Current Equipment

Ross Fluorescence Imaging Center Equipment Summary, including ACCM Leica SP8 confocal microscope (we manage for ACCM).

Reminder: All users need to make reservations in iLab for whatever instrument/service they are using. We need this for both billing and to track usage for our NIH P30 Center grant and other administrative purposes.

* upload your data to your JHU Microsoft OneDrive (myJH -> Cloud). no USB sticks allowed (subject to confisacation and discard in biosafety trash). Simit your web surfing to web sites/pages of JHU and critical vendors (not your personal web emasil etc) and especially no surfing the 'dark web' on image core computers.

  Information

click on entry in this column or scroll down this page.

  iLab Organizer

ACCM iLab (Leica SP8)

Ross Imaging Center iLab (all)

Workday hourly rate

JHU trained user

Olympus FV3000RS confocal Olympus FV3000RS info FV3000RS iLab $27 / hr
Leica SP8 confocal (ACCM confocal core) Leica SP8 info Leica SP8 iLab - ACCM confocal core $27 / hr
Olympus MPM multiphoton Olympus FV1000MPE info FV1000MPE iLab $50 / hr
IX83 FISHscope FISHscope info FISHscope iLab Scheduler $10 / hr
Keyence BZ-X710 Keyence BZ-X710 info BZ-X710 iLab $10 / hr
Zeiss AxioObserver.A1 inverted microscope & Olympus DP80 dual RGB and monochrome camera Zeiss AxioImager info Zeiss AxioImager iLab $10 / hr
Li-Cor Odyssey Li-Cor Odyssey CLx info Li-Cor Odyssey CLx iLab $4 / hr

Biotek absorbance plate reader ... has five filters (see the reader), simple to use.

(will add info page later) BioTek absorbance plate reader iLab $4 / hr
PTI QuantaMaster 40 Fluorimeter #1 PTI Fluorimeter #1 PTI Fluorimeter #1 iLab $4 / hr
PTI QuantaMaster 40 Fluorimeter #2 PTI Fluorimeter #2 PTI Fluorimeter #2 iLab $4 / hr

MetaMorph image analysis software 

3 licenses (USB license keys can float)

MetaMorph key #1 = 34135

Metamorph key #2 = 4646

MetaMorph key #3 = 34334

(#4 ex-Revolution X1 MM)

MM key #1 iLab

MM key #2 iLab

MM key #3 to be added

(#4 ex-Revolution X1 MM)

$4 / hr

$4 / hr

$4 / hr

...

See Usage Fees and Rates page for weekend, assisted and training pricing.

Legacy equipment (retired) idescribed briefly at bottom of this web page

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More information

Feature(s): tile scanning & image stitching and/or stage relocation

 Note: four of our microscopes can "do" tile scanning and image stitching AND/OR automate stage relocation for "cyclic" imaging (ex 12plex RNAscope 3 cycles of 4 probes)(save position list, reload when reutrn to slide - assumes you position the slide or imagign dish consistently). This can enable more efficient acquisition at optimum spatial resolution (i.e.high NA objective lens, wavelength limited pixel and Z-step size) (if the analysis computer can handle our 'data deluge') without you being present the whole time (a major benefit of automated microscopy):

  * ACCM Leica SP8 confocal microscope (standard tile scan or NAVIGATOR 'app' wizard)

  * Olympus FV3000RS confocal

  * FISHscope (Olympus IX83)

  * Keyence BZ-X710

Our Olympus FV1000MPE multiphoton microscope can also tile scan -- a user 'did' a 50 slide project of kidney tissue fibrosis in 2018 -- but currently (1/2021) very nfrequently used microscope (we'd be happy to see more users on it).

1/2021: we have also put on VueScan slide scanner for histology / brightfield IHC slides onto a PC in the image core and an iLab scheduler here. This produces images equivalent to a 4x or 5x objective lens 'nominal resolution' (though sometimes the slide scanner focus is not quite as good). Nice for acquiring large area quickley, and then making measurements of areas in Fiji ImageJ, MetaMorph, Photoshop, etc. If users find it useful, great; if not, we may remove it as a service and just have it at GM's PC. Scanner is a repurposed 35 mm film scanner. You are welcome to scan in your 35 mm film 'cassettes' or film strips, when VueScan is not being used for research.

