however, because detectors have a non-zero dark count rate and because they have a maximum detection rate before they saturate. Since dark counts and photon counts both contribute shot noise, if you try to image with a dwell time that isn’t much less than the inverse of the dark count rate, the darker pixels in the image will start to be dominated by dark shot noise rather than photon shot noise. In this case one should either image faster or get a detector with fewer dark counts. Te maximum detection rate similarly puts an upper bound on how fast once can image for a given SNR by limiting the number of photons any pixel can contain. For example, with a hypothetical detec- tor that has 1 dark count per microsecond on average and a maximum detection rate of 100 photons per microsecond, you would want to image with a dwell time of less than 1 microsec- ond, and would be limited to a maximum SNR of less than 10. Tis would be a very bad detector since its dark count rate and maximum detection rate are very close. For real photo multi- plier tubes (PMTs), usually the dark count rate is extremely low (KHz) while the maximum count rate is somewhere around a few billion, so you can image very slow without being lim- ited by dark counts, but if you try to image with dwell times of much less than a microsecond, the SNR pretty quickly drops down into the 20s or below and you either have to slow down or start averaging. Tis is why the default dwell time is usually around a few microseconds when using a PMT-based confocal or 2P. Michael Giacomelli

Te concern of one of the early posts in this thread was

fluorophore saturation and (superlinear) bleaching. Point scanning confocal is able to saturate the fluoro-

phores in the focus of the laser beam, so 1) the fluorescence intensity is lower than it should be and 2) bleaching may be accelerated (beyond the linear case analyzed in detail). With this in mind it makes a difference if a sample is scanned

slowly or quickly (+averaging) and the laser power used. Since fluorescence itself is a fast process taking only a few

nanoseconds it cannot be ‘outcompeted’ by fast scanning (i.e., a dwell time of a few ns per diffraction spot). Tis may not even be desirable if you want to collect the fluorescence before moving to the next spot. It seems to me that fluorophore saturation and the (earlier mentioned) excited state absorption and resulting bleaching can only be reduced by reducing the peak intensity. But there are other transient states of the molecules with microsecond - millisecond lifetimes that can lead to increased bleaching (when hit by yet another photon), and this is where faster scanning (+averaging) may help. I say *may* as I haven’t performed in-depth experimental testing of these effects, and they will be heavily sample-dependent. Zdenek Svindrych

My understanding has always been that fast scanning

would help, but we can’t go fast enough with current com- mercial equipment. Tis was shown by Stefan Hell’s lab when they used an electro-optic deflector (EOD) to obtain a pixel dwell time of ∼6 ns. Tis means a single excitation event/fluo- rophore/scan. Here are some relevant quotes from the paper (Schneider et al., Nature Methods 2015): “In confocal micro- scopes with approximately microsecond pixel dwell times, fluorophores typically face 10–1,000 excitation events until the illumination spot is moved, usually aſter a certain number of photons are collected on average. Tus, the excitation, detec- tion and bleaching events appear continuous despite the sto- chastic nature of these processes. Only if the dwell time of the moving illumination spot on a fluorophore is shorter than the

2020 January •

average pause between two excitation events will the molecule not be subjected to multiple events. In this case, the molecule will emit at most one photon per illumination cycle, preserv- ing the stochastic nature of the emission from the interrogated pixel. In the simple but common situation of excitation with relatively bright pulses that are shorter than the fluorescent state lifetime, the light exposure of a molecule typically must be shorter than the interval between two pulses.” “Terefore, our fast scanning approach reduces bleaching

and blinking. In fact, we compared the fluorescence yield of new ultrafast scanning with that of conventional (slow) scan- ning by imaging equally sized and dense areas for equal dura- tions. For many fluorophores and laser configurations, we observed that the total signal increased by 1.5- to 4.5-fold when ultrafast scanning was used (Supplementary Fig. 1).” Douglas Richardson

Light and Electron Microscopy Training

Microscopy Listserver Minimizing user error in a SEM laboratory My university is installing a new field emission SEM that

will be used by a wide range of faculty in biology, chemistry, physics, geology, archeology, and ceramics. Undergraduate stu- dents will use the instrument under the supervision of their fac- ulty advisors. An engineering physics professor and I (a geology professor) will be the primary managers of the facility. We are both reasonably competent, knowledgeable individuals, but we will have no dedicated technician and will never have a budget for one. I would like to minimize people accidentally screwing things up for the rest of us. I have protocols in mind to achieve this, but I know that people in this community are far, far more experienced than I am. Experience can be a good filter for iden- tifying effective ideas vs. folly. QUESTION: If you have experi- ence managing an SEM facility used by diverse users, would you please share some of your successful (and unsuccessful) ideas that you’ve tried to minimize user harm to the instrument? Tank you. Kurt Friehauf

We are a primarily undergraduate institution with no

recharge for in-house use. Our microscope (W-gun) is fully automated and is turbo-pumped, so our primary goal is to pro- tect the backscattered detector from damage or from someone popping the X-ray window by slamming the specimen drawer shut. If a PI wants his/her lab to use the SEM, I train the PI first. We have a basic Standard Operating Procedure (SOP) that requires use of a standard stub and holder, and requires that the specimen not extend more than 3 mm above the stub sur- face and not beyond the edge of the stub. Working distance is set at 20 mm nominal (about 17 mm

in practice), and not decreased (increase is okay, but the WD is set back to 20 mm at the end of the session). Once trained, the SOP is signed by the PI (whose department thereby assumes financial liability for damage), and the PI is allowed to use the microscope independently. Te PI is allowed to train his/her students (again with the same SOP); before the students are allowed to use the scope independently the PI has them sign off on the SOP, and he/she countersigns (again the PI’s dept. is responsible for any damage). I develop custom SOPs for PIs or students that need special conditions (specimen tilt, X-ray anal- ysis at 10 mm working distance, large specimens, etc.), but the same conditions apply—the PI and his/her department agree


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