. Olympus FV3000RS Confocal Microscope
Location: Ross 913
Olympus FV3000RS Confocal Microscope
Important News: July 24, 2020 policy update takes effect Monday August 3, 2020
FV3000RS scheduling policy change, as of week of August 3, 2020 ... lab groups limited to 3 half day sessions during workdays. This will also be posted on our web sites (confocal.jhu.edu and iLab).
To improve the usage efficiency for the Olympus FV3000RS Confocal microscope, we issued the following reservation rules.
Two prime sessions on each working days (Mon-Friday), $27/hour
1pm-2pm, Gap hours for empty room, per JHU research restart phase 1
Non-prime sessions: nights, weekend, holidays, $20/hour
Each lab has a quota of 3 prime sessions (3 half days) per week for reservation one week in advance. George will make the final count at 1pm on Friday for the next week.
If a reservation continues from morning to the afternoon session, it will be counted as two sessions against the lab quota.
Reservation can start before 9am for the morning session or end after 6pm for the afternoon one. So if you want longer session, you could also start earlier or end later.
Open prime session as of 1pm the previous Friday can be reserved irrespective of the lab quota.
Non—prime time is not counted to the quota at this time.
Definition of lab: A lab may be more than one faculty member, specific examples (not limited to currently named users):
Mark Donowitz includes Varsha Singh, Rafiq Sarker, Hong Yang.
Jay Pasricha includes Subhash Kulkarni, Monalee Saha, Qian Li.
Brian O’Rourke includes all his lab staff.
Please also note we will be monitoring overall use for time gaps between users (covid-19 phase 1 restart) as well as Frequent cancellation of reserved time without valid excuse.
20200708W: ZDC video at Olympus TruFocus Z Drift compensation
20191104M (November 4, 2019): we are now on JHU iLab for scheduling. See:
Our current equipment page for iLab scheduler links.
Our Olympus FV3000RS confocal microscope:
* IX83 inverted microscope stand.
* FV3000RS scanhead with galvo and resonant scanners.
Galvo scanner: 1x to 40x zoom.
Resonant scanner: 1x to 8x zoom.
* 6 position objective lens nosepiece turret, with:
* dry lenses: 2x (NA 0.08), 10x, 20x,
PLAPON2X; PLAN APO 2X NA 0.08,WD 6.2MM
UPLSAPO10X2; U PLANS S-APO 10X NA 0.40, WD 3.1MM
UPLSAPO20X; U PLAN S-APO 20X, NA 0.75, WD 0.6MM
* nice suite of three silicone oil objectives (30x, 40x, 100x) -- 1.405 refractive index. See Boothe et al 2017 ELife, https://elifesciences.org/articles/27240 for use with live cells and embryos.
UPLSAPO30XS; UPLSAPO N 30X SI OIL OBJ, NA 1.05, WD 0.8MM, W/CC
UPLSAPO40XS; UPLSAPO 40X SI OIL OBJ, NA 1.25, WD 0.3MM, W/CC
UPLSAPO100XS;UPLSAPO 100X SI OIL OBJ, NA 1.35, WD 0.20MM,W/CC
* 405nm, 445nm, 488nm, 514nm, 561nm, 640nm and 730nm lasers. the 405-640 nm lines are Coherent OBIS solid state lasers; the 730nm is special NIR laser.
|Description / comment|
|405nm||50mW||16.20 mW||14.60 mW||DAPI, BV421|
|445nm||75mW||0.24 mW (before fix)||4.80 mW (fixed)|
|488nm||20mW||13.56 mW||13.30 mW|
|514nm||__mW||0.75 mW (before fix)||11.20 mW (fixed)|
|561nm||20mW||13.65 mW||14.70 mW|
|640nm||40mW||19.70 mW||22.00 mW|
Table notes: (i) 20200709H fiber from 445/514nm laser launch to main (other 4 vis) launch had developed a problem, was replaced 20200716H.
* We typically operate with the "ND10" (10% transmission) filter in (engaged) and 0.1% to 1% power settign of the intensity slider, for each laser line. Can also attenuate power by using MBS with the "BS10/90", which is equivalent to our "RT10/90" (Reflection 10% / Transmission 90%) in our FISHscope (we also have a "R03/T97 beamsplitter in FISHscope).
