McTips

Tips & Procedures (web page)  http://confocal.jhu.edu/mctips/tipsprocedures   (please contact GM directly for PDF).

** 20240123U note: I am currently 20% time at Ross Image Center, so new McTips will be rare, and new 2024 page not worth organizing. I put an antibody solutions centrifugation tip near the top of thispage (just past the web links).

2021 PC Tips for microscope and analysis workstations http://confocal.jhu.edu/mctips/pc_tips_2021

2022 PC Tips for microscope and analysis workstations http://confocal.jhu.edu/mctips/pc_tips_2022

2022_part2 PC Tips (started 20220705U) http://confocal.jhu.edu/mctips/pc_tips_2022_part2

Confocal Sweetest Spots (web page) http://confocal.jhu.edu/mctips/confocalsweetestspot  

  Online Image Data Repositories - blog (20210427W web page)  image-data-repositories

    MPMicro "Multi-Probe Microscopy" (1500 pages - please do not print out) https://works.bepress.com/gmcnamara/2/ 

Multiplex fluorescence microscopy  http://confocal.jhu.edu/mctips/multiplex

     Alexa Fluor 610-X performance calculations wrt FISHscope http://confocal.jhu.edu/mctips/af610-x   (gm note: SulfoRhodamine 101 = SR101, Brightness=125 aboutthe same as AF610)

     StreamBio UK LinkBright CPNs labeling kits available (1/2003) from Sigma-Aldrich (hopefully low shipping and total costs) http://confocal.jhu.edu/mctips/streambiouk-linkbright-cpn-kits

McTips 2023 http://confocal.jhu.edu/mctips/mctips_2023 (no PDF)

McTips 2020 PDF go to: McTips 2020 download at https://works.bepress.com/gmcnamara/90

McTips 2019 PDF go to: McTips 2019 download at https://works.bepress.com/gmcnamara/85

McTips 2018 PDF go to: McTips 2018 download at https://works.bepress.com/gmcnamara/84

McTips 2017 PDF go to: McTips 2017  download at https://works.bepress.com/gmcnamara/81/

McTips 2017 Direct download is    https://works.bepress.com/gmcnamara/81/download 

Factoids (started 01/2022) http://confocal.jhu.edu/mctips/factoids

FISHscope Quick Tips (startup, close out session)  http://confocal.jhu.edu/mctips/fishscope-quick-tips -- FISHscope main page is   http://confocal.jhu.edu/current-equipment/fishscope 

* Quick Leica SP8 confocal microscope tip (see more info on our SP8 page from current equipment):

I recommend (we have Leica SP8 Klondike linear scanner - most SP8's are the same sub-model):

HyD detectors, Photon counting mode ... not max linear counting rate is 6 Mcps (million counts per second) = 6 counts per microsecond or 0.6 counts per 100 nanoseconds.

          At very high counts the safety interlock should trigger, resulting in all values ZERO until the user stops scanning (i.e. 1000x1000 x 100 planes Z-series could be lots of zeros).

600 Hz line scan rate (enables full range of zoom

10 Line accumulation as a starting point, more or less depending on user needs. Max is 16 line accumulation.

1 Frame accumulation as starting value, more if needed.

Modest laser power, usually 0.5% or 1% for 405nm laser, 1 or 2 percent for any of 488, 552, 638nm lasers, depending on what fluorophores are used (ex. DAPI, Alexa Fluor Plus 488, Alexa Fluor Plus 555, Alexa Fluor Plus 647).

Sequential scan tracks, usually ... can combine blue (DAPI) and NIR (AF647Plus) in moany experiments (3plex plus DAPI), such as: (i) AF555Plus, (ii) AF488Plus, (iii) DAPU + AF647Plus.

Always have detection bandpass AT LEAST 10nm away from any laser line - even if the laser line is not used in that wscan track. If no NIR, turn off the 638nm laser.

 

Alexa Fluor Plus secondary antibodies 3x brighter 1.3x more expensive

20240207W

Alexa Fluor Plus secondary antibodies 3x brighter 1.3x more expensive

Famous quote: "Time is money".

ThermoFisher (Molecular Probes) introduced circa 2023 Alxa Fluor Plus secondary antibodies.

https://www.thermofisher.com/us/en/home/life-science/antibodies/secondary-antibodies/fluorescent-secondary-antibodies/alexa-fluor-plus-secondary-antibodies.html#table

Donkey anti-mouse is $355 for 1 mg (1 mL)

https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Highly-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A32766

If you use 1:100 dilution, then 10 ug (10 uL) is $3.55 per coverglass, imaging dish, etc.

the "standard" prouct is 1.3 fold less, so $2.55.

the primary antibody would likely be used at the same amount, say $1 per specimen.

Confocal microscope time is $27/hr (work hours, our Leica SP8 or Olympus FV3000RS).

User time making specimens and imaging: $0 from the standpoint of the P.I. = already accounted for.

So 1 hour confocal time:

Plus: $3.55 + $1 + 27 = $31.55.

Regular: $2.55 + $1 + $27 = $30.55.

Or, since Plus is 3x brighter, could reduce confocal time to 1/3 hour ($9) and Plus slide same brightness as regular for 1 hour:

Plus at 20 minutes: $3.55 + $1 + $9 = $12.55.

My recommendation is choose the same acquisition time, get 3x brighter data.

I suepect many experiments could use lower concentration of secondary antibody to get "same or almost as bright" as the common 1:100 dilution = same more money, similar brightness. There is also potential to optimize incubation time of the primary and/or secondary antibodies, such as dilute further, incubate 24 hours (aking sure the specimens do not dry out), see (direct label flow cytometry experiments:

Whyte, C. E., Tumes, D. J., Liston, A., & Burton, O. T. (2022). Do more with less: Improving high parameter cytometry through overnight staining. Current Protocols, 2, e589. doi: 10.1002/cpz1.589

***

Big boost in signal: tyramide signal amplification (TSA) has been available since around 1990. Used optimally, can increase signal approximately 100x, while background is still close to zero. This could reduce imaging time by a lot (33 fold compared to "Plus" above) or increase signal for same scan time, or some combination. HRP is "easy to kill" the enzyme (Biocare Medical "PeroxAbolish" is one of the coolest name products for this), then multiplex,

Many companies now offer TSA reagents, see for examples (two of many):

https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/immunofluorescence/tyramide-signal-amplification-tsa.html

https://www.tocris.com/product-type/tyramide-signaling-amplification-tsa 

 

McTip 20240131 Centrifuge down antibodies

McTip 20240131 Centrifuge down antibodies

GM note1: use cold (stored 4 C) microfuge rotor - if use room temperature rotor, you will cook your antibodies - similar to frying egg whites. You could use ultrafiltration (i.e. 40nm Amicron filte) though some risk of your antibodies sticking to the filter (lose of titer).

