Welcome!
1. HPS Core - described below and at https://johnshopkins.ilab.agilent.com/service_center/5771/?tab=equipment - can use View Schedule at right to see availability - likely need to contact Geirge McNamara, PhD, HPS core manager, to do things (305-764-2081 cell).
2. Tecan M1000 plate reader, https://johnshopkins.ilab.agilent.com/schedules/546710#/schedule
High-Throughput Phenotypic Screening (HPS) is a specialized type of high throughput screening (HTS) and high content screening / imaging (HCS, HCI). All are typically chemical or genomic (i.e. CRISPR/Cas, RNAi) screens to identify molecules (drugs, genes) to kill cancer cells and/or make healthy cells healthier. Screens are often conducted in SBS (Society for Biomeolecular Screening) plates, i.e. 96-well, 384,well, 1536-well plates and standardized lab automation components that all work together. Major society is SLAS (wikipedia page). Recommended reading (or at least browing the table of contents): NIH NCATS Assay Guidance Manual online (open access).
HTS: typically use purified enzyme or live cells (tissue culture size, ~10 um length) to do drug screens ona multi-mode plate reader. Often many millions of compounds screened per campaign.
HCS: typically use live or fixed cells (tissue culture cells or small <500um diameter organoids) to do drug screens on a specialized microscope able to quickly read one or more SBS plates. Many thousands of compunds per campaign (say several libraries adding to 100,000 compounds).
HPS: typically use live organism -- ex. zebrafish larvae, Drosophila larvae (fruite fly), C.elegans larvae (roundworm), organoids (brain, retina, etc), cardiomyocytes (~125 um length), other large cells -- drug screens using multi-mode plate reader for high throughput, optionally other components -- we have a light sheet fluorescence microscope -- for follow-up examination. A simple "first gate" is if a chemical kills the organism, it is probably going to kill people, so not a drug candidate. Often the organism is genetically modified, such as Thierer et al 2019 (https://pubmed.ncbi.nlm.nih.gov/31366908/) -- see bottom of this page for scale of the screen (~300,000 larvae in ~14 work days) on our older equipment.
Example screen (added 20250209Sun) - 5 day post fertilization larvae on Mumm lab earlier verion of the platform - DJ Kelpsch, Liyun Zhang, James H Thierer, ... Jeff S Mumm, Steven A Farber 2025 (Jan 16, 2025) A whole-animal phenotypic drug screen identifies suppressors of atherogenic lipoproteins. bioRxiv https://www.biorxiv.org/content/10.1101/2024.11.14.623618v3 , PubMed PMID: 39605440 DOI: 10.1101/2024.11.14.623618 - used paraformaldehyde fixed larvae, Tecan M1000 plate reader (GM hypothesies and wants to test whether our HPS platform Union Biometrica Biosorter "large particle flow sorter" can by used to quantify NanoLuc intensity per larva (furimazine or cephalofurimazine substrate, ideally live larvae, fixed if necessary to permeabilize; probably 640nm laser for Extinction optical density profiles and triggering).
The High-Throughput Phenotypic Screening (HPS) Core seeks to support research teams throughout the Hopkins Community by providing access to a state-of-the-art phenotypic screening platform. The unified workstation includes components for sorting, plating, manipulating, imaging, and phenotyping individual cells, cultured organoids, or small animal models. We provide collaborative support for project/assay development, and fee-for-service charges based on robotics usage. We also facilitate access to statistical support from the Wilmer Biostatistics Core (WBC) as needed.
NIH HEI S10 grant abstract is at https://reporter.nih.gov/search/vZLinjtEzky4O76P0E_PkQ/project-details/9939116
Please cite the grant number in your manuscripts and grant proposals, 1S10OD026909-01A1
HPS is also known as ARQiVAST = ARQiv Automated Reporter Quantification in vivo, a plate reader-based approach enabling true HTS rates in vivo, and VAST Vertebrate Automated Screening Technology.
Rates are for JHU FY25 (July 1, 2024 - June 30, 2025). Note: the rate applies to both the entire HPS Platform (screens) and to single instrument(s), i.e. Tecan Spark multi-mode plate reader. Generally the rate is 1 hour intervals, with Dr. McNamara's assistance (Dr. McNamara's time is built in to the business hour rate). Every session will need an iLab reservation (George will usually make) with an Cost Object number (George will also reserve self Outlook calendar). Users are not to operate the HPS platform equipment on their own. Please beaware George sometimes takes vacation days (or out for illness, re covid-19 pandemic, flu etc) so please plan ahead and be aware postponement could sometimes be necessary.
