This instrument was decommissioned in May 2022 but this web page is here for historical information, general technical knowledge, methods in papers, etc.

 

Zeiss 710NLO
ACLSW 421  Badge Access Request Here

The Zeiss LSM 710 laser scanning confocal and multiphoton microscope (inverted) offers two imaging systems built on a Zeiss Axio Observer temperature-controlled microscope. An optional objective inverter allows it to function as an upright system. For confocal microscopy, there are lasers ranging from 405 to 633 nm and user selectable spectral detection from 420 to 740 nm. It offers a standard or spectral confocal and may also be used for photoactivation, fluorescence recovery after photobleaching (FRAP), and ratio and acceptor-photobleaching FRET. For multiphoton, excitation is provided by a Spectra-Physics Mai Tai® DeepSee™ and detection by five detectors ranging from 420 to 760 nm.


 

Instructions:  [Word doc]   [PDF]     How to do tiling.

To start Zen software, use Kiosk. [Word doc]   [PDF]

 

A few of the specifications:

  • 10X N.A. 0.30 Air Plan-NEOFLUAR SN 420340-9901
  • 20X N.A. 1.00 Water Plan-APOCHROMAT DIC VIS-IR (which is my favorite lens for imaging tissue)
  • 20X N.A. 0.75 planapochromat air
  • 25X N.A. 0.80 Oil-Glycerol-Water DIC (UV)VIS SN440842-9870
  • 63X N.A. 1.40 Oil Plan-APOCHROMAT DIC SN 420782-990
    People often ask, what about 100X? The numerical aperture defines the resolution, not the nominal magnification. The 63X N.A. 1.40 lens has the same spatial resolution at the 100X N.A. 1.4 lens when using effective optical zoom by scanning a smaller field slower or collecting a large number of pixels. It has benefits of being able to scan a larger field of view, longer working distance, and superior light collection.
  • Antivibration table: Newport ST Series with autotune damping and I2000 series legs 1500 mm X 1800 mm X 203 mm (71 X 59 X 8 inches), 805 lbs not including the legs.
  • All fluorescence detectors PMTs (3 internal for confocal and 5 external for multiphoton); additional transmitted light detector.

laser list
Please note, this scope is not good for Alexa Fluor 350 and 594. 350 will not work at all. High concentration 594 will provide signal but is excited inefficiently by the 543 nm laser.
Please review which fluorescent probes work with this microscope before purchasing for experiments.

Using the Mai Tai laser, the lowest practical wavelength is approximately 760 nm.

Excitation path has the following filters/mirrors in place to selectively deliver light to the sample and to allow for returning light to go to the detectors:
MBS position 1 MBC position 2

Rear
458 plate Fset38 wf
458/514 none Fset43 wf
458/543 -405 BS-MP 690+
none -445 BS-MP 760+
488 690+ rear
488/543 760+  
none -405/760+  
488/543/633 -445/760+  
T80/20 T80/20  
plate none  

Three internal detectors with user selectable spectral ranges from a little below 405 nm to to 740 nm. These may be used for standard confocal or for multiphoton. According to the software controls, the internal detectors collect up through 758 nm. However, testing with reflection mode with the longest wavelength physical gate, collection is practical only to 740 nm. After 740 nm collection intensity falls rapidly and is not detectable above 750 nm.

Standard Filters in External Detectors for nondescanned multi-photon. These filter blocks are easy to change if users have special needs and the blocks are purchased or borrowed.

  Wavelength (nm) Typical Uses
Channel 1 420 - 465 2nd Harmonic
Channel 2 465 - 500 Dapi, CFP
Channel 3 520 - 555 GFP, YFP, FITC
Channel 4 575 - 645 Cy3, DsRed, RFP, mCherry, Rhodamine, Qdots
Channel 5 660 - 760 Cy5, Qdots

Recommended alternative to water for using water immersion objectives.

General Confocal Best practices:

  • The pinhole is what makes the confocal a confocal. Always set at 1AU (which means 1 Airy unit) or smaller and reset this each time you change lenses.
    If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
    Except in exceptional live cell imaging where you understand that you are not being confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
  • Offset. Always use at 0 or 1.
    Other numbers are wrong.
  • Digital gain. The preset is 1. Leave it there.
  • Example of scan speed, averaging, filtering and noise click here.

