 100µm diam. at the image plane). Of course, you can only set the diaphragm properly if the scope is set to Kohler. Once it is set (to standardize the light path to that point) you can still tweak and condenser focus to approximate critical illumination. (i.e., make the image as bright as possible). The adjustment is inevitably a bit of a "fudge" because, as the arc is a 3D source rather than the planar object imagined by the Kohler Illumination diagram, it "cannot be focused into a plane".
Of course, there is still a problem: You probably want to use a hi-NA objective but above NA 0.5 more and more of the high-NA light will reflect back into the objective from its front surface.
Rays at >NA 1.0 will not escape into the air at all. Efforts to couple the sensor of your photometer to the objective will a drop of immersion oil will only work if there is no air-gap between the sensor window and the sensitive element. The options are:
1) To couple a small, strong plano-convex lens onto the back of the microscope slide with immersion oil to make the light beam less divergent or
2) To set up using an NA 0.75 air objective and hope that the difference in NA isn't too important or
3) Measure the fluorescent light signal at the CCD from a thin, uniform layer of fluorescent dye (It should be thin so that you don't end up focusing too far inside the layer, where SA and absorption may be variables you don't want).
So now you see why just measuring the output of the fibre seems easier.
Hope that this isn't too confusing. I am really theoretically very pro-LED (faster, cooler, just the light you want etc). Indeed, I think that Chapter 3 in the Handbook was one of the first places where LED microscope sources were discussed in any depth. I would just like to see a few more variables nailed down.
Cheers,
Jim Pawley
>
>
>Hi Phil,
>
>Current LED light sources can be brighter and (should have) more stable
>light output (and "instant" on/off, and less heat output and less ozone
>and no chance of the bulb exploding ...
>"do not look at top of arc lamp with remaining eye". Also many LEDs
>have precise - and reproducible - voltage control. Purchase price will
>eventually be made up in total cost of ownership.
>
>Brighter light sources enable selection of narrower wavelength range,
>for example, at the excitation peak of the desired fluorophore (and
>hopefully minima of unwanted fluorophores), leaving more room for
>emission wavelength range.
>
>George
>
>On 11/5/2013 11:56 AM, Philip Oshel wrote:
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>>
>>All,
>>
>>I had this question put to me by a new faculty member, and don't have
>>a ready answer:
>>"Is there a ballpark percentage for how much less bright an LED vs a
>>standard mercury lamp light?"
>>This is for regular epifluorescence, not confocal.
>>
>>This is in the realm of arm-waving over a picture of beer (a good,
>>dark stout), ignoring brands, how old the Hg bulb is, ex/em cubes,
>>which part of the spectrum is used, and all that. Personally, I'd
>>think the answer is more like, "Doesn't matter, the dimmer system is
>>still too bright to use all the available light and not damage the
>>specimen." But ... ?
>>
>>Phil
>
>
>--
>
>
>
>George McNamara, Ph.D.
>Single Cells Analyst
>L.J.N. Cooper Lab
>University of Texas M.D. Anderson Cancer Center Houston, TX 77054
>Tattletales http://works.bepress.com/gmcnamara/26/
--
James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0, Phone 604-885-0840, email <[log in to unmask]> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
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