Rates are subject to change(s)
Internal
$ 75/hr HPS assay development
$ 75/hr HPS assay optimization.
$150/hr HPS screening - business hours.
$ 75/hr Robotics usage - off hours. Example: automated unattended diluting drugs into plates overnight during non-supervised hours.
$ 75/hr Image analysis - Zeiss Arivis 4DVision software training and use.
$ 75/hr Data analysis.
JH Partners (FastFarward)
$ 90/hr HPS assay development
$ 90/hr HPS assay optimization.
$180/hr HPS screening - business hours.
$ 90/hr Robotics usage - off hours. Example: automated unattended diluting drugs into plates overnight during non-supervised hours.
$ 79/hr Image analysis - Zeiss Arivis 4DVision software training and use.
$ 79/hr Data analysis.
External - Non-Profit
$ 75/hr HPS assay development
$ 75/hr HPS assay optimization.
$150/hr HPS screening - business hours.
$ 75/hr Robotics usage - off hours. Example: automated unattended diluting drugs into plates overnight during non-supervised hours.
$ 75/hr Image analysis - Zeiss Arivis 4DVision software training and use.
$ 75/hr Data analysis.
External - For Profit
$129/hr HPS assay development
$129/hr HPS assay optimization.
$258/hr HPS screening - business hours.
$129/hr Robotics usage - off hours. Example: automated unattended diluting drugs into plates overnight during non-supervised hours.
$129/hr Image analysis - Zeiss Arivis 4DVision software training and use.
$129/hr Data analysis.
We bill through iLab: George McNamara will typically make the iLab reservations - need the P.I. and/or user to be in iLab with at least one Cost Object - and inform George (usually by email) what cost object to use for a session. Providing data: small datasets can be emailed by George to the user and optionally the P.I. Larger datasets we will probably have the user log in to myJH on one of the HPS computers and have them upload the data to their JH OneDrive. We normally do NOT allow USB drives on our PCs. External users: email for small datasets, we will make arrangements to get large datasets to you -- probably best if you are able to log in to your company Microsoft OneDrive account and upload your data.
George McNamara (305-764-2081, gmcnamara@jhmi.edu) one of our key instruments is the Union Biometrica BioSorter - large object sorter. We "do" mostly zebrafish larvae since readily available from Jeff Mumm (P.I.) lab. Happy to discuss testing your specimens. withrespect to hourly rates, George's self training is no charge. The Biosorter uses a 750 mL sample cup, with minimal volume 50 mL (below which air will get into the sample line and mess up the run). Because of the layout of the HPS platform it is not practical to use the alternative 50 mL sample cup on our Platform.
January2025 update: installation of our BSL-2 biosafety level 2 enclosure around the entire HPS platform is done and all components re-connected and plate cranes aligned to "handoffs" (Mon Jan 28, 2025). The BSL-2 enclosure Fire Suppression System and emergency power cutoff to the HEPA filter fans have not yet been installed (as of March 31, 2025) - we are using the Platform without it (the risk of fire is very low).
October 2024 update: installed vibration isolation table for VAST-LSFM and replacement "end table" for UnionBio Biosorter and Tecan Spark. (ii) .
August 2023 update: Tecan Spark multi-mode plate reader has been moved from Smith 4001H (Mumm lab) to HPS platform. Hudson Robotics have calibrated the SciClops plate crane robot and written scripts to load and unload a plate (it is possible to reach over the space between the LPSD/VAST Bioimager instruments and the VAST capillary-LSFM, but the script will be better. The Spark requires ~1 minute from power on using the switch in the back (the "power' button on the front is Standby. not power - back switch is better).
February 15, 2023 update: Room renovation done early December. Installations of several components mid-December 2022 and mid-January 2023. George McNamara (manager) started getting training early Feb 2023 (more to install and learn!).
Oct 10, 2022: Smith Bldg room renovation started - JHU facilities, vendors, Jeff Mumm and I (GM) are estimating "early December (2022)" for most components to be installed and integrated - that is "first light".