GM note 2: Also, if you use BD/BioLegends Brilliant Violets (BV421, etc), Brilliant Ultraviolets (BUV395 etc) you should use BD's Brilliant buffer to avoid aggregation of the Brilliants; quantum dots may or not play well in Brilliant Buffer and vice versa. I also note the authors used several QDot antibodies - quantum dot antibodies originally (first generation thermoFisher/Molecular Probes, acquired QDot Corp circa 2005) were highly prone to aggregation - may or not have been corrected in current (2024) versions. QDots have "blinking issues" which may be good for PALm/STORM/SMLM super-resolution microscopy and/or bad for standard "brighter is better" fluorescence microscopy.

Fun fact: Authors used many Brilliants (BUV, BV, BB) and several quantum dots. Brilliants technology won Nobel Prize in Chemistry in 2000 "conductive polymers" (see Sirigen history page - acquired by BD); Quantum dots won Nobel Prize in Chemistry in 2023 (more for light sources for televisions and computer screens than for fluorescence). 

bioRxiv preprint doi: https://doi.org/10.1101/2023.12.14.571745

50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells
Andrew J. Konecny, Peter Mage, Aaron J. Tyznik, Martin Prlic, Florian Mair

Antibody mixes were centrifuged for 10,000 x g for 5 minutes immediately prior to use to remove antibody aggregates. We have observed a decrease in aggregation of Qdot 605 :: CD2, BUV615 :: CD141, Qdot 625 :: NKp46 (CD335), Pacific Blue :: CD1c, NovaFluor Blue 555 :: CD8a, NovaFluor Blue 585 CD4, and RB744 :: CD127. An example for such aggregates before and after centrifugation is shown in Online Figure 18.

Network Address Tip

 

Apple Mac users networking SMB://networkname is equivalent to Windows PC \\networkname

(the above line item is from McTips 2018 index).

for example, if a server is \\TikiGoddess on a Windows PC, then SMB://TikiGoddess on a Mac.We do not currently have a PC or server called TikiGoddess - to see Tiki_Goddess, see http://confocal.jhu.edu/gallery   .

Note: You may need to be logged in as a local user, not any JHU SOM Domaion login (ex: WIN domain). Local login cannot see the Domain assets for security. You can easily log in/out or "switch users" between a local and a Domain login.

Please do not give out the names of our file server or any image core workstations to anyone.

**

You can create a network "share" on YOUR PC. Your office/lab PC is owned by JHU, and managed by your PI, so you should get permission before creating a "shared" folder -- and if you do not fully understand networking and computer security you should probably NOT crete any shartes.

For example, C:\TikiGoddess\Share could be made visible on the campus network (may be restricted to being on -- or not on -- the Domain). Then you could transfer files from our acquisition PC to your PC "share", and as soon as you get back to your lab, move the contents from your "Share" folder to a secure (not visible not network) folder. 

We recommend you upload from our acquisition PC(s) to your JHU OneDrive - this is simpler (though usually slower) than network share drives. MyJHU --> Cloud --> JH OneDrive (can be made a favorite in MyJHU), upload your new data (i.e. save to a folder with today's date, so your new data is segregated from previous data), then sign out of your OneDrive, wait for browser page to update, then sign out of MyJHU, wait for browser page to update, then close the web broswer.

Reminders:

* No USB drives on any of our image core PCs.

* no checking your email (JHU or personal) on any of our PCs. Besides your privacy, if you get an email with a computer virus it could infect our PCs and JHU network (which could lead to you being fired for cause). 

* No surfing the "Dark Net" on any of our PCs - and please use MyJHU just to access OneDrive (not email, not other content ... not your daily covid-19 "click"). 

* Scroll down for some more tips.

McTips 2018 PDF (as of 201810002) go to: McTips 2018 

https://works.bepress.com/gmcnamara/84

The McTips 2018 includes some of my thoughts with respect to Fast Photon Counting (FPC) to make fluorescence confocal microscopy both faster and more quantitative than is now practiced by most biomedical researchers (i.e. twiddle the HV gain and offset values until someone proves their boss' hypothesis ... especially when they are using 'Santa Crap' antibodies and don't bother with controls).

George's Quick Tip on Optimizing Confocal Microscope Image Acquisition

20190603Mon (ok, not that quick ... please be patient)

Applies to: Olympus FV3000RS (analog PMTs), Leica SP8 (photon ounting HyDs), and other point scanning confocal microscopes (that is, whatever instrument you are using, whether at JHU or elsewhere).

Goals - sequential!!!:

1. acquire optimally, then explore your data (initially at the confocal PC, then at your office PC) visualization and quantitation.

2. compare acquired image data to your experiment requirements -- what hypothesis(es) are you testing? how close to resolution limit(s) do you need to be to acquire the data to critically test your hypothesis(es) - then figure out what settings to use in the future.

Quick Summary for 1.4 NA objective lens:

  • Use pixel size 60 nm, Z-step size 180 nm.
  • Use fast pixel dwell time, ex. 2 microseconds per pixel on FV3000RS ... small pixel dwell time minimizes photobleaching and phototoxicity.
  • Use "a few" line averaging, try 3 line avg on FV3000RS, so total pixel dwell time (t-PDT) = 6 microseconds (2 us/pixel * 3 line average). More averaging if needed.
  • FV3000RS: low laser power, HV 500 mV, offset = 0 (can subtract later), always keep "Gain" at 1.0 ... Leica SP8 is simple: use HyD photon counting mode.
  • Unidrectional scanning -- because (i) less prone to jaggedness artefacts, and (ii) every pixel gets identical "dark time", since flyback is dark. That is, in bidirectional scanning, the end pixels get re-illuminated immediately, whereas middle has delay. For more on jaggedness, and a fix, see Papiez et al 2019.
  • I generally operate FV3000RS in galvo mode, 2 microsecond pixel dwell time ("PDT") ... resonant scanner mode is faster (typically average more to get usable "total Pixel Dweel Time") ... in fact, RS mode likely to benefit the most from my proposal below.