* Master beamsplitters ("MBS"): Most users use the standard quad DM405/488/561/640 with the four designated laser lines. We have found that using an "off target" laser line works fine, just need to crank up the laser power in the software (and possibly switch from ND10 to no ND). Our current set of MBS, between decks, and within decks are shown below. The "fiber port" path goes to our two external NIR1 CH1 and NIR2 CH2 GaAs PMTs (see graph below for QE curves for GaAsP and GaAs PMTs (the checkbox next to Fiber port sets the between decks beamsplitters to Glass, we typically leave this UNchecked, and use SDM685 between deck 2 and external ... with multiple scan tracks we could have the whatever beamsplitters and internal and external PMTs we want).
* Four internal "spectral" GaAsP fluorescence photomultiplier tube (PMT) detectors (most '3000s have two GaAsP, two 'standard' PMTs).
* Two external GaAs near infrared fluorescence photomultiplier tube (NIR PMT) detectors (nearly all '3000s lack any external detectors). Standard HV is 500 mV.
Comparison of GaAsP and GaAs PMTs quantum efficiencies from Wang 2016 IEEE Photonics Journal.
[GaAsP vs GaAs ... transition wavelength 712 nm]
NIR dichroics and emission filters:
T685LPXR ... T685lpxr; Long-pass dichroic 685nm 12.7x2mm unmounted (inside scanhead) ... simultaneous 6plex fluorescence: enables all 4 internal + the 2 external detectors to be used simultaneously.
T760LPXR; 760nm DICHROIC BEAMSPLITTER 26MMX38MM (standard Olympus filter cube splitting NIR1 and NIR2 PMTs)
ET720/60M ... NIR1 GaAs PMT emission filter.
ET780LP ... NIR2 GaAs PMT emission filter.
* Transmitted light detector.
* RS = Resonant Scanner (high speed).
* Standard confocal galvo scanner also installed (i.e. large field of view, high pixel count).
* laser autofocus (ZDC2 = zero drift compensation).
* SSU "ultrasonic" gliding stage (much nicer than a typical motorized stage).
* OKO Lab stage top incubator.
* Advanced software set
* 3D DECONVOLUTION (license moved 7/2019 to our new FISHscope).
* COUNT & MEASURE FULL
* CELLSENS DIMENSION DESKTOP
We have both galvanometer (various image formats, some examples are 512x512, 1024x1024, 2048x2048, 4096x4096 pixels) and resonant scanner modes.
Olympus FV3000RS Resonant Linescan Mode
8000 lines per second, 512 x 1 pixels.
Resonant scan mode is 10-bit dynamic range, 0 .. 1023.
Need to use Zoom = 1
Go live, click on Line ROI, click in image window … position horizontal line.
Low laser power prudent to not kill your cells.
Good to check that the master beamsplitter and all dichroics are set correctly.
Detector emission bands should be at least 10 nm from any laser line (protect our PMTs!).
1.0 Airy units is ‘standard’ confocal pinhole size. See below for more on this.
HV 500 mV is ‘standard of care’ for our GaAsP internal and GaAs external PMTs.
Gain = 1.0 (always).
Offset … typically 1 or 2%… goal is to have zero light be above zero intensity (~50 is good).
Optional: You can increase dynamic range by turning on TWO or more detectors, with adjacent bandpasses, assuming your fluorophore and a detector pair dichroic splits the light path, such as 540nm split to PMT1 and PMT2. (I would like to see an option for polarizing beamsplitter or neutral density filter, and have FluoView ‘do the math’) (concept adapted from Ralph Weissleder "HDR" confocal publications - they use 90/10 beamsplitters, we think 'spectral splitting' makes more sense and is safer for the detectors). You do the math later, i.e. Fiji ImageJ or MetaMorph to add the channels together.
2. Scanning times at 512x512 pixel format, no averaging (multiple by 2, 3, etc, up to 16, if averaging).
Resonant Scan Pixel dwell time line frame Typical
Uni-directional 0.067 usec 0.127 msec 65.729 msec 16 line average --> 1 second for single scan track, 512x512 pixels, zoom to Nyquist sampling pixel size.