Aug 9, 2022 iLab issues - yes, I realize you or a colleague needed to get this far in iLab to see tis message -
issues related to iLab for end users and Core Managers, JHU has a full set of simplified help documents on our Core in the Box Website. See link below. The available help docs on this page may not always solve the issue, but please feel free to send the link along to your users that may have questions.
https://www.hopkinsmedicine.org/research/resources/synergy/core-in-a-box/ilab-support.html
Also, emails for JHU contacts are: gmcnamara@jhmi.edu (me - for cores I am involved in), Shawn Franckowiak sfrancko@jhmi.edu , Jeffrey Smith smithje@jhmi.edu , Melanie Branagan mbranag1@jhmi.edu
Prof. Jeff Mumm | PhD Director | HPSCore@jhmi.edu
George McNamara, PhD, HPS Core Manager | gmcnamara@jhmi.edu | 305-764-2081 cell
Monday - Friday
9am - 5pm, by appointment (we anticipate doing some drug dispensing overnight, unattended)
]**
20221010M notes - some features
* Hudson Robotics - https://hudsonrobotics.com/ two SciClops plate cranes. Solo pipetting station, two Micro10x dispensers and software to integrate all components. SoftLinx software to integrate all components. Plate cranes, drug dispenser, more.
* Tecan Spark multi-mode plate reader (state of the art ... bioluminescence, fluorescence, absorption ... best luciferase is Promega's NanoLuc, as in Jay Thierer & Steve Farber's LipoGlo zebrafish papers). NanoLuc-->HaloTag BRET (bioluminescence resonance energy transfer) - see Hellweg ...Johnsson, Hiblot 2023 PMID 37291200 for NanoLuc->GFP->HaloTag(NIRF). GM is a big fan of NanoLuc and HaloTag. Also SNAP-tag and new (9/2024) SNAP-tag2 as potentially brighter alternatives to fluorescent proteins (speaking of which: I encourage replacing EGFP with tdAausFP1 or (monomeric)StayGold green fluorescent proteins (see www.fpbase.org).
https://lifesciences.tecan.com/multimode-plate-reader
Tecan Spark measurements - plate - detection - action - kinetic
Kinetic Loop - available - set up first if using (loop detections)(can move assays 'sections' in GUI if add Kinetic Loop later).
Detection
Detection modes Comments
Absorbance
Absorbance Scan spectrum
Alpha Technology PerkinElmer Alpha Screen kits or similar (homogeneous assay; "one time read") - Also Biosignal2 Omega See note below
Fluorescence Intensity usually use the dual monochromators, has some filters
Fluorescence Intensity Scan spectrum or spectra
Fluorescence Polarization FPol also known as Fluorescence Anisotropy ("FA")
TR Fluorescence Intensity TRF Time resolved fluorescence, Tb2+ ions donor or Eu3+ (or other lanthanide), aka TR-FRET Time Resolved Fluorescence Resonance Energy Transfer.
Also known as HTRF - George has a Tecan "High Class HTRF Performance ..." application note (PDF) for using the Spark (ex: Cisbio -> PerkinElmer/Revvity LANCE cAMP assay).
We did LANCE cyclic AMP TR-FRET assays 9/2024 using the dual monochromators - worked fine. Uses Europium (Eu3+) chelate to Alexa Fluor 647 (2 channels) acquisition.
The HTRF appnote also states (1) use PMT in its linear detector range of 80 - 220 , (2) the HTRF assays are typically done in "half area" plates (which conserves reagents). GM prefers manually setting PMT gain instead of appnote's "optimal" recommendation.
Luminescence Luciferase Bioluminescence (BLI) NanoLuc:Furimazine brightest (in vitro - see Cephalofurimazine for in vivo mouse imaging - zebrafish brains not tested); Chemiluminescence (HRP:Lumigen etc).
Occasionally new luciferases and/or substrates are published, also brioluminescence resonance energy transfer (BRET) fusions.
Luminescence Multi Color i.e. multiple luciferases:luciferins (NanoLuc:Furimazine may be 100x brighter than FLuc, RLuc, etc)
Luminescence Scan Spectrum 19 Longpass and 19 Shortpass filters, usually used in pairs to generate Bandpasses.
Actions
Wait
Comment
User Intervention
Shaking
Condition
Injector
Move Plate
Temperature
Gas
----
Tecan Spark PMT linear detector range is 80 - 220 (maximum is 255) - gain settings outside this range would require a calibration curve (curve may be useful for any setting).