Manual stitching tips (can also be useful if the acquisition machine stitching does not work perfectly or is not "time effective" to do at the acquisition PC) ...

* when possible, move in one axis only; watch 'marker' as you move and stop before the marker goes out of view on the other side.

* Typically you should acquire with 10% or more overlap of tiles. Sometimes 20% -- i.e. test 10% vs 15% vs 20%. You might put small pieces of sticky note(s) on the monitor as a visual guide for how close to the edge(s) 10% 915%, 20%) is.

* Photobleaching is bad: You may want to turn down the epi-illumnation intensity (the "big dial" on the Zeiss inverted microscope lamp ... be sure to reset the dial to 100% for the next user) and longer exposure time. Photobleaching is "non-linear": higher intensity illumination causes disproprotionally more photobleaching.

* Photobleachign is under your control with sample preparation: use the right mounting medium [consider testing Prolong Glass, buy without DAPI; if need DAPI, put it in with primary and/or secodnary antibodies, and wash extensively to get black background outside cells/tissue] and right fluorophores; Tip: Tyramide signal amplification (TSA) can make fluorescence signal 100x brighter than standard immunofluorescence, enabling shorter imaging time (effectively no photobleachng). Also possible to multiplex TSA.

* Sparse specimen stitching: most stitching software relies on "features" to stitch, so if very sparse, may nto work. 

* Zero overlap stitching (automated microscopes): the field of view can be known precisely (from image settings) so you COULD acquire ADJACENT fields of view and use MetaMorph's "Montage Stack" to stitch (limit: 30,000x30,000 pixels, which is pretty big). We have Metamorph on severl PCs.

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Liu JT 2021 Harnessing non-destructive 3D pathology . Nature Biomedical Engineering (Feb 15, 2021 epub). https://doi.org/10.1038/s41551-020-00681-x

Image stitching. A key step in the acquisition pipeline for 3D microscopy data is the efficient assembly of large numbers of 2D image tiles into seamless volumetric datasets. A number of commercial software packages (such as Imaris Stitcher and Volocity) as well as popular open source tools (such as TeraStitcher ref 142,143 and BigStitcher ref 144) have been developed to address this challenge. Some software tools (in particular, BigStitcher) are designed to correct for deformation and registration artefacts through affine transformations, including chromatic shifts between wavelength channels.

==> GM notes:

* QuPath https://doi.org/10.1038/s41551-020-00681-x (Bankhead 2017 Sci Reports) does not appear to have image tile stitching, may still be of use for image analysis. 

* Adobe Photoshop (CS1 and later) Automate/Batch has image stitcher.

* Microsoft Research - Image Composite Editor (ICE 2.0) free image stitcher

https://www.microsoft.com/en-us/research/product/computational-photography-applications/image-composite-editor/

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Feb 22, 2021: "Work in progress" - we have set up a histology/immunohistochemistry brightfield slide scanner, we call "VueScan". Image quality is about the same as a microscope 5x objective lens, but "VueScan" acquires a much bigger field of view in under 2 minutes. 5x lens ~2x2 mm, VueScan ~36x24 mm. You can then use the Vuescanned images to guide your imaging on our other microscopes (i.e. upload to your Microsoft OneDrive, and either print out images or organize your project into PowerPoint etc, download to the microscope PC or bring your own laptop, and reference the "overview" data). VueScan is in iLab - please contact George for training. The "VueScan" is named for the image acquisition software (Hamrick VueScan Pro, www.hamrick.com), the scanner is a Plustek OpticFilm 8200ai 35mm film scanner with one of the film strip holders modifed by George to hold a microscope slide. I also note that if you have a flatbed scanner in your lab, ideally with transparency lid, you could acquire in your own lab (VueScan would be good software to use).