Bi-directional 0.067 usec 0.063 usec 33.333 msec
Galvo Scan Pixel dwell time line frame
Uni-directional 2.000 usec 2.116 msec 1.086 seconds 5 line average --> scan time depends on image format (max 4096x4096 pixels).
Bi-directional 0.500 usec 0.488 msec 0.250 seconds
Pinhole setting: 1 Airy Unit is standard, you can choose a different value (and is different for same diameter, different emission wavelength bands). Brightness is area of a circle (pi * r^2), relative to 1 Airy unit, and implies one subresolution fluorescent volume. Examples:
0.5 Airy unit … 1.2x better resolution per axis, but (0.5)^2 = 0.25 brightness.
0.6 – 0.7 A.U. … ~1.1x better, (0.6)^2 = 0.36, to (0.7)^2 = 0.49, “confocal sweet spot” – Jeff Reece, NIH.
0.666 A.U. … ~1.1x better, (0.666)^2 = 0.44, “confocal sweetest spot” – GM single value alternative to J.R.’s range.
1.0 A.U. … 1x … please note that this diameter pretty much matches the excitation laser spot size.
1.5 A.U. … more light, both from adjacent XY and above and below.
2.0 A.U. … even more light, both from adjacent XY and above and below.
3.0 A.U. … yet more light, both from adjacent XY and above and below.
>> A.U. … quasi widefield of spot illumination. Can be helpful finding right focus of specimen (if careful).
** 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 acknowledge NIH shared instrumentation grant 1S10OD025244-01 (Prof. Brian O'Rourke and Prof. Mark Donowitz).
For details on our award, please see
Our recommendation for Acknowledgement section of your manuscript is:
The Olympus FV3000RS confocal microscope was acquired with NIH shared instrumentation grant 1S10OD025244-01 (Prof. Brian O'Rourke and Prof. Mark Donowitz) and used in the Ross Fluorescence Imaging Center, Hopkins Conte Digestive Diseases Basic & Translational Research Core Center.
Please note that our G.I. Center supplements its members use.
* We thank:
Jason Brenner and John Gibas, Olympus, for demonstrating the system.
Prof. Brian O'Rourke, PI of the NIH grant (and Associate Director of our core).
Prof. Mark Donowitz, S10 grant co-author, P.I. NIH P30DK089502 grant funded Hopkins Digestive Diseases Basic and Translational Research Core Center, and much more.
Prof. Olga Kovbasnjuk - former core Director, now at University of New Mexico.
All the 8 Major and 2 minor project P.I.'s and users, for making demo(s) successful.
NIH and U.S., taxpayers, for the grant; NIH S10 shared instrumentation 'confocal microscopes' study section members, and NIH Council, for our award.
Olympus for accepting our Zeiss LSM510META confocal microscope scanhead for credit toward our new microscope.
The NIH S10 grant proposal projects that enabled our funding of the FV3000RS -- these, and newer-than-S10 pilot projects, are our priorities for year 1 (Sept 2018-August 2019), and we seek to enable their ongoing research.
Major Users projects:
1) Mark Donowitz, MD, Coordinated Regulation of Intestinal NaCl Absorption and Anion Secretion in Health and Disease.
2) Pankaj Pasricha, MBBS, MD/Subash Kulharni, PhD, Imaging the structure of the Enteric Nervous System and its associated cells.
3) Cynthia Sears, MD, Bacterial Biofirms and Colon Cancer, with Emphasis on Toxigenic B. Fragilis.
4) Nicholas Zachos, PhD, Mechanisms of Diarrhea Examined by Live Cell Imaging of Human Enteroids.
5) Brian O'Rourke, PhD, Project Title: Mitochondrial dysfunction as a source of cardiac arrhythmias and heat failure.
Minor Users projects:
6) Joanna Melia, MD, ZIP8 as a Mediator of Intestinal Inflmmation and Intracellular Zinc Homeostasis (Zinc ion micronutrient transporter in Inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis).
7) Jennifer Foulke-Abel, PhD, Enterotoxigenic E. coli pathogenesis and human enteroids (ETEC),
8) David A. Kass, MD, Leveraging Protein Kinase G-1 Nanodomain Control and Molecular Targeting to Enhance its Therapeutic Use Against Myocardial Disease.
Two of our Minor Users moved to University New Mexico (not shown) - we hope the time made available will help support our GI Center's pilot projects, and additional users from all over.