When using zebrafish larvae we always optimize the Z-position {top icon bar in SparkControl} to get the brightest signal from the zebrafish larvae (or similar objects) - this is different from homogenous solutions the Spark was designed for. Best practice is to also have the user look at each well to check for "floaters" (which would be several millimeters from the bottom of the well of a black round bottom plate, and therefore dim). Ideally each fish should be
(1) optional: use Micro10x dispenser to put (say) 100 uL E3 (no anesthesia) into each well (HPS platform could go Carousel -> Micro10x -> Handoff B2 -> Biosorter).
(2) load plate: Biosorter droplet is 25 uL, 17.5uL sheath fluid, 7.5 uL sample cup Ed media + fish, can use Biosorter Sheath carboy with 100 mL of 100x E3 then add 9.9 Liter PicoTap water to make 1x E3.
(3) add drug(s) etc (maybe to 250 uL per well total), incubate (room temp. in Carousel) 24-48 hours.
(4) add appropriate Tricaine (i.e. 3x or 4x in E3) to reach 1x tricaine in E3, "top off" the well to achieve flat meniscus for best Tecan Spark plate top read (yes this matters).
(5) optimize Z-position (several wells on first plate; should be same for all later plates in a run).
Tecan Spark fluorescence is often done with the dual monochromators (2 ex, 2em), can do mixed mono and filter. The monochromators provide much more flexibility. THe Tecan SparkControl software issues a warning in top right of the control screen if the excitation and emission monochromator slits are too close together - and will not allow you to acquire until you separate them (shift wavelength and/or narrow slit width). This is to protect the PMT detector from saturation and to enable you to routinely acquire high quality data. Single monochromator extinction ~3 O.D., two in series is ~6 O.D., equivalent to the blocking of high quality interference filters (~6 O.D.) listed below for the Spark (interference filters are the major type of filter on modern fluorescence microscopes). Since excitation and emission are different wavelengths (and SparkControl software prevents bands being too close) excitation-> emission blocking is (in theory) ~12 O.D., with the major source of background being autofluorescence at the selected emission wavelength band.
Tecan Spark fluorescence filters ... Center(Bandwidth)
Excitation: 400(20), 430(20), 495(10), 510(10), 560(10), 590(10)
Emission: 460(20), 485(20), 520(10), 535(10), 590(10), 620(20)
Beamsplitters: Dichroic 625, Dichroic 560, Dichroic 510, 50% mirror, Automatic [the Tecan M1000 only had 50% mirror; Dichroic enables ~2x improvement in signal, if appropriate for fluorophore). Any of these can be used with the monochromators and with the excitation/emission filters.
* Fluorescence aperture: The Fluor beam diameter for Tecan Spark and M1000 is 2mm for plates up to 384 wells. It's 1 mm for 1536 well plates (the Mumm lab has a Tecan M1000 plate reader). A day 5-8 zebrafish larva (unfed) is ~600 um diameter * ~1500 um length (I realize a larva is not a cylinder, but simple description of rough dimensions).
----
Tecan Spark luminescence filters (3 modes; 2 different settings)
Luminescence and Luminescence Multi Color modes
360 385 400 415 430 445 460 475 490 505 520 535 550 565 580 595 610 625 640 665 700 Bandwidth min 35nm (adjacent values) to max 340nm (700nm-260nm=340nm)
Luminescence Scan central wavelength
398 413 428 443 458 473 488 503 518 533 548 563 578 593 608 623 638 653 (bandwidth 25nm, step size 15nm) (max 18 measurements)
----
GM note: Hudson Robotics SoftLinx software will control Tecan Spark and all other HPS components.
GM 20230824 note on Alpha screen
"One time read" (no kinetics, which confused me the first time we hosted an assay ... Tecan disables kinetic reads with Alpha for this reason)
expensive assay kit
high enegery laser (which we have, and "zaps" the sample, resulting in "one time read")
GM 20240528 note on Biosignal2's Omega screen
--------------------------------------------------------------------
* Union Biometrica ... https://www.unionbio.com see https://www.unionbio.com/documents/BioSorter_Brochure.pdf
* BioSorter (improvements compared to older "COPAS"; BioSorter (FlowPilot III software) includes Profiler II line profiles and size [time of flight = length], transmitted light (optical density) and up to 3 color fluorescence gating). sort into 96 or 384 well plate.