Always save files as LSM or CZI. These files can be opened directly into inmage analysis software. These files retain instrument settings, channel integrity, bit depth, and spatial scale that may be necessary for image analysis.

If you save files as TIF or other formats, the inetgrity of of color channels may be lost and you will have no metadata regarding instrument settings and spatial scale.

 

Other Stuff

Example publications using this microscope:

  1. Bréart B, Ramos-Perez WD, Mendoza A, Salous AK, Gobert M, Huang Y, Adams RH, Lafaille JJ, Escalante-Alcalde D, Morris AJ, Schwab SR. Lipid phosphate phosphatase 3 enables efficient thymic egress. J Exp Med. 2011 Jun 6;208(6):1267-78. doi: 10.1084/jem.20102551. PMID: 21576386, PMCID: PMC3173249
  2. Chou WC, Takeo M, Rabbani P, Hu H, Lee W, Chung YR, Carucci J, Overbeek P, Ito M. Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling. Nature Medicine. 2013 Jul; 19(7): 10.1038/nm.3194 PMCID: PMC3859297, PMID: 23749232, DOI: 10.1038/nm.3194
  3. Fang V, Chaluvadi VS, Ramos-Perez WD, Mendoza A, Baeyens A, Rivera R, Chun J, Cammer M, Schwab SR. Gradients of the signaling lipid S1P in lymph nodes position natural killer cells and regulate their interferon-γ response. Nature Immunology. Nat Immunol. 2017 Jan;18(1):15-25. doi: 10.1038/ni.3619 PMID: 27841869
  4. Fooksman DR, Nussenzweig MC, Dustin ML. Myeloid cells limit production of antibody-secreting cells after immunization in the lymph node. J Immunol. 2014 Feb 1;192(3):1004-12. doi: 10.4049/jimmunol.1300977. Epub 2013 Dec 27.
  5. Fooksman DR, Schwickert TA, Victora GD, Dustin ML, Nussenzweig MC, Skokos D. Development and migration of plasma cells in the mouse lymph node. Immunity. 2010 Jul 23;33(1):118-27. doi: 10.1016/j.immuni.2010.06.015. Epub 2010 Jul 8. PMID: 20619695
  6. Hofmann H, Logue EC, Bloch N, Daddacha W, Polsky SB, Schultz ML, Kim B, Landau NR. The Vpx lentiviral accessory protein targets SAMHD1 for degradation in the nucleus. J Virol. 2012 Dec;86(23):12552-60. doi: 10.1128/JVI.01657-12. PMID: 22973040, PMCID: PMC3497686
  7. Hubbard-Lucey VM, Shono Y, Maurer K, West ML, Singer NV, Ziegler CG, Lezcano C, Motta AC, Schmid K, Levi SM, Murphy GF, Liu C, Winkler JD, Amaravadi RK, Rogler G, Dickinson AM, Holler E, van den Brink MR, Cadwell K. Autophagy gene Atg16L1 prevents lethal T cell alloreactivity mediated by dendritic cells. Immunity. 2014 Oct 16;41(4):579-91. doi: 10.1016/j.immuni.2014.09.011. Epub 2014 Oct 9.
  8. Jang JE, Hajdu CH, Liot C, Miller G, Dustin ML, Bar-Sagi D. Crosstalk between Regulatory T Cells and Tumor-Associated Dendritic Cells Negates Anti-tumor Immunity in Pancreatic Cancer. Cell Rep. 2017 Jul 18;20(3):558-571. doi: 10.1016/j.celrep.2017.06.062. PMID: 28723561.
  9. Liese J, Rooijakkers SH, van Strijp JA, Novick RP, Dustin ML. Intravital two-photon microscopy of host-pathogen interactions in a mouse model of Staphylococcus aureus skin abscess formation. Cell Microbiol. 2013 Jun;15(6):891-909. doi: 10.1111/cmi.12085.
  10. Logue EC, Taylor KT, Goff PH, Landau NR. The cargo-binding domain of transportin 3 is required for lentivirus nuclear import. J Virol. 2011 Dec;85(24):12950-61. doi: 10.1128/JVI.05384-11.PMID: 21976643, PMCID: PMC3233122