Please note the news items sometime include policy statements.
June 26, 2020: free OlyVIA viewer, download at https://www.olympus-lifescience.com/en/image-sharing-made-easy-meet-olyvia/ or look on our file server (OlyVIA).
June 19, 2020 FV3000RS usage tip for very bright labeling (ex. fluorescent Pharlloidin to label F-actin)l;
FV3000RS confocal microscope - avoid fluorophore saturation of very bright signals:
"even lower" laser power than 0.01% laser power by using a different Master Beamsplitter … if use an “MBS” that does not list the laser line, then laser at specimen is ~1% of the slider value … so if slider is 5% then ‘wrong MBS à ~0.05%
Standard of care PMT “HV” (high voltage) is 500 mV (500 in software). Users can use lower values, ex. 450 or 400, but Hamamatsu (PMT manufacturer) and Olympus (confocal) have informed us 500 mV is optimal for these detectors.
October 4, 2019
1. we are moving to Agilent iLab Organizer ... went live November 4, 2019.
April 22, 2019: Use of our two NIR GaAs photomultipliers (PMTs):
Internal & External T685LPXR ... <685nm internal PMTs 3 and 4, >685nm external NIR1 and NIR2 PMTs.
External dichroic T760LPXR ... Split light for NIR1 and NIR2 PMTs.
NIR1 emission filter ET720/60M ... 690-750nm
NIR2 emission filter ET780LP ... 780+ nm (GaAs sensitivity limit ~860nm).
Critical safety issue: our 730nm laser line is within the emission band of NIR1 emission filter. It is critical that no user sends 730nm laser light to NIR1 (also true for the other laser lines and internal detectors).
- the optimum "HV" = 500, and are controlled by knobs on each of the two power supplies above the electronics cabinet (the front panels are: 0.500 Volts = 500 mV). 500 according to Jason Brenner, our Olympus confocal salesperson. GM is ok with user's evaluating other settings (i.e. 600, 700), note that the saturation warning (HiLo software LUT) cangive way to a protection circuit ... upshot is do NOT saturate the NIR PMTs (or our GaAsP internal PMTs).
- these GaAs PMTs have higher quantum efficiency (QE) above 712 nm, compared to using the four internal GaAsP PMTs -- see Wang 2016 IEEEPhotonics.graph later on this web page.
- We plan to purchase Spherotech UltraRainbow fluorescent beads, fluoresce from UV to NIR, for testing --> our thanks to Olympus tech support for suggestion.
- We expect Li-Cor Dyes IRDye680 (or IRDye700) on "CH1" (EXTERNAL pmt #1) and IRDye800 on "CH2" (external PMT #2) will work well ... compatibility with Donowitz lab (and many other research labs) Li-Cor Odyssey Western blots 'gel documentation & quantitation' systems (NIR fluorescence works nicely for Western blots inpart because lower autofluorescence of the support matrix, glass, etc).
- Li-Cor dyes are not the only option: some AlexaFluor, Abberior, ATTO, CyDyes, could be used. GM is especially hoping that chlorins and bacteriochlorins will work well (i.e. antibody or oligonucleotides), since extremely narrow emission spectrum -- NIRvana Sciences has graphed 8 dyes emission 600-800 nm range (see background graphic for Bruce Pitner, CSO, NIRvana Sciences, on linkedin). Of course 8 bacteriochlorins would be best imaged simultaneously, maybe all four internal GaAs (600-700 nm, if FV3000RS dichroics were optimized for this), plus FOUR external (we have two now): i.e. one GaAsP for 700-725 nm dye, three GaAs PMTs. Of course if NIRvana and/or other can "do" 8 dyes in 600-800nm, maybe could "do" an additional four in 800-900 nm, implying an additional four external detectors (for 8 total, plus the four internal). I also note that chlorins and bacteriochlorins might work well as tandem acceptors of Brilliant Ultraviolet, Brilliant Violet, Brilliant Blue, and Brilliant Green-Yellow, for potentially 4 donors * 12 acceptors ==> 48plex NIR + the four donors = 52plex (plus spatial resolution could be of the photons from the donors, since not 100% transfer to acceptors ... see my linkedin blog, "Resolution Blues", https://www.linkedin.com/pulse/resolution-blues-meets-21plex-salute-fluorescence-basic-mcnamara/ and Brilliants in graphic table in March 29, 2019, news immediately below.
