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Dear List,

I have mentioned this (below) several times - sorry for boring you up.

I have tested Hamamatsu 16 um pixel EMCCD back in 2004 versus Hamamatsu 
EB-CCD.

I was testing HIV particles labeled with mRFP1 - a Red Fluorescent 
Fluorescent Protein.

To my surprise, the EB-CCD outperformed EM-CCD. By eye I could hardly see 
signal above background, same was true for the EM-CCD, only sparklings were 
seen. EB-CCD was still giving a pleasant image.

However, recent tests revealed that EB-CCD is still too noisy for the noisy 
channels, e.g. CFP, CFP-YFP FRET channels. In the yellow-orange-red (till 
640 nm) EB-CCD is a good camera. I might be wrong, but the first impression 
is that EMCCDs seem to be over pushed by Marketing Departments.

As for many others, my preference is still Hamamatsu Orca ER (or Orca II), 
2x2 binning gives ca. 13 um, similar to EB-CCD, and visibly smaller than 16 
um EM-CCDs.

Still, there are no reliable, statistically exciting, comparative data 
between all the available "cool" cameras in relation to sensitivity, noise, 
etc. under conditions of proper sampling (oversampling); fluorescent beads 
of uniform intensities would be very helpful, and R&D Departments could 
start working or reveal their "classified" data (Leica is a good example for 
not revealing their QE curves for their average Axiocam cameras - Even 
though they fixed many problems).

.....even though biological samples are quite noisy due to autofluorescence, 
sensitivity is of a great concern, and high transmission narrow bandpass 
filters (10-20 nm) is one of the possible solutions providing that camera is 
sensitive and noise-calm.

So far, we have low sensitivity, low noise, GOOD cameras (2x2 binning 
helps), and high sensitivity, high noise, "suspicious" cameras with no chip 
specs on the pixel-by-pixel basis for those scratched in the back, that 
makes single pixel quantitative imaging pseudo-quantitative.

Hamamatsu, Roper, Andor have lots of work to do to truly impress the LIST.

Blah-blah-blah is the easiest way to nowhere.

Cheers,

Vitaly
NCI-Frederick


----- Original Message ----- 
From: "Hugo.Ostermann" <[log in to unmask]>
To: <[log in to unmask]>
Sent: Wednesday, July 11, 2007 2:55 PM
Subject: AW: AW: Hamamatsu EMCCD


> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Listers,
>
> I have tested this Scott's approach
>
> "2X magnifier with a 1k x 1k backthinned EMCCD that has 13 um pixels to 
> give
> you an effective pixel size of 6.5um."
>
>
> with a Cascade II 512x512 16µm pixel on a DeltaVision Microscope.
>
> I found that I got 4 times less light to each pixel so the net effect was
> unvisible. The Standard Roper HQ Camera gave much nicer and less noisy
> images.
>
> What do the specialists say to this "post objective lens magnification"
>
> Best regards
>
> Hugo Ostermann
> [log in to unmask]
>
> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List [mailto:[log in to unmask]] Im
> Auftrag von Scott Phillips
> Gesendet: Mittwoch, 11. Juli 2007 18:12
> An: [log in to unmask]
> Betreff: Re: AW: Hamamatsu EMCCD
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> NOTE: Commercial Interest
>
> Dear Kathy,
>
> I agree with what Arne has said, especially the part about practical
> testing of different cameras on your system and seeing how they work on
> your samples.  All the cameras companies are happy to loan you a camera to
> make these tests.
>
> For dendritic spines, I understand the desire for small pixel sizes.  The
> Orca ER has 6.45um pixels, which is great for getting the detail of these
> small objects.  Andor offers EMCCDs in a variety of flavors with pixel
> sizes at 8um, 10um, 13um, 16 um and 24um.  In particular we offer a system
> that combines a 2X magnifier with a 1k x 1k backthinned EMCCD that has 13
> um pixels to give you an effective pixel size of 6.5um.  In this way you
> have the advantages of the EMCCD technology combined with resolution.
>
> The beauty of the EMCCD is its ability to boost the signal before it gets
> to the readout amplifier. In this way one is no longer limited by readout
> noise and is able to get a good signal with fewer photons.  These cameras
> do have a larger read-noise than some of the conventional CCD cameras, but
> this can be off-set by using a little bit of gain to effectively negate
> that read noise.  Too often users crank the EM gain way up and complain of
> increased noise.  It is true that there is an inherent noise to these
> cameras (CIC, or Clock-induced Charge) but it is related to the strength
> of the gain.  Using an EMCCD at gains less than 300x makes them a very
> effective imaging tool.  The ability to image with fewer photons means
> that you can image your sample longer with less damage.
>
> One last note of the use of the Optivar in conjuction with the Yokogawa
> spining disk.  Since the  excitation light is also passing through the
> optovar, you are expanding the incoming light beyond the back aperture of
> the objective and throwing away some of your excitation light.  I was
> speaking with representatives from Yokogawa at Jim Pawley's course a
> couple of weeks ago and they concurred that if you need more magnification
> it is best to change objectives.  The alternative is to place a magnifier
> in front of the camera so as not to affect the excitation path, as offered
> by Andor.
>
> Cheers,
>
> Scott Phillips
> Imaging Applications Specialist
> [log in to unmask]
> 206-280-5597
> Andor Technology
> discover new ways of seeing
> Web: www.andor.com
> Main US Office: 860-290-9211
>