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Hi everybody,
Working with Leica SP5 and SP8 equipped with HyD detector, I have characterized the HyD mode of operation to help our users to obtain the best with the HyD. If it is helpful, I could share an application note that I wrote for internal use in our facility (https://seafile.igbmc.fr/smart-link/b4d36de8-550f-45e2-9ec5-5b20dda7191d/)
I agree that " HyD detector is working in the same conditions independently of gain settings or mode. What differs is the processing of the HyD output signal."
The photon counting mode is the HyD basal mode of operating and we need to be careful about "saturation" ( that means that the Hyd detector is no longer linear when the signal reaches 60 MHz or more than 60 counts/µs, so the signal may be underestimated). This can be a problem for intensity quantification.
The standard mode is just a processed mode of the output of the hyD with first a correction for the non-linearity and the implementation of a gain (that is quite artificial in the sense that the gain is a "digital" gain, no action on the detector itself, the output signal remains the same - just a processing operation). In a simple way, changing the gain from 10 to 100 % just multiply by a factor 10 the count value.
So you will not damage the HyD by using a high level gain, but you can easily create a false saturation or have only few discrete and separated intensity levels in your histogram so little information in your image (knowing that the gain can increase until 500 %).
Damage will occur if the detector receives a too intense fluorescence signal and is overloaded many times.
Whereas, when new users are trained on our SP5-SP8 systems , we recommend to use the HyD in the standard mode gain 10 % (non-linearity correction is applied but no artificial gain).
If the count levels is too low, then apply an accumulation to fill the 8bit depth in a better way.
Best
Elvire
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Elvire GUIOT
Imaging Center of IGBMC
https://www.igbmc.fr/plateformes-technologiques/microscopie-photonique
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-----Message d'origine-----
De : Confocal Microscopy List <[log in to unmask]> De la part de Khoroshyy Petro
Envoyé : vendredi 2 février 2024 10:11
À : [log in to unmask]
Objet : Re: Question about proper use of HyD detectors
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Hi everybody.
I'm afraid I have to disagree with this statement.:
-------------
- Does routinely using the detectors at 100% damage them?: High gain
settings, especially if used routinely, can lead to faster wear and tear of
the detectors. While HyD detectors are robust, like any electronic
component, they can suffer from degradation over time if consistently.
------------------
HyD detector is working in the same conditions independently of gain settings or or mode. What differs is the processing of the HyD output signal.
So our ability to kill the HyD detector is the same in one mode or another.
Best.
Petro.
On Thu, 1 Feb 2024 at 18:43, Catalin Pavel <[log in to unmask]> wrote:
> *****
> To join or leave the confocal microscopy listserv or to change your
> email address, go to:
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> *****
>
> Hi Michael,
>
>
> Your inquiry touches on several critical aspects of advanced
> microscopy techniques, particularly the use of Hybrid Detectors (HyD)
> on Leica SP8 and Stellaris confocal systems. Your observations and
> challenges are common in settings where users have varying levels of
> experience and training with these sophisticated imaging tools. Let's
> address your concerns
> systematically:
>
> Proper Use of HyD Detectors and Image Optimization
>
> 1. Gain Settings and Bit Depth Adjustment: Increasing the gain to
> 80%-100% and switching the bit depth to 12 bits can indeed lead to
> better
> image visibility but at the cost of increased noise, which can
> compromise
> image quality. Your approach to educating users on the importance of
> balancing gain and bit depth to optimize signal without unnecessarily
> increasing noise is correct. The key is to capture as much signal as
> possible without saturating the detector, which requires
> understanding the
> system's dynamic range and the sample being imaged.
> 2. Understanding Histograms and Dynamic Range: The use of histograms to
> adjust the dynamic range and avoid saturating the entire 8-bit
> spectrum is
> an excellent practice. Encouraging users to focus on collecting
> high-quality images with low noise by not fully utilizing the 8-bit
> range
> (unless necessary) helps in achieving better image quality. The
> insistence
> on using the glow-over display mode to focus on these aspects is
> commendable.