Proposal (written here 20190603Mon): Acquire onto MULTIPLE DETECTORS, by setting the detectors to ADJACENT wavelength bands, equal area under the emission spectrum curve (on FV3000RS assumes dichroic beamsplitters enable optimal split). 

FV3000RS: 4 internal GaAsP detectors (but also dichroic beamplitters that may not be optimal for your fluorophore ... consider switching to fluorophores that are ... and/or help us buy optimal). Example:

   Standard scan: 2 microsecond pixel dwell time. one detector, 500-560 nm, 8 line average.

    VS

   Proposed scan 2 microsecond pixel dwell time. FOUR detectors, 500-515, 515-530, 530-545, 545-560 nm, 2 line average. Note: FV3000RS (currently., 6/2019) does not have optimized dichroics to take advantage of this hypothetical setting. It would be interesting for Olympus to (i) install 50/50 beamsplitters, and (ii) add to FluoView software the image math to combine (add, 12-bit 0 ... 4095 each --> 14-bit 0 ... 16380) the signal of all four detectors, (iii) optimize C.I. deconvolution to take aqdvantage of extended dynamic range. Bonus: "discard outlier" option when operating PMT's in high HV mode (13-bit if always throw out most extreme value ... Max Krummel has punlished similar idea). I also note ISS.com may have electronics (and software) to enable "fast photon counting" with current FV3000RS PMTs, and also pulsed laser to enable "fast FLIM". going from current analog (0 ... 4095) to photon counts (photon counts!) would be a "game changer" for our FV3000RS in terms of simplfying quantitation (the deconvolution module would also need to be optimized for photon counts).

 

Leica SP8: 2 HyD photon countng hybrid detectors. Can split the emission spectrum (area under the curve) 50:50, add together ... enables image acquisition in half the time (or get 2x more photons for 'full time). Reminder: scan as fast as possible for whatever zoom you ae using. 600 Hz enables full field of view (0.75x zoom by Leica convention). Example: 600 Hz with 10 Line Accumulation is superior to 60 Hz no accumulation, because less photobleaching and less phototoxicity for 600 Hz. SP8's fastest speed is 1800 Hz (requires 7.5x zoom or greater). Note: SP8 has spacefor two more internal HyD detectors AND Leica introduced (Spring 2018) third generation "SMD HyDs" (cooled, so less thermal noise, resulting in fewer dark counts, and faster counting by about 2x than our 2nd gen HyDs, so effectively bigger dynamic range per pixel dwell time), see below for all four seeing one fluorophore together ... would be great if you could provide us the $$$ to get a full set of four SMD HyD's (trade in our current two) + chiller + a (yes, even more $$ for) pulsed laser to enable the FALCON (Leica Fluorescence lifetime contrast ... generically "Fast FLIM") + PC and Leica LAS X software upgrade. 

  • if we can get our Leica SP8 upgraded to four SMD HyD's (per above) we would be able to go 8x faster than now (single 2nd gen HyD), by the ~2x faster photon counting rate of SMD HyD * using all four SMD HyDs to observe a single fluorophore. 
  • SMD HyD's count at 80 MHz, that is 1 photon is detected in each 12.5 nanosecond reptitition.

 

Related References:

Vinegoni ... Weissleder 2016 Nature Communications https://www.nature.com/articles/ncomms11077   

whose high dynamic range by multiple detectors featured 50/50 beamsplitters and neutral density filters (ND filters kill photons: how dumb!), did have supplemental figure on 'asymmetric' beamsplitters of 90%/10%, so PMT1 = 90%, PMT2 = 10%, PMT3 = 1% (ignoring tiny losses on each optical surface). One unknown to me is how damaging to PMT1 is zapping it with a lot of light in bright area ... ex: at the limit, entire field of view might be bright, so saturate PMT1 the entire time (our Leica SP8 HyD's have a safety cutoff, so prolonged saturatin would simply cutoff the experiment until the user stops scanning).

Pinkard ... Krummel 2016 PloS One https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150430 

Nice approach ... rank filter 50% = median filter, so rank filter 75% could be used to "pick" second highest value of the FV3000RS's internal PMTs ... I suggest "discard outlier" (usually highest intensity) and ACCUMULATE the other three PMTs values (i.e. 3500, 1000, 1000, 1000 --> discard 3500, accumulate 1000+1000+1000 = 3000, data is 16-bit anyway), would work nicely. If doing averaging, PC electronics now fast enough (and RAM cheap enough) that Olympus could keep each value in RAM, wait until that pixel is done acquiring, then rank filter (Max K) or 'discard outlier(s)' and accumulate. Optionally always rescale as if all values were good, i.e. for four data points, discard one, accumulate, multiply by 4/3 ... for 12 data points, if discard two, accumulate ten, multiply by 12/10. 

Papiez et al 2019 IEEETBE https://www.ncbi.nlm.nih.gov/pubmed/31034401 and https://ieeexplore.ieee.org/document/8701671 

Back story:

Assumes:

  • you have optimally labeled fluorescence specimens, including negative controls (labeling details is outside the scope of this quick tip).
  • You are interested in seeing what you have on your specimen, and have time to explore the resolution limits of the specimen and optics.
  • You have 'good' understanding of your microscope and/or can work with an expert (ex. me), and have the time and interest (and money) in getting your confocal imaging right.
  • Operating at high NA (yes, also assumes you know what NA is!).
  • operation at confocal pinhole 1 A.U. (yes, assumes you know what A.U. is). I mention briefly 0.5 A.U. below -- the penalty of 0.5 A.U. is pinhole is 1/4 area, so ~1/4 the number of photons gets through (most confocals can set pinhole to ~0.31, implying 10% area and number of photons compared to 1.0 A.U.).
  • your primary interest is in a green fluorophore, emission 520 nm, ex. Alexa Fluor 488, EGFP, mNeonGreen, "mXX" (Nathan Shaner's 5/2019 alias for his new 6x brighter than EGFP green fluorescent protein ... also has new bright YFP). I note that shorter wavelength enables proportionally better resolution, ex. BV421 (em center wavelength 430 nm) enables 1.2 fold better resolution than emission 520 nm (520/430 = 1.2, that is, 520 nm is 1.2 fold worse, so 430 nm is 1.2 fold better).
  • Using fluorescence ... I note that reflection from appropriate nanoparticle(s) could have advantages (I would like to see FL-Nanogold tested on our confocals! See www.nanoprobes.com and check for distributors).
  • Access to spatial deconvolution algorithm, with GPU accelaration (Olympus: Cellsens "C.I." decovnvolution; Leica: HyVolution = Leica HyD detectors -> Huygens software). With excellent specimens and optimal acquisition settings, this increases resolution by ~10% (if pixel size AND signal to noise ratio are each optimal) and improves contrast and dynamic range. GPU enables (near) instant gratification.
  • Ignoring for here AiryScan, STED, etc, optical and engineering 'tricks', just exploring standard point scanning confocal microscope.
  • ==>Assumes the microscope is performing well, vibration isolation table is floating (and operating correctly), no air drafts blowing on the specimen or stage.