- The current filter cube splitting the light onto CH1 = 690-750 and CH2 = 780LP, is exchangable ... if someone would like to invest in, and donate to the image core more cubes (Olympus IX83 standard format cubes), or convince us to spend our core money on cube(s) [we think simpler to gain experience with current cube).
March 29, 2019: Brilliant Violet BV421 works fine on our confocal microscopes (used twice on SP8 for live cells).
When will confocal microscope users start using Brilliant Violets and Brilliant Blues and/or SuperBrights?
BD Fluorophores Table
More of my thoughts on Brilliants, and similar (SuperBrights, NIRvana prototypes, etc) at my linkedin Pulse blog pages:
and BD Biosciences is now at 21plex for all Brilliants
BUV ... 7plex ... note: we do not have a UV laser (and have not tested -- yet -- whether our CW 640 nm or 730 nm lasers will result in 2-photon excitation of BUVs or BVs).
BV ... 9plex (!!!) of which BV510 is wide emission spectrum (in principle, not an issue if acquisition settings and spectral unmixing algorithms 'deal well with it' ... probably best to use BV510 on some 'dump channel').
BB ... 5plex.
BYG ... 1plex (as of 20190331), should be possible for BD Biosciences to make more.
So, on our FV3000RS, potential for 9 BV + 5 BB + 1 BYG = 15plex.
Brilliant's are typically direct label antibodies, so, if $1 per plex per microscope slide or imaging dish, $15 for one assay. I suggest staining cells in suspension, doing flow cytometry, and bringing 'left over' cells to the imagecore(please use minimal biohazard cells!!!).
Note :Biolegend has 8 Brilliant Violets available:
www.Phitonex.com ... 19plex with 488, 561, 640 nm laser lines (and may have more by end of 2020).
[FACSSymphony Reagents - Table]
March 15, 2019: ZDC830 installed = Olympus Zero Drift Compensation unit wavelength changed from 730nm (temporary unit shipped with installation) to 830nm.
Please note: 830nm is in the wavelength range of External PMT #2 ("ePMT#2" sixth pmt on the confocal).
Depending on your settings, the ZDC 830 nm signal might get to "ePMT#2", so please turn off "continuous ZDC" if using ePMT#2.
December 20, 2018: Tip - transferring files/folders to Microsoft OneDrive:
I suggest using 7-ZIP to package all of your session's files&folders into a single zip file (ex. Mary Elizabeth Garrett 20181220Thur awesome FV3000RS expt.zip).
Every JHU staff (employee, student) gets 5 Terabytes of Microsoft OneDrive space (MyJHU -> Cloud -> OneDrive), so nice way to back up all your data.
November 27, 2018: Our OkoLab inserts are not a good match to LabTek chamber slides (even though oneinsert is for LabTek).
We recommend using 35 mm imaging dishes. For example, www.cellvis.com 35 mm imaging dishes are $1 to $2 per dish
($100 to $200 per case of 100 dishes), depending on format (and offer other formats).
Other companies have imaging dishes, such as Mattek (35 mm formats) and WPI (FluoroDishes).
November 13, 2018: 7 and 6 !!! 730nm laser and GaAs NIR PMTs are here bringing us to 7 lasers and 6 PMTs.
The NIR PMTs should be used at 0.500 HV (just leave the external boxes as is). The internal FluoView software controls do not do anything, just ignoe them.
It is possible to have all 7 laser lines active by enabling all seven detectors (HSD1-HSD4, TD, CH1, CH2) and selecting different laser lines for each.
October 19, 2018: OkoLab incubator policy:
Temperature: if you are using, turn on one hour before your session (if needed add one hour to reservation time since the current user can keep it off through the end of their session). Turn OkoLab unit off when you are done.
CO2: if you do not need it, set the controller to zero (0%), so the unit does not beep at you or anyone else.
Openings: No openings! Cover all the openings if it is possible. ex. 2 slide holder: if you use one slide, fill the other slide position with a glass slide. For the few people with special chambers that cannot fill the opening, ok, you are special.
