Leica SP8 (see iLab)

Leica SP8 Confocal Microscope
Location: Ross 9th Floor

The Leica SP8 confocal microscope is on a Leica DMi8 inverted microscope stand (DMi8CEL). 

*** ACCM Confocal Microscope will be #1 at JHU SOM Core Showcase, October 11, 2018, http://grcf.jhmi.edu/wp-content/uploads/2018/09/Program_CoreShowcase_2018_FINAL.pdf

June 1, 2018 was the start of the iLabs Organizer reservations for our Leica SP8 confocal microscope:

ACCM Leica SP8 Confocal Microscope (ACCM Confocal Microscope)



This microscope is owned by the Division of Anesthesiology & Critical Care Medicine (ACCM) and managed by Ross Fluorescence Imaging Center, George McNamara, PhD, 305-764-2081 (cell), gmcnama2@jhmi.edu.

Our Lecia SP8 confocal microscope was purchased on a budget, so between the price and capabilities of a 'baby confocal' like Leica SPE, and a 'fully equipped' confocal microscope (example: our SP8 has only 3 detectors, no incubator ... with 3 detectors, can acquire many channels by sequential scanning multiple tracks). The two Leica HyD detectors are best operated in "counting mode" = photon counting. This enables much simpler quantitation of fluorescence signals than conventional PMTs whose digitizers output values that depend on both the High Voltage ("HV") gain and the "offset". 

The Division of Anesthesiology & Critical Care Medicine (ACCM) users are aiming to use 20 hours per week, that is, 50% of JHU business hours access (it's ok if ACCM uses more). It is a 'win' for everyone if we can maximize efficient use of the SP8 since more revenue helps pay for the instrument and service contract. For now, we will try to manage this ACCM 'preferred access' informally. (i) If ACCM users get above 20 hours per work week: great. (ii) if non-ACCM users add up to more than 20 hours in any work week and ACCM users did not need 20 hours: great for everyone. (iii) We encourage ACCM users to reserve well in advance (i.e.1+ week). 

   This is a state of the science/engineering confocal microscope - we want everyone who can benefit from the LeicaSP8 confocal microscope to be able to get excellent data with it.

Rates all users are:

    * $27/hour for fully trained expert users, during JHMI business hours (Mon-Fri 9am-5pm).

.    * $20/hour for fully trained expert users, outside JHMI business hours (outside Mon-Fri 9am-5pm).

    * $70/hr for users, with Dr. McNamara's oversight.

    * $120/hr for Dr. McNamara to provide training and/or to operate the confocal microscope for users who need an operator.

    ==> Dr. McNamara reviews sessions on the sign-in sheet when doing the 'confirm' step in iLab Organizer.

Common 2*2 immunofluorescence experiment:

     scan track 1: 488 nm and 638 nm lasers, AF488 on HyD1, AF647 on HyD3.

     scan track 2: 405 nm and 552 nm lasers, DAPI   on HyD1, AF568 on HyD3.

     HyD's in photon Counting mode, usually 10 line accumulation, no frame accumulation. If dim signal: 16 line accumulation and 'whatever needed' frame accumulation you need to get good data (overnight or over weekend if necessary ... though you should think about better fluorophores, more laser power, tyramide signal amplification, etc, to get better signal).

     For 20x/0.75NA objective lens: 1 Airy unit pinhole size, 120 nm XY pixel size, 360 nm Z-step size (GM's simple XY has three times better spatial resolution than Z).

     For 63x/1.4NA objective lens -- with HyVolution2 set to 'aggressive', improvement is for each axis, so 1.2^3 = 1.728 or 1.414^ = 2.82, for volume): 

          "standard confocal" resolution: 1.0 Airy unit pinhole size, 50 nm XY pixel size, 150 nm Z-step size.

          "super-resolution" resolution (~1.2x improvement):    0.5 Airy unit pinhole size, 40 nm XY pixel size, 120 nm Z-step size. Note: 0.5 AU and "Area = pi r^2"  implies 25% intensity.

          "super-duper resolution" resolution (1.414x improvement):    0.3 Airy unit pinhole size, 40 nm XY pixel size, 120 nm Z-step size. Note: 0.3 AU and "Area = pi r^2"  implies 9% intensity.

             Reminder: the HyD detectors photon counting mode greatly facilitates quantitation of these settings. You can evaluate each by enabling sequential scan tracks with each pinhole setting.

Equipment Summary:

Scanner speed 1 to 1800 Hz, default 400 Hz, typically used at 600 Hz to enable full field imaging (zoom 0.75x). Hz = scan lines per second.

    Faster speeds require zooming. For example, 1800 Hz requires zoom 7.5x or more (you control number of pixels). We encourage evaluating using "max speed" 1800 Hz with summing the HyD photon counts (line accumulation = 10, for example). Max speed decreases photobleaching (speed and scanning area interact).

     Hardware zoom 0.75x - 48x (by changing confocal scanning area with the XY scanning mirrors).