Theoretical limit of widefield microscope: dxy = 0.61 * Lambda / NA ... dz ~ 3 * dxy. So: dxy = 0.61 * 520 nm / 1.4 NA = 214 nm.

Theoretical limit Confocal 1.0 A.U. microscope: dxy = 0.51 * Lambda / NA ... dz ~ 3 * dxy. So: dxy = 0.51 * 520 nm / 1.4 NA = 189 nm.

Theoretical limit Confocal 0.5 A.U. microscope: dxy = (0.51 * Lambda / NA)/1.2 ... dz ~ 3 * dxy. So: dxy = (0.51 * 520 nm / 1.4 NA)/1.2 = 158 nm.

George's Quick Rules of Confocal Settings:

  • Refractive index match your specimen mounting medium to the microscope objective lens immersion oil
    • Olympuws FV3000RS: 1.405 R.I. silicone oil (see Boothe 2018 ELife for use of OptiPrep if using live cells).
    • Leica SP8: 1.518 R.I. standard immersion oil (we use leica oil).
    • Tip: I encourage all specimens to be imaged in 35 mm (or larger) imaging dishes, non-fluorescent glass ... ex. Mattek.com (~$2/dish), WPI FluoroDishes, CellVis (~$/dish, though the latter may have 'autofluorescent stuff' on it, obliterating the $1/dish cost savings).
  • use 1.4 NA objective lens (or close as possible on the specific microscope ... if you would like to donate a 1.45 NA lens for our confocals, that would improve resolution by 3% (1.45/1.40 = 1.03) -- which may be quantifyable if you perfectly refractive index match. Note: High NA objective lenses are designed for imaging at the coverglass, and have limited working distance (usually less than 100 um). Can extend working distance by ~50 um by using #0 thickness coverglass (nominally ~120 um) instead of standard #1.5 coverglass (nominally 170 um thick), assuming you refractive index match (i.e. 1.518 R.I.) and/or deconvolution software can correct for thin coverglass (don't count on it).
  • George's take on Nyquist Sampling Theorem (which was developed for sine waves, and states 2.2 data points needed for one wave) ... confocal images are acquired with 2D pixels, or 3D voxels, and specimens are not usually simple sine waves oriented along one axis, SO: Pixel size = oversample by 3 fold from the dxy above. Optionally, oversample even more (maybe 3.5 fold, unlikely to benefit from 4.0 fold, but if you find >3 fold useful, please share with me).
    • For 1.0 Airy unit, fluorescence emission 520 nm (and photostable fluorophore, optimally matched mounting medium):
      • for dxy = 189 nm, George recommends   60 nm XY pixel size ... optional: SVI.nl Huygens suggests 43nm (see below).
      • for dz   = 3 * dxy,  use 180 nm Z step size (ok to use 200 nm)... optional: SVI.nl Huygens suggests 131 nm.
      • I note that 60/43 = 1.395 and 1.395^2 = 1.947, so my recommended setting results in ~2-fold more photons and 2-fold less time, tthan SVI's recommendation ... assuming identical pixel dwell time (ex. 2 microsecond per pixel * 3 line averaging on FV3000RS = 6 microseconds 'total pixel dwell time').
      • You are not required by any law to use 1.0 Airy unit: bigger gets you more light (and tradeoff is more depth in Z) or smaller gets you "somewhat better" resolution, as described in "confocal sweetest spot" web page http://confocal.jhu.edu/mctips/confocalsweetestspot 
      • More info below screen shot
      • SVI.nl Huygens - online calculator https://svi.nl/NyquistCalculator:

                                  

  • To get the most benefit from deconvolution -- as with SVI.nl Huygens settigns above, you need:
    • Bright labeling = optimize fluorophore or fluorophores, such as BV421, Alexa Fluor 610-X (Mailard 2020 Chem Sci reported 610-X brightest dye of 42 they tested), optimized mounting medium (refractive index match, potentially replace H2O with D2O per Maillard, for red and NIR fluorophores).
    • Optimize acquisition parameters (pinhole size, detector choice ... for Leica STELLARIS confocal, HyD S SiPM's may be superior over most of spectral range to HyD X and R -- X maybe better in "blue', such as BV421).
    • Shorter wavelength excitation and emission enables better spatial resolution (unless you have STED capability, then optimal STED fluorophore[s] and depletion laser power can do better): Premier fluorophore is BV421 (antibodies available from BD Biosciences, BioLegend; BV421-streptavidin available from BD, BioLegend, Jackson Immunoresearch).
    • If you can do tyramide signal amplification (TSA) you can get ~100 fluorophores into confocal resolution spot vs typically 3 for direct label or ~10 for secodnary antibody labeling (each numgber is "on average", but could be wide distributions). For example, Alexa Fluor 488-tyramide likely 20x brighter than standard primary + secondary antibody labeling. Tyramides also highly amenable to multiplexing (in 2021 pathology labs using IHC autostainers are at 8plex - see Janis Taube, JHU, publications and AstroPath collaboration.
  • x

 

 



https://www.fpbase.org - Fluorescent Proteins Database - including Spectral Viewer and FRET Ro Calculator

Spectral Viewer https://www.fpbase.org/spectra/ 

FRET Calculator https://www.fpbase.org/fret/ 

FRET equation from above:

QY = Quantum Yield, EC = Extinction Coefficient, J(λ) = Overlap Integral, R0 = Förster Radius, �� = refractive index, κ2 = orientation factor
Wu & Brand (1994). Resonance Energy Transfer: Methods and Applications. Analytical Biochem. 218 

 

Note: GM also has a FRET calculator Excel file inside PubSpectra ZIP file.

PubSpectra web page          https://works.bepress.com/gmcnamara/9

PubSpectra download link    https://works.bepress.com/gmcnamara/9/download

20190528U: stay tuned late 2019 for Nathan Shaner's improved GFP and YFP. The GFP is 2x brighter than his mNeonGreen, so 6x brighter than EGFP. Time has come to retire EGFP!!!