Standard "sample cup" is 750 mL - has "rotor" to keep the large objects in suspension [bidirectional mixer setting works best], we pause at ~50 mL to add more volume; we can also use a rotor with a 50 mL centrifuge tube (ThermoFisher tubes good; some other tubes bad), but not trivial to switch between 750 mL and 50 mL. We note that ~4 fish per mL (4-8 days post fertilization) works well for sorting, so 4 * 700 mL = 2800 fish. Please make sure there is no debris in your fish suspensions before putting into the Biosorter sample cup (or other specimens. Any clog greatly decreases throughput and risk completely clogging the tubing or flow cell. Any debris, fish fragments, chorion fragments, food (paramecia or brine shrimp) are "objects" which can show up as events (flow cytometry terminology) and cause the Biosorter to not sort your "event" (to some extent controllable with "purity" vs "enrichment" sort decision options). If low fish density, you could "pre-sort" into a 50 mL tube (each fish droplet is 25 uL, so 1000 fish would be 25 mL) then decant the counted fish back into the 750 mL Sample cup at optimum density (i.e. 4 larvae per mL) -- note that pre-sort from low density may take 30+ minutes (do not let sample cup go below 50 mL!!!).
For measurements, without sorting, we could use >10 objects per mL. Data is text files (comma or tab delimited, re: CSV files for Microsoft Excel). User could obtain a quote from our salesperson, Yongwoon Kim, PhD, eastern region sales, Union Biometrica, for an analysis license of Flowpilot III analysis software - the main feature is that you can review the Profiler data, which is not in the text logs.
Four flow cells ("FOCA"): FOCA-250, FOCA-500, FOCA-1000, FOCA-2000
(UnionBio also offers FOCA-500 metal free - we do not have this currently; could discuss with user how to pay for this or other accessories we do not have).
lasers 514, 561, 640nm (can use all three, any two, or one – normally 514nm is the laser for the “Extinction” [aka forward scatter] detector, with 1.5 O.D. neutral density to attenuate the laser light).
detection (8/2023 default):
514nm Extinction (forward scatter), 512/24 bp ("side" scatter), 543/22 (yfp, mNG), 615/24 bp (mScarlet3, mScarlet, mCherry, HaloTag etc), and NIR 680/42 bp
Alternative red: 586/20 bp (typically in place of 615/24).
-->see below for VAST-LSFM wavelengths
* LPSC/VAST/VASTomography ... large object particle sorter and classifier and imaging.
* Our standard capillary is 650 um diameter (10 um thin glass walls), which works nicely for day ~6 to ~10 zebrafish larvae (length is ~2 mm, face on these fish fit the capillary; younger have too much yolk to risk clog; day 6 and younger not tested as of 1/2024).
We can also image organoids (that are the right size and promise to not clog the capillary or tubing, or C. elegans larvae.
We have 600um, 650um and 700um diameter capillaries - these are difficult to swap out so we expect to stay with 650um diameter.
We normally use a 20x/1.0 Water immersion objective lens for the camera(s), 672x672 um field of view, so a typical zebrafish larva could be imaged in about 3 views: head, trunk, tail, with some overlap.
The ASI piezo-Z has a range of 500 um, so we can acquire most of the dimension across the 650 um capillary.
For zebrafish larvae, please use PTU to inhibit melanin production; if practical, also use the mutation(s) that prevent pigment formation, though we note these are more difficult to maintain in the lab (lack of pigment in the RPE may make hunting difficult - Jay in Jeff Mumm's lab is evaluating a diet for easy feeding). Potential future option: a mouse lab replaced the Tyrosinase albino mutant allele (mutants are albino and have retina morphology problems) with tyrosine hydroxylase gene to make L-DOPA but not melanin pathway molecules - GM suggests doing the same in zebrafish.
* Applied Scientific Instrumentation https://www.asiimaging.com/ ... Light Sheet Fluorescence Microscope (LSFM) integrated with LPSC/VAST/VASTomography ... single sided SPIM (selective plane illumination microscopy, aka LSM/LSFM light sheef fluorescence microscopy)
- Goal: similar capability to Union's VASTomography (our JHU collaborators are working on the details)
VAST-LSFM light paths
Vortran lasers 445nm 514nm 561nm 639nm
ASI dichroic (standard) 561nm (large cube between emission light paths). If this is removed, then Top_Camera gets all the light (emission filter wheel before it).
Each of Top and Bottom filter wheels have identical set of four emission filters:
Emission: 474/27, 532/18, 593/40, 680/42, block (could buy additional filters for wheels), empty (laser sheet scattered light - which is much brighter than fluorescence) ... the cameras can also use the VAST tray light.