  11. Longman RS, Diehl GE, Victorio DA, Huh JR, Galan C, Miraldi ER, Swaminath A, Bonneau R, Scherl EJ, Littman DR. CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22. J Exp Med. 2014 Jul 14. pii: jem.20140678.
  12. Maurer K, Reyes-Robles T, Alonzo F 3rd, Durbin J, Torres VJ, Cadwell K. Autophagy Mediates Tolerance to Staphylococcus aureus Alpha-Toxin. Cell Host Microbe. 2015 Apr 8;17(4):429-40. doi: 10.1016/j.chom.2015.03.001. PMID: 25816775
  13. Mendoza A, Fang V, Chen C, Serasinghe M, Verma A, Muller J, Chaluvadi VS, Dustin ML, Hla T, Elemento O, Chipuk JE, Schwab SR. Lymphatic endothelial S1P promotes mitochondrial function and survival in naive T cells. Nature. 2017 May 24. doi: 10.1038/nature22352. PMID: 28538737
  14. Ménager MM, Littman DR. Actin Dynamics Regulates Dendritic Cell-Mediated Transfer of HIV-1 to T Cells. Cell. 2016 Jan 27. pii: S0092-8674(15)01700-6. doi: 10.1016/j.cell.2015.12.036.
  15. Rabbani P, Takeo M, Chou W, Myung P, Bosenberg M, Chin L, Taketo MM, Ito M. Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration. Cell. 2011 Jun 10;145(6):941-55. doi: 10.1016/j.cell.2011.05.004. PMID: 21663796, PMCID: PMC3962257
  16. Ramos-Perez, WD, Fang, V, Escalante-Alcalde, D, Cammer, M, Schwab, SR. A map of sphingosine 1-phosphate distribution in the spleen. Nature Immunology. 2015 Dec;16(12):1245-52. doi: 10.1038/ni.3296. PMID: 26502404
  17. Ruocco MG, Pilones KA, Kawashima N, Cammer M, Huang J, Babb JS, Liu M, Formenti SC, Dustin ML, Demaria S. Suppressing T cell motility induced by anti-CTLA-4 monotherapy improves antitumor effects. J Clin Invest. 2012 Oct 1;122(10):3718-30. doi: 10.1172/JCI61931. PMID: 22945631
  18. Takeo M, Chou WC, Sun Q, Lee W, Rabbani P, Loomis C, Taketo MM, Ito M. Wnt activation in nail epithelium couples nail growth to digit regeneration. Nature. 2013 Jul 11; 499(7457): 228-232 PMCID: PMC3936678, PMID: 23760480, DOI: 10.1038/nature12214
  19. Takeo M, Lee W, Rabbani P, Sun Q, Hu H, Lim CH, Manga P, Ito M. EdnrB Governs Regenerative Response of Melanocyte Stem Cells by Crosstalk with Wnt Signaling. Cell Rep. 2016 Apr 27. pii: S2211-1247(16)30405-3. doi: 10.1016/j.celrep.2016.04.006. PMID: 27134165
  20. Waite JC, Leiner I, Lauer P, Rae CS, Barbet G, Zheng H, Portnoy DA, Pamer EG, Dustin ML. Dynamic imaging of the effector immune response to listeria infection in vivo. PLoS Pathog. 2011 Mar;7(3):e1001326. doi: 10.1371/journal.ppat.1001326. PMID: 21455492
  21. Waite JC, Vardhana S, Shaw PJ, Jang JE, McCarl CA, Cameron TO, Feske S, Dustin ML. Interference with Ca(2+) release activated Ca(2+) (CRAC) channel function delays T-cell arrest in vivo. Eur J Immunol. 2013 Dec;43(12):3343-54. doi: 10.1002/eji.201243255. PMID: 23939929
  22. Williams CR, Dustin ML, Sauer JD. Inflammasome-mediated inhibition of Listeria monocytogenes-stimulated immunity is independent of myelomonocytic function. PLoS One. 2013 Dec 9;8(12):e83191. doi: 10.1371/journal.pone.0083191.


 


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