ThermoFisher Prolong Glass  (without DAPI)     is now the best choice, if imaging fixed specimens with oil immersion objective lens. 

==> Or- from Marker Gene Technologies (see MGT web site for more options for each product):           * 

           * Opti-Bryt (fixed cells) https://www.markergene.com/opti-bryt-trade-perm-antifade-mount.html

           * Opti-Klear (live cells) https://www.markergene.com/opti-klear-live-cell-imaging-buffer-5x.html

Prolong Glass info states needs to cure for 30+ hours.

My advice:

* grow cells in imaging dishes (mattek or WPI-Inc ... or ibidi imaging quality coverglass chambers)

** at no time should cells be allowed to "air dry" = keep submerged.

* fix (i.e. formaldehyde), permeabilize if needed.

* immunofluorescence (i.e. http://www.nano-tag.com 2ndary nanobodies with each mouse mAb) ... can include DAPI and/or other counterstains here (example: fluorescent phalloidin).

* wash extensively (but quickly).

* "drip on" some Prolong Glass with imaging dish tilted, so that it forces aqueous media away ... pipet out the "run off", drop more (but not too much $) Prolong Glass ... goal is ~100% Prolong Glass, ~0% aqueous.

* allow to "cure" 30+ hours, in the dark, at room temperature, no lid, large volume of air (i.e. not small sealed box) to let volatiles escape.​ ... Probably simplest to go closer to 48 hours (and would be nice to be consistent in experiments).

 

20180803Fri ... connecting to our file server from Windows (win 7).

* ask George for the name of our file server - and please do not give out the name or IP address of our server.

* you are welcome to set up your own 'share drive to transfer your files (and can we please have 42 Terabytes of space on yours?).

Some Windows PC's are able to see our file server. Some are not. Today we -- "we" being 99% Jim Potter and 1% GM -- were able to trouble shoot the network acess issue. 

0. assumptions:

      (i) windows PC (win7 or ideally win10)

      (ii) plugged into the JHU SOM network (Ethernet cable).

      (iii) you have administrator privileges on the PC (does not need to be 'Administrator' login name).

1. Use JHARS to connect to JHU network (if you have not already done so) https://jhars.nts.jhu.edu/

  1a. after setting  up (or confirming) JHARS, probably useful to power off the PC, wait a few seconds, then power up and log in.

2. enable all the items in the Local Area Connection Properties dialog box (it is probably ok to enable more, but at minimum you need IPv6 and IPv4 and probably more).

3. using CMD prompt -> IPconfig / all (2nd screen shot below) ... see that DHCP Server 10p15.76.226

local network

 

Windows Start menu ... cmd ... ipconfig / all

    ==> DHCP Server 10.15.176.226

    ==> Subnet mask 255.255.255.0     (if this is not correct, you may not be able to see JHU network at all!).

ipconfig all

 

 

 

 

January 15, 2019 (20190115U) new book and eBook:

Basic Confocal Microscopy second edition
https://link.springer.com/book/10.1007%2F978-3-319-97454-5
W. Gray (Jay) Jerome, Robert L. Price 2018

Chapter 1 includes:
Our Ten Commandments of confocal imaging are as follows.
1. The Perfect Microscope and the Perfect Microscopist Do Not Exist
2. Confocal Microscopy Is More Than a Confocal Microscope
3. During Specimen Processing the Integrity of the Specimen Must Be Maintained as Much as Possible
4. Photons Are Your Friends and Signal-to-Noise Ratio (SNR) Is King (GM note: and Queen and President and Premier ... and Dean, milliDean, microDean, nanoDean)
5. Quantification of Fluorescence in a Confocal Micrograph Is a Challenge and at Best Is Only Semiquantitative 
6. Scientific Digital Imaging and Normal Digital Imaging (Family Photography) Are Not the Same
7. Your Image Is Your Data: Garbage in Will Result in Garbage Out
8. The Resolution and Bit Depth Present in a Digital Image Are a One-Way Street
9. The JPEG (Joint Photographic Experts Group) Image File Format Is EVIL but Useful
10. Storage Media Is Essentially Free and Infinite
Notes:
* JHU staff can download eBook PDF for 'free' (that is, JHU has a subscription to publisher's content ... which comes out of NIH and other grants indirect overhead).
* Springer ofters MyCopy softcover edition $24.99 see "Buy" button at top right of https://link.springer.com/book/10.1007%2F978-3-319-97454-5

GM comments:

#1. reminds me of my high school's National Honor Society slogan (which I'm paraphrasing here): "Those of you who think you're perfect, amuse those of us who are". (our NHS T-shirt had the original slogan and Mr. Wampole's face ... Mr. Wampolewas the advanced math teacher in addition to neing NHS chapter advisor).

#5. fluorescence intensity is 'at best only semiquantitative' ... this is sad & true

  * (I blame it on 30+ years of researchers not requiring 'good intensity quantitation' of manufacturers AND manufacturers not making quantitation easy and reasonably priced).

  * I suggest ACCM's Leica SP8 confocal microscope HyD detectors in photon counting mode enables users to come close to quantitation. There are still issues of laser performance (most lasers fluctuate in power), Z-drift and XY-drift (not a big deal for sngle focus plane, single field measurements), and specimen refractive index induced issues (if any mismatch in R.I., then Z affects intensity -- see Staudt and Hell "TDE" paper and Olympus silicone oil graph).

  * Fast FLIM and - simpler and less expensive to get going and less data deluge - "fast photon counting" (FPC) can be implemented on any PMT or Hybrid or APD based point scanning confocal microscope. Re: Becker&Hickl fast FLIM or ISS FastFLIM" (and either would be less expensive to add to our FV3000RS confocal microscope than buying a new fully loaded Leica SP8 Falcon Fast FLIM ... bonus: Wolfgang Becker correctly disses Leica's featuring 'fast lifetime contrast' (FALCON) over fast TCSPC data).