Sept 28, 2018: Extended imaging sessions: we ask that users who require a lot of time in one day (i.e. >6 hours) work out how to conduct the imaging to additional user(s) to image the same workday, whenever possible. For example, rather than book 9am-5pm, get to know the instrument and your specimens performance on it, so you can acquire overnight from say 4pm-12midnight (unattended, if no liquid perfusion involved). For very long experiments, please build up your skill set(s) to run from (say) 4pm Friday to 9am Monday. The Olympus ZDC (zero drift compensation) device works well and enables stable focus -- especially in conjunction with the OkoLab stage top + shroud environmental control unit to maintain constant temperature (and humidity, and CO2 control for experiments needing these).
The main exception is the major project that proposed to do 24 hour imaging sessions, every week, for 6 months. If the users end up conducting over weekends, great; if their experiiment will work best for them during work week, that is fine: the experiment was part of the reason we were awarded the S10 grant.
Sept 25, 2018:
* John Gibas optimized the network connection between our FV3000RS computer and our file server so that 10 Gbit Ethernet direct connection is working well. Thanks John! Our server is connected to the campus network by 1 Gbit (the Ross Bldg network). As part of our teaching image core users, we explain how to access our file server from JHU computers (we continue our longstanding policy of no usb drives on our computers ... we provide login capability on specific image core computers to enable users to transfer from our server to their JHU Microsoft OneDrive 5 Terabyte per staff member space for those labs whose PC's or Mac's cannot see our file server).
* OkoLab stage top and shroud incubator unit is up and running. We do have a CO2 tank available in the room. Please note that all experiments involving OkoLab incubator MUST have all openings in the insert covered to protect the "microscope insides" (objective lens turret etc) from humidity. We have placed 35 mm and 60 mm dishes, and slides, on the top of the cabinet, where all the inserts are also kept, to cover the openings.
Sept 5, 2018: Late arriving accessories status:
Oko Lab incubator expected to be installed mid-September.
730 nm laser and two external GaAs photomultiplier tube (PMT) detectors expected to be installed late October 2018.
Sept 4, 2018: Our FV3000RS was delivered and installed mid-August 2018. We anticipate image core manager George McNamara will be training the first cohort of users in late August through September 2018. Jason Brenner of Olympus is greatly helping by training users who need "advanced applications", such as FRAP time series (fluorescence recovery after photobleaching).
Priorities: the microscope is open access with prioritization for year 1 (Sept 2018-Aug 2019) primarily for the S10 projects it was purchased for.
Priority in year 1 is to enable our S10 grant Major and Minor project users to do the work the grant proposal was written for. We will accomodate additional projects (from M&M users labs, new pilot projects funded by the G.I. Center after the S10 submission), and additional users, if time slots are available. We recognize that many researchers need new data to help get new funding, so in addition to supporting our G.I. pilot project, we will do our best to help all researchers, whether on this FV3000RS or our other microscopes or even just advice. We also manage the ACCM confocal Microscope http://confocal.jhu.edu/current-equipment/leica-sp8-confocal-microscope so if FV3000RS is too busy with year 1 M&M Projects, please discuss with us use of the ACCM Confocal microscope - a very nice Leica SP8 confocal DMi8 inverted microscope.
Published Olympus FV3000 Confocal image data --> Cellsens Deconvolvution
Hinman SS, Wang Y, Allbritton NL. Photopatterned Membranes and Chemical Gradients Enable Scalable Phenotypic Organization of Primary Human Colon Epithelial Models. Anal Chem. 2019 Dec 3;91(23):15240-15247. doi: 10.1021/acs.analchem.9b04217. PMID: 31692334
Figure below from Hinman et al, supplemental file, annotated by GM. "Advanced Maximum likelihood" (advmle) is an option in Olympus Cellsens "constrained iterative" (C.I.) deconvolution module (extra cost from Cellsens basic ... our advmle license was purchased on our FV3000RS NIH S10 grant and moved to our NIH P30 supplement co-founded FISHscope PC. So: you should cite both our S10 and P30 grant numbers if you publish data with our deconvolution software.
Our thanks (again) to our local Olympus representatives:
Jason Brenner email@example.com ... as of summer 2019 regional manager.
John Gibas, Olympus, firstname.lastname@example.org
Bo Faust, PhD (James J. Faust), Olympus confocal and multiphoton sales ... email tbd.