     Max image resolution 8192 x 8192 pixels (64 megapixels). Max number of scan tracks is ~40 in LAS X (9/2018 version). Since we have four laser lines, generally two, three or four scan tracks is sufficient. There is an channel unmixing "matrix" to let you unmix 'spectral overlaps'.

4 lasers:

   405 nm (50 mW)

   488 nm (20 mW)

   552 nm (20 mW)

   638 nm (30 mW)

   Note: HyD's detection must be at least 5 nm from laser lines, for simplicity, please restrict your HyD emission bands to 10 nm from any laser line. Example, 500-540 nm is away from 488 and 552 nm laser lines.

TCS LIAchroic beamsplitters

3 epi-illumination detectors: 2 HyD, one conventional PMT ... used for fluorescence. Tghe PMT can be used for reflected light (the HyD's need to be protected from reflected light).

  Two HyD's = hybrid GaAsP faceplate PMT front end, Aavalanche photodiode (APD) back end), 2x quantum efficiency compared to standard PMTs.

  • ==> The HyD's are photon counting devices. This provides a straightforward path for intensity quantitation compared to conventional PMTs.
  • We typically recommend 10 line acumulation, resulting in 'typical' pixel (voxel) counts of 5 to 100 vs "empty" pixel dounts of zero or one or two. A photon count of 10 looks small, but compared to 'background' of zero is quite high. With a standard PMT, it is very difficult to 'translate' the intensity value -- the value depends on the detector gain and offset values (each can be chosen poorly, re https://www.youtube.com/watch?v=VA7J0KkanzM ) -- obtained to either (i) photon counts, (ii) number of fluorophores, or (iii) number of antibody molecules (immunofluorescence) or oligo probes (single molecule RNA FISH. 
  • On our SP8, you can sit with Dr. McNamara (and/or Leica representatives) and evaluate 'on image core time' the HyD's against each other (HyD1 vs HyD3) and our standard PMT ("PMT2") with different settings (sequential track mode makes this easy). 

  One PMT (Hamamatsu R9624) with low dark current, 40 MHz sampling of ADC digitizer.

1 transmitted light detector (brightfield, DIC).

Motorized XY scanning stage (control by remotes or software, do not touch the yellow motors).


HyVolution2 quantitative deconvolution software using NVidia M6000 GPU card ("instant gratification"). "Super-resolution" possible when using the combination of:

    small pinhole (<1.0 Airy Unit ... usually 0.5 Airy unit, can 'go down to' 0.28 Airy unit, signal is proportional to area, so 0.5 Airy unit implies 0.25 photons detected per scan, and 0.3 A.U. is 0.09 photons detected, if other parameters constant)

    HyD detector(s): photon counting mode please!

    Huygens Essential deconvolution (SVI Hyugens Essential intregrated into Leica LAS X software).

    Combination is rated for 140 nm XY resolution, compared to "conventional" confocal ~210 nm XY resolution (at 500 nm emission wavelength ... 405 nm excitation of any of BUV395, BV421, SB436 may enable better resolution). 

Confocal quality objectives: HC PL APO 20x/0.75 CS2 and HC PL APO 63x/1.40 Oil CS2  We also moved a pair of Leica 'non-confocal' objective lenses onto the microscope to facilitate 'by eye' searching. 

LAS X 3D visualization.

Please note:

* the Leica SP8 confocal microscope does not currently have an environmental control incubator system. This simplfies specimen access, whle implying most users will work with fixed specimens on microscope slides or 35 mm imaging dishes. GM recommends for growing cells in Mattek or similar 35 mm imaging dishes (#1.5 or precision 170 um coverglass bottom), using minimal amounts of expensive reagents for labeling (antibodies, single molecule RNA FISH), large volume (2 mL) for inexpensive reagent wash steps.

* Having 3 epi-illumination detectors (2 HyD, one conventional) means maximum 3 fluorescence channels per excitation "scan track". The SP8 does enable sequential scan tracks, so you can image more than "3plex". 

* We are very open to labs purchasing -- in exchange for time credit -- additional components for the Leica SP8 microscope. For example, 37 C environmental unit (that works on SP8 / DMi8) and two more PMT detectors to "max out" the SP8 scan head. the Leica "laser box" can accept one more laser line (695 nm, possibly ~350 nm). Long term, the SP8 scanhead does not have an X1 port -- this is upgradable in the field. We would love to have 2, 4 or 8 APDs on the X1 port, to enable simultaneous 13plex (5 internal, 8 on X1) acquisition per laser line. APDs are 70% quantum efficiency in the red and near infrared (~600 - 900 nm emission), which would enable further multiplexing, discussed at:



   We would especially like to see 13plex acquisition per laser line + multiple laser lines + "joint" spatial deconvolution and spectral unmixing, optioanally spectral phasors, per: Hoppe (2008 and 2016), Valm&Borisy (120plex 2016), Fraser ('spectral phasors' 2017) groups publications on parts of these. 