 

Grey and Price 2018  Table 1.1:

Reference: 

Pawley J (2000) The 39 steps: A cautionary tale of quantitative 3-D fluorescence microscopy. BioTechniques 28:884–888

https://www.future-science.com/doi/abs/10.2144/00285bt01 

 

Leica Microsystems - THUNDER Imager Tour at JHU SOM 3/3019

https://www.leica-microsystems.com/jhu-tour/ 

Leica - LIGHTNING and THUNDER

Leica THUNDER Tour - See Through the Haze with Computational Clearing
Johns Hopkins University, School of Medicine, Ross Fluorescence Imaging Center
* Summary: alternative to 'optical clearing' (which requires fixation and chemical clearing) for your fluorescent specimens.
* Demonstrations March 12-15, 2019. Two instruments:
   1. "Model organisms": Leica M205, 5x 0.5NA objective lens ... large field of view.
   2. "Live cells": Leica DMi8 inverted microscope, environmental controls (37 C, 5%CO2) available, full set of objective lenses.
* Seminar: THUNDER Imagers – Decode 3D Biology in Real-time.
RSVP please.
Computational Clearing – available exclusively on Leica Microsystems THUNDER Imagers – offers groundbreaking ease of use, throughput, speed and sensitivity for 3D tissue, live cell and model organism imaging. Unparalleled image quality from stereo, upright and inverted live cell microscopes, with no special sample preparation needed.

THUNDER Imager - how it works (pdf) ... two imaging systems platforms.

https://www.leica-microsystems.com/science-lab/thunder-technology-note/ 

LIGHTNING info (web link below - pdf download at bottom of that page) ... 'adaptive deconvolution' (GPU enabled).

https://www.leica-microsystems.com/science-lab/how-to-extract-image-information-by-adaptive-deconvolution

20190712Fri - JHU Data Repository (Repositories)

==> JHU now (2019) uses Microsoft OneDrive for single user backup and sharing, ex. a graduate student or postdoc can share specific OneDrive folders with JHU colleagues and P.I. (not sharable outside JHU) Each JHU employee or student gets 5 Terabytes (5 Tb) OneDrive storage for free. Additional space should be arrangable through I.T. 

***

For published data:

Craedl - Collaborative Research Administration Environment & Data Library 

https://craedl.org/docs/

Craedl is hosted by MARCC Maryland Advanced Research Computing Center https://www.marcc.jhu.edu  

***

See also:

JHU Sheridan Libraries 

https://guides.library.jhu.edu/dataservices/data/analyze

* Data Services   https://archive.data.jhu.edu/

* more info from https://guides.library.jhu.edu/dataservices/publish-and-share/how-to 

 

Find a Repository

Data Archiving

Research data can be archived into the JHU Data Archive. Research data is made discoverable and publicly-accessible, assigned a unique, persistent identifier (such as DOI) for accurate citation by others, and managed to ensure that they are usable in the future. JHU Data Archive is available to all JHU researchers, including graduate students. Archiving services for projects under 1 TB are FREE. For archiving larger datasets, please contact us to discuss fees that may apply. More information about data sharing and archiving is available on our Archiving page.

University Archives and Records Management

The Ferdinand Hamburger University Archives preserves the history of the Johns Hopkins University in print and digital form, including the privately-held records of JHU faculty.

Selecting a Repository for Data Deposit

Tips and set of questions researchers can use in determining whether a particular research data repository will work for their circumstances.

DSpace Repository

DSpace preserves digital materials generated related to Johns Hopkins research. It is also a place for Electronic Theses and Dissertations (ETD) for Johns Hopkins students (If you need help with the thesis/dissertation submission, you can find the instruction here, including an 1-hour recorded ETD workshop video).

Inter-university Consortium for Political and Social Research (ICPSR)

One of the largest archives of datasets in the world, the Inter-university Consortium for Political and Social Research, (ICPSR) has a vast collection of social, political, and behavioral science datasets. These datasets can be used in a multitude of fields such as sociology, political science, history, business, public health, economics, and education. You might consider archiving with ICPSR if your work falls into one of these categories or other related subjects and/or if you are looking for an archive that will support sensitive, restricted use data.

Registry of Research Data Repositories (re3data.org)

A global registry of data repositories that covers research data repositories from different academic disciplines. Use re3data.org to search for data repositories in a specified discipline and download and/or deposit data there.

 

 

20210510M Excitation power at the specimen for widefield (Lumencor SPECTRA III) for Olympus IX83 and confocal (Olympus FV3000RS)

Note: numbers are approximate to simplify the math. Also going to ignore usual S.I. units (Joules, etc)

Widefield:  http://confocal.jhu.edu/current-equipment/fishscope

confocal:   http://confocal.jhu.edu/current-equipment/fv3000 

Our Lumencor lamp has ~500 mW per channel (8 channels, 368-747nm). Entire eyepiece field of view (and objective lens field of view) is illuminated usually [a square aperture COULD be used to block the area outside the camera field, is not), imaging area is ~50%, so "power to the camera field of view is ~240 mW per channel for field of view for 1 second (and normally only one channel on, since monochrome camera ... Olympus cellSens "Process Manager" lists up to 20 acquisition settings, lamp channel --> filter cube --> Sutter 10 position filter wheel). .Maximum exposure time on ORCA FLASH4.0LT is 10 seconds. Typically acquire between 100 ms and 1000 ms (1 sec). Will use 1 second here. Pixel size for the 60x/1.4NA objective lens --> camera is 108nm, round to 100nm.

Our Olympus confocal has ~20 mW per laser lines (7 laser lines, 405-730nm). Fastest pixel dwell time: 2 microseconds, if 5 line average, then 10 microsecond "total pixel dwell time". For ~4 million pixels (2048x2048), laser(s) on for 40 seconds (actual scan time approximately double sincewe usually use uni-directional scanning). While I typically acquire at 50nm pixel size for 60x/1.4NA (with GPU deconvolution), I use 100 nm here to match the camera rounded pixel size above. The confocal spot size is NA and wavelength dependent (and is "Airy" or roughly Gaussian), will simplify to in focus pixel is getting ~50% of the excitation photons, so 10 mW per channel per pixel..

 

100 nm pixel size * 2000x2000 pixels = 200x200 um image size

Widefield: every pixel 1 second exposure (simultaneously). 240 mW / 4,000,000 pixels = 0.00006 mW = 0.06 uW = 60 nW (1 second). 

Confocal: 10 mW for 10 usec (focused spot, 'typical' confocal pixel dwell time). In practice, probably <=50% of the power reaches the specimen (light loss in coupling to the scanhead, in the scanhead, through objective lens back aperture). so would be ~5 mW for 10 usec ... but typically use 0.1% laser power so ~5 uW for 10 usec.

Real-life excitation power is much lower than the "max power" above.

Lumencor on FISHscope: we typically use 10% of maximum, so 500 mW --> 50 mW, etc. ... so with 10% power, 6 nW / pixel for 1 second.