Scientific Image Ethics - a few links

==> does an instrument/services page seem like a weird place to put a section on image ethics? No! If you want to use our core, please plan to, and do, behave ethically.

JHU has an online data repository - for journals that do not enable you to (cost effectively) post all your data as "supplementary" information 9in original data format, with MetaData, and your complete methods, including any image processing and analysis 'scripts/macros/journals'), you could post it at CRAEDL, and include the DOI with your paper. Hopefully they do - or will -- enable preprint data to be posted. preprints both give you academic credit (that is, you have a date you can claim a discovery) and nable getting feedback on your work. See also ELife's 2020 editorial on "moving to preprint reviewing" model, https://elifesciences.org/for-the-press/a4dc2f54/elife-shifting-to-exclusively-reviewing-preprints 

https://craedl.org/docs
Craedl, the Collaborative Research Administration Environment and Data Library. Craedl is a research data management service that understands the way academic research gets done.

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HHS (NIH's parent organization) Guidelines for Best Practices in Image Processing 2008

https://ori.hhs.gov/education/products/RIandImages/guidelines/list.html

(web page has live links to Photshop videos for each item)

  • Guideline1 Treating Images as Data: Digital scientific images should be treated as data
  • Guideline2 Saving the Original: Manipulations of digital images should always be done on a copy of the raw image data. The original must be retained.
  • Guideline3 Making Simple Adjustments: Simple adjustments to the entire image are usually acceptable.
  • Guideline4 Cropping is usually OK: Cropping an image is usually acceptable.
  • Guideline5 Comparing Images: Digital images that will be compared to one another should be acquired under identical conditions.
  • Guideline6 Manipulating the Entire Image: Manipulations that are specific to one area of an image and are not performed on other areas are questionable.
  • Guideline7 Filters Degrade Data: Use of software filters to improve image quality is usually not recommended for biological images.
  • Guideline8 Cloning Degrades Data: Cloning objects into an image or from other parts of the image is very questionable.
  • Guideline9 Making Intensity Measurements: Intensity measurements of digital images should be performed on raw data and the data should be calibrated to a known standard.
  • Guideline10 Lossy Compression Degrades Data: Avoid the use of lossy compression.
  • Guideline11 Issues With Magnification: Magnification and resolution issues are important.
  • Guideline12 Issues With Pixels: Be careful when changing the size (in pixels) of a digital image.
     

 

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which was cited by:

Brocher 202102 BioVoxxel preprint - Seeing the Big Picture - Scientific Image Integrity under Inspection - Project Image Integrity Assessment

* https://www.researchgate.net/publication/349521202
* see also  https://www.linkedin.com/pulse/seeing-big-picture-scientific-image-integrity-under-dr-jan-brocher
* key acronym: GSP = good scientific practice

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I am a big fan of Jerry Sedgwick's (2008) section on microscopy image ethics in:

https://www.amazon.com/Scientific-Imaging-Photoshop-Measurement-2008-06-02/dp/B01JXNG5IK

Scientific Imaging with Photoshop: Methods, Measurement, and Output by Jerry Sedgewick

Notes:

* don't pay a crazy price for the book (post covid-19 andemic you could borrow mine). The JHU library might have a print you could check out.

* disclosure: I wrote a favorable review/comment of the book on Jerry's request. 

 

 

 

 

PIs: to set up a new user and cost center, see our iLab instructions page


Monday June 15, 2020 we re-opened as part of JHU-Research's covid-19 phase 1 re-opening. 

All users must read - and follow - our Covid-19 Phase 1 re-opening policies

http://confocal.jhu.edu/covid-19-policies

Quick summary:

1. Obey room occupancy limit (posted at door). 12/2020: 1 person per 200 square feet, so small rooms occupancy = 1.

2. wear your face mask (cover nose and mouth), and new lab gloves (bring your own supply) when touching microscope, mouse, keyboard, etc. If you handle a specimen for biosafety reasons, discard those gloves in biosafety trash and put on new lab gloves for microscope etc.We provide 'saran wrap' at each station - you are welcome to discard old wrap and apply new wrap at the start (and if needed during) your session. typically keyboard, you can also cover mouse, eyepieces.