> 3. Resistance to Best Practices: Resistance from experienced users to
> adopt optimal imaging practices is not uncommon. This resistance often
> stems from habits developed over years of using different systems
> or under
> different training regimes.
>
> Addressing Your Questions:
>
> - Is there a right way?: Yes, the right way involves optimizing image
> quality while minimizing noise and avoiding detector saturation.
> This often
> means not maxing out the gain or unnecessarily increasing bit depth and
> instead focusing on the proper use of the system's dynamic range.
> - Does routinely using the detectors at 100% damage them?: High gain
> settings, especially if used routinely, can lead to faster wear and
> tear of
> the detectors. While HyD detectors are robust, like any electronic
> component, they can suffer from degradation over time if consistently
> pushed to their limits.
> - Suggestions for Reeducation:
> - Workshops and Seminars: Organizing regular training sessions that
> emphasize the scientific rationale behind optimal imaging
> settings can be
> beneficial. Use real examples to show the impact of different
> settings on
> image quality.
> - One-on-One Training: Some users may benefit from personalized
> training sessions where they can see firsthand the effects of
> their usual
> settings vs. optimized settings.
> - Documentation and Guides: Providing clear, accessible guides that
> explain the principles of image acquisition and optimization can
> help users
> understand why certain practices are recommended.
>
> Final Thoughts:
>
> It's understandable to feel concerned about the misuse of
> sophisticated equipment, especially when it impacts research quality.
> Your efforts to educate and guide users are vital for maintaining the
> integrity of scientific imaging. While it can be challenging to change
> ingrained habits, consistent education and demonstration of best
> practices are key. It's also essential to balance guiding users
> towards optimal practices and allowing them some autonomy to explore
> their methods, as long as it doesn't compromise the equipment or the data quality.
>
> Considering your role and expertise, your approach to reeducation
> should continue to be firm but flexible, providing evidence-based
> guidance while remaining open to discussions on individual techniques and preferences.
> Ultimately, the goal is to ensure that all users can achieve the
> highest quality images possible with the technology available,
> contributing to the advancement of research and scientific knowledge.
>
> On Wed, Jan 31, 2024 at 6:51 PM Cammer, Michael <
> [log in to unmask]> wrote:
>
> > *****
> > To join or leave the confocal microscopy listserv or to change your
> > email address, go to:
> > https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> >
> >
> > I am writing to seek advice and knowledge regarding properly using
> > HyD detectors on Leica SP8 and Stellaris systems.
> >
> > This week I have tried to train & troubleshoot issues on two SP8s
> > and one Stellaris where I found that people had turned the gain up
> > to 80% to 100% and in one case switched the bit depth to 12 bits to get better images.
> > This appears to be routine common sense.
> >
> > In all cases I tried to explain that when the histogram peaks become
> > discrete, turning up gain (and bit depth) adds no information and
> probably
> > adds more noise than simply adjusting the LUT.
> >
> > People are unwilling to wait for line averaging.
> >
> > Accumulation to build an image is both slow and challenges their
> > visceral need to see the image immediately.
> >
> > When I train people who have never used a confocal before, I find I
> > can guide people to collect images that don't fill the whole 8 bits
> > with low noise high quality images. I insist they use glow over
> > display mode and focus on the histogram to define the dynamic range
> > which doesn't need to fill all 8 bits.
> >
> > But people who have used confocals in the past are highly resistant
> > to
> any
> > of this. They crank the gain until images have saturated spots and
> insist
> > that this is how they were trained in the past. (And don't get me
> started
> > on the snapshot mode...)
> >
> > Is there really a right way? (Other than photon counting mode.)
> >
> > Does routinely using the detectors at 100% damage them?
> >
> > Do you have suggestions how to reeducate or should I just relax
> > about
> this?
> >
> > I'm interested in any an all statements of fact and opinion
> > regarding
> this.
> >
> > Best regards-
> >
> >
> >
> > Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
> >
> > NYU Langone Health, New York, NY 10016
> >
> > [log in to unmask]<mailto:[log in to unmask]>
> >
> > http://nyulmc.org/micros http://microscopynotes.com/
> >
> > Cell voice (no text) 1-914-309-3270
> >
> > Office 1-646-501-0567
> >
>
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