Top camera: >561nm use with 593/40 or 680/42 (default lut: Magenta)
Bottom camera: <561nm use with 474/27 or 532/18 (default lut: Cyan)
- ASI LSFM SPIM components ... adding Spring 2023: 500um range pizeo-Z for detection objective lens, so can do conventional Z-series, rotate VASTomography capillary, acquire multiple angles (work out muti-view fusion spatial deconvolution later).
Typical experiment could be to image dorsal, left, ventral, and right views (wrt detection objective lens); Jan Huisken published optimal multiview may not be orthogonal directions, ex. ignore yolk sac or imaging through retinal pigmented epithelial cell layer
He & Huisken 2020 Nat Commun - Image quality guided smart rotation improves coverage in microscopy https://www.nature.com/articles/s41467-019-13821-y
- Excitation: Thorlabs Water Dipping Excitation Objective, 20x/0.60 NA, 5.5 mm WD.
- excitation alternative (not on LSFM): Multi-immersion objective, 10mm WD, NA 0.7 @ RI 1.45, EFL 8.4mm @ RI 1.45. (R.I. range 1.33 to 1.56)
| At RI = 1.33, NA = 0.64; XY resolution = 470 nm, Z resolution = 3.2 um |
| EFL = 9.2 mm so with 200 mm FL tube lens, Magnification ~21.7x, FOV (672 um)^2 |
- Detection: Olympus XLUMPLFLN 20x Water NA 1.0 WD 2.0mm.
- dual Hamamatsu ORCA-Fusion (front illuminated) sCMOS cameras with CameraLink frame grabber cards (i.e. 2 fluorescence channels simultaneously) (note: "BI" back illuminated Fusion was not available when the grant was submitted).
20x lens Field of view is 672 x 672 um, camera format is 2308x2308 pixels, 291x291 nm pixel size.
Excitation beam thickness: this is under our control, within limits.
The light travels through the glass (10um wall) or FEP plastic (thicker wall) AND the LSFM has adjustment controls. The sheet generated by a MEMS scanner that (1) produces a narrow beam, and then (2) scans the beam to create a sheet [in our alignments, we turn off the scan direction and inspect and adjust the beam). The narrow beam does not behave like a focused point of a laser scanning confocal microscope. We are using a 0.6 NA objective lens, so if we ignore beam details and calculate for widefield, we get https://svi.nl/Nyquist-Calculator recommended pixel size of XY = 216nm -- our beam is thicker than this. Note: we can at any time measure the beam waist.
XY resolution on detection arm: Our 291x291nm pixel size (see above) is far larger than the https://svi.nl/Nyquist-Calculator calculation of recommendated XY 130nm pixel size.
(recommended Z step 274nm), which is OK since larger pixels get us more light per pixel. We typically use Z=2um step size, so 500um Z-series is 250 planes.
- Data: Micro-Manager OME-TIFF file format.
- Data responsibility: the data is yours and YOU & your P.I. are responsible for transferring your data to YOUR storage site(s), i.e. user PC and lab storage
-- HPS Core does not have 'infinite storage" and do NOT archive user data.
-- see 20241114H (11/14/2024) video: Research Integrity (RI) Colloquia | Data Management Plans and Ethical Use of Data - Stuart Ray (Prof.
- Data transfer: the ASI PC can "see" some other computers on the JHU network; you may want to "expose" a share folder on one of your PCs such that we can see it ... and then transfer to a secure storage.
crude and slow, but easy, is your myJH --> JH OneDrive (not ideal - since slow - best used as a transfer method to your PC / lab storage). No USB sticks or drives, since risks computer viruses.
20241118M update: JHU Research I.T. is rolling out SafeStor, up to 10 Terabytes per Project, free for first 3 years. I suggest "one cost object => one Project"
- ASI uses micro-manager.org to control all the components on th light sheet sub-system (which in turn would be triggered by Hudson Robotics).
- acquisition PC - nice workstation (including 10 Gbe Ethernet).
- analysis software: Zeiss arivis Vision4D.
- Possible future plans: we may add GPU enabled spatial deconvolution (was not budgeted in proposal) - our priority is counting and measuring objects, such as fluorescent protein expressing rod cells or cone cells (or cone sub-type), retinal ganglion cells, etc, not prettying up Z-series.
