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Dear All,
I would like to add some comments to this discussion.

Guy, you are right, we've had a discussion about CLEM about a year ago (and 
I'm sure, this will not be the last one). 

Michael, I think there is misunderstanding about data can be obtained with 
CLEM. For each individual pixel the optimal exposure time is determined. 
The integrated fluorescence signal is extrapolated at the end of each 
pixel. So, you are right, this extrapolation is a kind of correction 
procedure, but this happens internally in the CLEM electronics and you do 
not notice the difference between CLEM and non-CLEM when you look at the 
images. So, this means that the extrapolated pixel intensity is unbiased. 
The only difference is that the noise on the signal is increased for pixels 
that allow a shorter exposure time. So, CLEM reduces the exposure time in 
very bright pixels (S/N is too good in non-CLEM) and in dark background 
pixels (why would you want to have a good S/N if there is no signal!). This 
reduced S/N in parts of the image where a good s/n is not needed, is the 
price for a reduction in photobleaching and phototoxicity. The signal does 
NOT change by CLEM.

This brings me to the point of quantitative imaging with CLEM:
I do really not agree that CLEM should be switched off when you want to do 
quantitative imaging as suggested by Stephen. As I explained: CLEM does not 
change the measured gray values. There is only one reason why people might 
think that CLEM does not give quantitative data and that is photobleaching. 
I will here argue that CLEM does give images that are not less quantitative 
than non-CLEM images. 

So, now we come to the point of photobleaching.
Since CLEM uses different exposure times for different pixels, the sample 
is non-uniformly exposed to light. This causes non-uniform photobleaching. 
So, if you scan your sample several times, you cannot use a standard method 
for photobleaching correction. So, Ron Hoebe and I developed a dedicated 
correction procedure for CLEM images (which we will make available later). 
This algorithm calculates how much light each pixels in a 3D volume has 
received before scanning that pixel and corrects for bleaching. Since we 
know of every pixel how much the exposure time was, we can correct. This is 
not difficult, it is only a little bit more complicated. We also measured 
the difference between this dedicated procedure and a standard correction 
methods and found out that the difference is not much (only a few 
percents). Ron did a lot of computer simulations to prove that his program 
works, by simulating every single photon for every single pixel (lot of 
computation time...).

So, Stephen, you might say: Well when I want to have quantitative images, I 
just turn of the CLEM. Let's see what would happen. When you do turn it 
off, your photobleaching will increase again to its non-CLEM value (about 5 
to 10 fold) So you will end-up with a strongly bleached image and a dead 
cell! Great! You do a nice quantitative analysis of an image of a dead cell 
that you can hardly see anymore! And you do this only because you are 
afraid that the non-uniform bleaching is not corrected optimally?

And what about non-uniform photobleaching in non-CLEM confocal images. Does 
anyone realize that photobleaching is stronger in the middle of an image 
than more to the edges? Since we illuminate the sample with a cone of 
light, the pixels more to the edges of the volume get a lower light dose at 
the end of a scan. And I have never seen photobleaching correction 
procedures that take this into account. So, how quantitative is your 
imaging right now (without CLEM)

Finally, we should realize that bleaching is far from linear (especially in 
confocal and TPE microscopy. All bleaching correction procedures assume 
linear bleaching (what else can you do??). So, with this in mind: How 
quantitative are your images now. I prefer a 7-fold reduction of 
photobleaching so that I do not need any correction procedures!!

Aryeh, you are right: CLEM also increases the dynamic range of the system. 
However, your argument "the detector can be operated with a more linear 
response to intensity changes" is not fully correct. We do not reduce the 
intensity of laser light, we only reduce the light exposure time. But since 
the exposure time is reduced for very bright pixels, clipping of the 
(integrated) signal is history. This makes that you can see details in the 
weakly stained parts while not saturating the brightly stained parts. --> 
larger dynamic range.

Stephan asked: "can someone who has used the CLEM device please confirm..."
So far, only a hand full of people have used CLEM. Some developers of Nikon 
rested CLEM and Ron and I as the inventors of CLEM have tested it, but so 
far only one person seriously applied CLEM for his biological research. 
Winnok de Vos from the group of Patrick Van Oostveld from Ghent, Belgium 
visited our lab for months and months to do his live cell imagine. He spent 
many nights and days behind the CLEM microscope. To his experience, cells 
can cope with a certain amount of trouble (ROS, light damage). As long as 
your exposure is under this critical threshold, you are fine. When the 
light is too much, you are in trouble. By using CLEM he succeeded to stay 
under this threshold and he just could go on imaging. Without CLEM it was 
about half an hour; with CLEM for more than 24 hours.

So, we should not only focus on the bleaching reduction by CLEM (although, 
where do you find a anti-bleaching reagent that reduces bleaching by a 
factor of 5 to 10 ????) but also to the reduction of phototoxicity. 
 
At this moment there is only one working CLEM microscope here at the 
University of Amsterdam and some prototypes of the Nikon-CLEM. But soon 
there will be much more now Nikon implemented CLEM as a standard option in 
the new A1. As soon as I have my Nikon-A1, I will give my comments on my 
findings, especially on my first A1-CLEM experiences. I expect a better 
CLEM than our own experimental set-up. We'll see...

I think this comment satisfies you for the coming few months....

Kind regards, Erik




---------------
E.M.M. Manders, PhD
Ass. prof. Molecular Cytology
Manager Centre for Advanced Microscopy

Centre for Advanced Microscopy
Swammerdam Institute for Life Sciences
Faculty of Science
University of Amsterdam		
E-mail:    [log in to unmask]
Tel:       +31-(0)20-5256225
Fax:       +31-(0)20-5257934
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