Olympus FV3000RS confocal: we typically operate the confocal with the "10% Neutral Density" option selected, limiting power control range to 0.01 - 10% power (zero is also available) AND "typical" power setting range is 0.02% to 1.00%, and 0.10% is common, hence the ~5 uW for 10 usec.

Less than maximum power: We do this to minimize photobleaching (users can contribute to this by using optimal fluorophores and op[timcal mounting medium).

***

background info on fluorescence imaging:

Widefield camera: ~80% peak quantum efficiency.

Olympus confocal GaAsP detectors: ~40% peak quantum efficiency (of face plate, and will ignore a lot of details that may lower the effective QE, since the poibnt of a PMT is to multiply the photon signal, and at typical HV "gain", one photon --> 100,000 electrons, Analog-to-Digital Converter produces 'nice' signal to the software).

Widefield: in plus ALL the out of focus light ... Z-series followed by GPU deconvolution can produce "confocal like" results.

  Widefield XY resolution: dxy = 0.61 * Lambda / NA = 0.61 * 500nm / 1.4 = 214 nm

   Widelield plus deconvoluton: ~10% improvement, so 214 - 21 = 193 nm.

   GM interprets Nyquist sampling theorem for 2D as needing ~3 to ~3.5 pixels across resolution, so 214 / ~3 is ~70nm pixels. On FISHscope, we deliberately chose to purchase a 60x/1.4NA objective lens so more light would reach a pixel (108x108nm of specimen = ~10,000 nm^2) than if we purchased a 100x/1.4NA lens (~65x65nm, 4,225nm^2, which is 42% as many photons for same exposure time, implying need ~2.5x longer exposure time ... and assuming all the excitation power of the lamp reaches the specimen).

Confocal, at standard of care 1.0 Airy unit pinhole: optical section, mostly blocking the out of focus light, so only "in focus plus lamost in focus" light  detected. For same field of view as our FLASH4.0LT, acquisition time takes a lot longer (nominally 40 seconds, see above). Each focus plane is "clean" (in and nearly in focus light, no out of focus light), that is, every photon is a "good" photon. 

  Confocal XY resolution (1.0 Airy unit): dxy = 0.51 * Lambda / NA = 0.51 * 500 / 1.4 = 182 nm.. (if pixel size is ~50nm XY).

    Confocal XY resolution (1.0 Airy unit) plus deconvolution: ~10% improvement, so 182 - 18 = 164nm. (if pixel size is ~50nm XY).

Confocal can be used at SMALLER pinhole size (Zeiss has a nice appnote with the details), one example here: 0.5 airy unit

  Confocal XY resolution (0.5 Airy unit): dxy = (0.51 * Lambda / NA) / 1.11 = 0(.51 * 500 / 1.4 ) / 1.1 = 165 nm.. (if pixel size is ~50nm XY, maybe useful to use 40nm).

  Confocal XY resolution (0.5 Airy unit) plus deconvolution): 165 - 16 = 149nm (if appropriate pixel size). 

Leica appnote claims "not muich loss of fluorescence emission" at oderately small pinhole size (vs 1.0 airy unit). Best to do the test yourself, using out Leica confocal and HyD detector(s) in photon counting mode (converting PMT --> ADC values to photon counts is not trivial, and most publications that report confocal PMT data do so in "Arbitrary Units" -- quite literal, since no one "proves" that 1000 digitiizer counts is double that of 500 ... on Leicawith HyD in photon counting mode, the counts are the photon counts)..

Nyquist sampling microscopy data - good calculator at https://svi.nl/NyquistCalculator

Better resolution: use shorter wavelength fluorophores, such as BUV395 or BV421 or SB436 or SuperNova v428 (assuming microscope has proper excitation wavelength, filters, good detector QE in blue, good transmission through objective lens), and no background fluorescence (from specimen or optics). 

Conclusion: both widefield and confocal are useful. I strongly encourage decovnolving each.Good to great data if you use each correctly. 

*   Widefield: usually faster acquisition, limited flexibility in acquisition field of view (wrt illumnination field). 

*   Confocal: massively flexible in acquisition settings (more on Leica SP8 than FV3000RS).

There is a saying in the restaurant business (and most other retail businesses) 3 keys to success: "Location, location, location". 

Fluorescence microscopy success, assuming good widefield and/or confocal microscope available (and the user knows what they are doing, or gets help from the image core manager); has 3 keys to success: "sample preparation, sample preparation, sample preparation".

Last thoughts:

* Bring a nicely labeled specimen (i.e. 4 color fluorescence microscope slide-sample-coverglass) with you and image at the START of every session (can be single in focus plane, modest field of view if confocal, so not take long). Helps make sure microscope (and user) are operatign correctly. Sometimes a lase may be "dim", or oil on the 20x dry lens, or 'just some weird shit' (though more likely previous user did something wrong).

* Every experiment (every replicate) needs an appropriate negative control or controls. If using "old school" primary + secondary antibodies, usual key cotrol is "no primary, ALL secodnary" antibodies. Can be "quick look by eye, then small field of view acquisition" (of the brightest relevant area you see by eye, and focus with the instrument). The hope is to document that the negative control worked -- but if the cotnrol is 'surprisingly bright' better to find out at the start of the session, and troubleshoot (and think!), than to assume life is good. I note that users of labeled primary antibodies (ex: Brilliant Violet 421 anti-CD8 monoclonal antibody from the flow cytometry world) need to think through their cotnrols. Ideally: cell linke(s) with the gene of interested transfected/transduced in or knocked out. For many cells types and tissues, controls can be other cells, such as for T-cells, most blood, lympgh node, or spllen T-cells are CD3+ and either CD4+ or CD8+ (assduming you block FcReceptors and are aware that a few t-cells are CD4+ CD8+, some are CD4- CD8-, some macrophages are CD4+, some dendritic cells are CD8alpha positive, and some cells trogocytose (eat and/or nibble on) other cells; also G.I. (gut) T-cells are ~50% alpha-beta TCR (either CD4+ or CD8+) and the other half are gamma-delta TCR (may be CD4- CD8-, but could be positive ... "cells do not read Cell, Immunity, Nature, Science or ELife").

* "Old antibodies die, please through them away". I now offer the chicken egg rule: if youi put a (raw) chicken egg into a refrigerator the same time you put a new antibody in a lab refrigerator, and left each for 1 year (ex. March 1, 2020, before covid-19 pandemic labs shutdown to now, May 2021), would you eat the egg? 

 

20220422Fri - some of the software I find useful (copy and paste any URL's that I did not make hyperlinks or search company/product names)

Software Tools

IrfanView ... shareware image viewer.