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** Please note that image core management sometimes has to cancel/postpone user sessions due to required service visits or other reasons. When this happens we will try to make the user's next imaging session be "no charge" (even if longer than the cancelled session). 

Please Reserve Your Instrument!!! Ideally 24 or more hours in advance; if not in advance, then right after you fill in the paper sign-in sheet. Reservatons policty is online at

http://confocal.jhu.edu/reservations-policy

Please read - and follow - our Biosafety Policy 

http://confocal.jhu.edu/biosafety-policy 

Quick biosafety in image core tips: 

* "safety is job #1". If you are not using our equipment in a safe manner, then do not use our equipment.

* do not touch our microscopes,keyboards, computer mice, door knobs, light switches, etc, with lab gloves. 

* Yes, handle your SBS plate, imaging dish, other biohazard specimen, with a new lab glove(s) ... after adjusting -- without glove -- the microscope condenser arm, etc, to recieve/remove your specimen.

* Yes, biohazard spills have happened here at JHU/JHMI - see July 2018 tuberculosis spill national news. 

* Transport your specimen(s) in a spill resistant tray(s) and/or box(es), i.e. on a lab cart. Do not touch the passenger elevator door buttons, or stairwell handles, with lab gloves. 

* our major users include 6 different models of infectious diarrhea, and potentially clinical specimens from G.I., with "C. diff" and/or novel infrectious agents, and Prof. Cindy Sears lab, who have shown that essentially all human colorectal cancers involve contributions from infectious agents (800,000 new cases per year woldwide). It is critical that all users conduct experiments in a manner that is safe for both the user, other users, core staff, custodial staff, and visitors. 

* Dr. McNamara is the image core manager and the biosafety officer for the microscope rooms. If you are3 unwilling to follow our biosafety rules, either you and your P.I. should explain to Dr. McNamara and Image Core Director Prof. Bin Wu, how to improve our biosafety policies, or you should not do experiments here.

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Data transfer:

Plan A (best): your MyJH -> your Microsoft OneDrive "JHOneDrive": Nearly all our PC's can see "The Web" and so can see MyJH. Now (mid-2018) that every JHU staff member gets 5 Terabytes of Microsoft Onedrive "cloud" storage space, you can transfer from the acquisition PC or our server to your OneDrive account. Be sure to logout ("sign off") of your OneDrive session here when you are done -- you need to do this manually. You also need to log out of your MyJH session.

Plan B: Many JHU SOM computers can see our file server. This is the simplest way to transfer files for those with access. Do not give out the name or I.P. address of our server. We prefer you use your JH OneDrive (plan A above). 

Plan C (risky): You / your lab could 'expose' some "share folder" on your PC or server and transfer from our PC/server to your share. You should only do this with explicit permission of your P.I. and network administrator, since setting up your 'share' incorrectly could expose your PC/server to attack. 

NOT Permitted: USB drives are NOT permitted on our computers. We will dispose of them in the Biosafety trash. 

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Legacy equipment (retired) include:

Andor spinning disk confocal microscope.

http://confocal.jhu.edu/current-equipment/andor-spinning-disk 

Zeiss AxioImager.M2 automated upright microscope, with PTI DeltaRAM high speed random access monochromator, RatioMaster software, MetaMorph license #4646 (repurposed for analysis), Photometrics Coolsnap HQ CCD, standard metal halide lamp (interchangable with DeltaRAM). Was used for Fura-2 ratio imaging Ca++ ions - a method mostly obsolete with GCaMP6 and similar fluorescent protein biosensors and image on our Olympus FV3000RS confocal microscope, with resonant scanner mode available 512x512 pixels (1 fps at 15 line average), also "kymograph" line scan acquisition. 

http://confocal.jhu.edu/current-equipment/zeiss-upright-scope-w-pti-ratiomaster

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Vendors contact information for our instruments is online at http://confocal.jhu.edu/vendors-contact-information