* Liconic incubator - www.liconic.com incubate multiple SBS plates - with internal plate crane to move a plate in and out of incubator (sliding door on top). Holds up to 42 plates (21 per rack), top loaded with a Liconic plate arm. The Liconic incubator loading and unloading is done through the Hudson Robotics SoftLinx software that integrates the entire HPS Platform. The Hudson SoftLinx program has a "relatively manual step" in instructing the Liconic to load or unload - so using the Liconic incubator may require clicking in SoftLinx to trigger some steps.
Thierer et al 2019 (https://pubmed.ncbi.nlm.nih.gov/31366908, https://www.nature.com/articles/s41467-019-11259-w); screening manuscript submitted (11/2024) likely published in peer reviewed journal in 2025 -- scale of the screen (~300,000 larvae in ~14 work days) on our older equipment. NanoLuciferase gene knocked in to the ApoB gene (ApoB-NanoLuc fusion protein) zebrafish larvae plus high fat diet to model atherosclerotic cardiovascular disease -- and drug discovery to decrease "heart disease" lipids ... 14,000 compound screen to be published. The larvae were 5 dpf (days post fertilization), unfed. NanoLuc + furimazine (substrate, with O2 being co-substrate) on Tecan M1000 plate reader could report increases and decreases in NanoLuc activity, proportional to ApoB expression level which is proportional to lipid level. Of 14,000 compounds, 29 validated as ApoB-changing hits in secondary screens plus one compound inhibited NanoLuc enzyme activity.
Part 1: 3,000 compounds assayed. Each 96-well plate had two compounds, so 48 wells per compound. For each compound, there were 4 concentrations with 8 larvae each (32) + 4 DMSO solvent control + 4 positive control (drug) = 48 wells. 48 * 3000 = 144,000 larvae.
Part 2: 10,000 compounds, 16 larvae per compound, 160,000 larvae (calculations ignoring negative and positive control wells).
Total: 13,000 compounds, ~300,000 larvae, 29 validated as ApoB level changing (some up, some down), infer lipid levels changing the same way.
* as mentioned at top (20250209Sun addition) - 3000 drug screen preprint: DJ Kelpsch, Liyun Zhang, James H Thierer, ... Jeff S Mumm, Steven A Farber 2025 (Jan 16, 2025) A whole-animal phenotypic drug screen identifies suppressors of atherogenic lipoproteins. bioRxiv https://www.biorxiv.org/content/10.1101/2024.11.14.623618v3 PubMed PMID: 39605440 DOI: 10.1101/2024.11.14.623618
| Name | Role | Phone | Location | |
|---|---|---|---|---|
| Prof. Jeff Mumm |
HPS Core Director
|
HPSCore@jhmi.edu
|
Smith Bldg
|
|
| George McNamara, PhD |
HPS Core Manager
|
305-764-2081 cell
|
gmcnamara@jhmi.edu
|
HPS Core, Smith Bldg
|
| Service list |
| ► WBC Hourly Rate (2) | |||
| Name | Description | Price | |
|---|---|---|---|
| Established Hourly Rate |
For hours >20 |
Inquire | |
| Startup Hourly Rate |
For hours >10 up to 20 |
Inquire | |
Smith Bldg is on southwest corner of JHU SOM campus. Accessible from "main" hospital part of campus using tunnel under Broadway to JHOC - brief walk to Smith Bldg outside or through employee-only tunnel. https://www.google.com/maps/place/400+N+Broadway,+Baltimore,+MD+21231/@39.2962553,-76.5970508,17z Wilmer Bendann Surgical Pavilion (Smith Bldg) 400 N. Broadway, Baltimore MD 21231 Research entrance is the northeast corner, facing N. Broadway and a driveway. Broadway & Orleans St intersection is at the southeast corner of Smith Bldg. Public parking: (1) McElderry garage (short walk to/from Smith Bldg - can arrange to meet George at the closer JHOC Outpatiant Bldg and escort through JHU tunnels to Smith); (2) Orleans Garage (main hospital visitor parking). (3) "patient parking" between Smith and CRB - in principle for Wilmer Eye Institute patients. GM discourages on street parking. Loading dock for crates -- please contact George to help with all deliveries - both arrival and getting items to Smith 3074 (gmcnamara@jhmi.edu and 305-764-2081 cell) 1550 Orleans St, Baltimore, MD 21287 This is the Koch Cancer Research Building's Loading Dock. Please note that Orleans street is a divided highway - trucks need to be heading west (will go past JHH main entrance). Larry Chase (CRB loading Dock Supervisor) lchase2@jhmi.edu) (667) 776-4876