  Please note that Z-step size is the physical amount the Z-motor moves the objective lens. That is, if imaging air (R.I. 1.0), physical step and "optical step" in the specimen are the same. If you image through a 400 um thick object object that is R.I. 1.600, the 'apparent Z' is 250 um ... that is, the Z-motor will travel 250 um. Example: hemacytometer coverglass is 400 um thick (we measured the thickness 'edge on' by standing the coverglass on edge ... 'mind the gap' ... the hemacytometer counting gap height is 100 um).


Leica SP8 scanhead schematic - We have LIAchroic (not AOBS) and two 2nd generation HyD detectors (we have rotation, we do not currently have X1 port). We would love to have a full set four internal SMD HyD detectors (each 2x faster than our 2nd gen, and lower noise because of chiller) and FALCON software upgrade to be able to add multiple detector outputs ... could enable 8x faster imaging ("fast photon counting"), and in future could add AOBS and pulsed laser (ex. Leica "WLL" white light laser [super-continuum laser, 80 MHZ = 12.5 ns interval], to enable precised excitation wavelengths (ex. Nathan Shaner's 6/2019 bioRxiv preprint on AausFP1 bright new GFP could be excited precisely at excitation maximum, AOBS emission side precisely cover emission peak ... and same for the YFP varient).

from https://downloads.leica-microsystems.com/Leica%20TCS%20SP8/Brochures/Leica%20TCS%20SP8%20Scan%20Head-Flyer_EN.pdf 

Leica SP8 scanhead schematic



January 31, 2019: Ross S910 room hosting the ACCM Leica SP8 confocal microscope has been renovated with (i) blackout curtains for each microscope station, (ii) three pairs of track lights for each station, railings for the air ducts for HEPA filters.

Summer 2018

Leica Workshops hosted by Ross Fluorescence Image Core and ACCM Confocal Microscope Core

* FALCON = FAst Lifetime CONtrast (Fluorescence Lifetime on HyD photon counting detectors) ==> potential future upgrade of SP8).

Leica LAS X Navigator and TCS SP8 FALCON
GPU-based navigation, FLIM and Super-resolution

APPLICATION TALKS - June 19, 2018, 12:00-1:15 PM
Turner Concourse G-007, Darner Room

For more information or to reserve a demonstration time,
visit http://www2.leica-microsystems.com/e/149501/jhu-lasx-/3tsp5w/258951420

FALCON - Seminar, Geoff Daniels, Monday August 13.

For more information, visit http://www2.leica-microsystems.com/e/149501/jhu-lasx-/3tsp5w/258951420


For Confocal applications, call or email Geoff Daniels (senior sales):

Dvir Blivis (new regional confocal salesperson).


For Widefield applications, call or email Paula Cranfill:

Regional leica team members include:

Sarah Crowe - confocal applications

Jessica Calafati - regional manager.

Several field service engineers.


George's Conclusions:

* The new 3rd generation HyD detectors count faster than our SP8's "2nd gen" HyD's ... we would love if someone (SOM, DoM, philanthropy, anyone is welcome ... I note we would need a lot of user hours to pay for both annual service contract and buy 3rd gen HyDs from the daytime hourly rate of $27/hr) could provide money to (i) buy one or more of the 3rd gen HyDs. the Leica SP8 scanhead can use four HyDs,so ideally buy FOUR new 3rd gen HyDs and hopefully get Leica to accept the two current 2nd gen in trade-in).

* FALCON concept is interesting ... Fast FLIM data implies -- to me -- a big "data deluge" AND requires at least one pulsed laser (yes, we would love for someone to donate money for one or more pulsed lasers, also new, more powerful computer to deal with the data).

* I propose "Fast Photon Counting" (FPC) for now: 3rd gen HyDs, used with our current lasers, get data acquired a lot more quickly. The "photon counting speed advantage" of 3rd gen HyD's vs current 2nd gen is non-trivial to figure out under real use conditions. I estimate 10x advantage for each 3rd gen HyD at less than 2x the cost of each 2nd gen HyD. THis should not be seen as 10x / 2x = 5x advantage, but rather: one time investment ---> 7+ years of user's benefiting from 10x greater throughput (I estimate lifetime of SP8 is 8+ years,could be even longer).


June 13, 2018: Leica SP8 is now in Ross S910A (S = Service corridor). Access is by scan card reader and SP8 is for fully tained users or users getting training from Dr. McNamara (or fee for service to have Dr. McNamara 'do' the imaging).

June 1, 2018: (see also above) Leica SP8 calendar/scheduler moved from Ross FIC (this site) to iLab Organizer as of June 1, 2018 (this scheduler booked all the time or calendar removed).***

  We are also adding billing data per month per lab starting Feb 1, 2018 - May 31, 2018. Each P.I. will be billed a block of time each month (ex. Prof. Cyrus David Mintz, March 2018, 51 hours, in one time block).

January 5, 2018 news: ACCM's image core will be activated on iLab Organizer. Congratulations! We will continue to manage and host the microscope. The scheduling will move to iLab when the ACCM's iLab page is activated.

Reserve Equipment