Hamrick VueScan Pro (www.hamrick.com) – universal scanner software, far better than the junky software Epson etc bundle. Pro version has lifetime updates (3 PC licenses per purchase).

Belarc Advisor ... scans all hardware on a PC (also software and Windows info) and generates a report. Sponsored by a RAM manufacturer so one feature is to show RAM slots and RAMs on board, and easy to then buy more.

crucial.com ... ram company that makes it easy to find RAM to upgrade PC.

crystaldiskmark (64-bit) and crystaldiskinfo (64-bit drive info such as temperature) ... -  drive performance - typical: HDD 100 MB/sec, SATA-6 SSD 600 MB/sec, NVMe M.2 SSD 3,000 MB/sec (PCIe3 x4), NVMe_PCIe4 6,000 MB/sec (some drives in right PC may reach 7,000 MB/sec)(potentially faster than DDR2 etc RAM in older PCs') ... late 2022 NVMe PCIe5 should reach 15,000 MB/sec. 

(64-bit standard edition) https://crystalmark.info/en/download/#CrystalDiskMark 
                or direct download link   https://osdn.net/projects/crystaldiskmark/downloads/75540/CrystalDiskMark8_0_4a.exe

CrystalDiskInfo useful to check on drives (HDD, SATA SSD, NVMe SSDs) with respect to error messages, warnings, temperature. If you see warnings, strongly consider buying a new drive and cloning your current drive (ex. could use MiniTool Partition Wizard -- I like Sabrent NVMe drives because includes Acronis TueImage for Sabrent cloning software).  

more NVMe tips

20220606M - More NVMe tips:

* always use a cooler, such as a Copper heat sink,

https://www.amazon.com/dp/B08MV44567

Awxlumv M.2 Heatsink Pure Copper NVMe M2 2280 SSD DIY 7 Fins Cooler with Thermal Pad for Desk Computer(2 Pcs)

... $12.99 for pair or $7.99 for single (prices 20220605S)  -- Copper supposed to be better than the slightly less expensive Aluminum.

* am going to start checking EVERY NVMe drive "monthly" for its health and temperature according to crystaldiskinfo - also informs on SATA-6 SSDs, HDDs. Examples:

Good NVMe drive 1TB (SPCC = Silicon Power - I now 6/2022 mostly use Sabrent NVMe drives)

crystaldiskinfo good NVMe

not good: "caution" on an HDD

crystaldiskinfo caution 3TB HDD

One of my Samsung NVMe drives at home read "Health Status 0%" - this was bad, and was overheating. I switched to a better heat sink, purchased a new Sabrent PCIe4 2TB NVMe drive, cloned it using an Orico NVMe drive duplicator (amazon.com $150 - see below), put on an even better heat sink - Copper heat sink near top of this box -- and now have 100% healthy, relatively cool drive. Old Samsung was PCIe3 ~3000 MB/sec read/write, new is PCIe4, 6190 MB/sec   write 6773 MB/sec new Sabrent Rocket 4.0 Plus 2TB (on MicroCenter PowerSpec G509 PC with PCIe4 NVMe slots on motherboard). 

**

Orico NVMe duplicaor info

https://www.amazon.com/Dual-Bay-ORICO-Tool-Free-Enclosure-Duplicator/dp/B08CTZPLH4

Dual-Bay NVME Docking Station, ORICO Tool-Free USB C to NVME SSD Enclosure for M Key PCIe 2242 2260 2280 22110 M.2 SSDs, Support Offline Clone Duplicator Function Up to 10Gbps(SSD NOT Include)-M2P2

$149 amazon price (20220605S).

Can also act as NVMe --> USB-C external drive. Duplication does not require a PC (and is simpler). $150 is not cheap, but I've had to clone (often also upsize) NVMe drives to pay for themselves (in my opionion - have one at home, one at office) ... For comparison, simple enclosure with fan $37, https://www.amazon.com/gp/product/B0827NHNNY or simple aluminum USB enclosure $19 https://www.amazon.com/gp/product/B07MNFH1PX [benefits from Copper or aluminum heat sinks on both faces.

MetaMorph ... much more capable for many image analysis needs than the "free, you get what you pay for" Fiji ImageJ.

MetaFluor ... high speed ratiometric image acquisition and analysis software (stripped down for speed and task compared to MetaMorph - some customers buy combined MM/MF). Classically used for Fura-2 ratio imaging of Ca++ ion concentration (dynamic range ~20x with narrow excitation 340 and 380 nm excitation ratioing -- much better than 2022 standard of care intensity fluorescent proteins in GCaMP# series or Fluor-3 or Fluo-4 dyes). Capable of Fura-2 & BECF dual ratio imaging - BCECF calssic ratiometric cytoplasmic (cytosolic) pH indicator. 

Fiji ImageJ ... image opening/saving (including movies to/from TIFF etc), image processing, image analysis ...  "free, you get what you pay for". 

Tomshardware.com ... web site info on new PC hardware

MiniTool Partition Wizard ... modest price for Pro version, only need to resuce one old HDD etc to more than pay for it. Example, on 20220601W I put an HDD into a Sabrent drive duplicator (USB3). The drive had an "invalid ..." error. Following instructions at https://www.partitionwizard.com/partitionmagic/dynamic-disk-invalid.html "MTPW" was able to make the contents visible and enable me to save to another drive (I used a new WD Elements 18TB drive as destination -- always good to have backup space available). 

Glary Disk Cleaner ... frees up space (often Gigabyte per week with Windows 10 and Google Chrome junk). Free (shareware) - avoid installing their games and other tools. ... old versions work fine and are less likely to sneak their free-you-getwhat-you-did-not-pay-for junky other programs.

Glary Undelete ... need to install on PC before you have need to undelete files. Free (shareware) - avoid installing their games and other tools. 

7zip ... utility for unzipping ZIP giles ... also works on "gz" and "tar" archive files (whah are beloved by Linux operating system).

Spatial deconvolution -- All three support GPU acceleration, usually NVidia GPU (tip: more cores, more ram, faster PCIe interface better):

  • svi.nl Huygens (on Leica SP8 HyVolution2)
  • Microvolution (check out my image in their image gallery)
  • Media Cybernetics - AutoQuant (first company with "blind" deconvolution)
  • Andor/Imaris deconvolution (both companies part of Oxford Instruments)

Microscope companies often now offer Spatial Deconvolution, as optional extra cost modules, examples: