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OlympusBX43_Microscope_general.md

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Using the Olympus BX43 Microscope

These are some general notes for using the Olympus BX 43 "upright" microscope in room 413LSB.

General Microscopy links (primers and such)

  1. Microscopy Resource center. From very basic to very detailed. You could spend weeks on this site! Also these sites here and here
  2. Basics of light microscopy guide
  3. Guide to image acquisition

##General notes This scope was purchased and optimized for two purposes.

  1. Getting very high quality images at relatively low magnification (2X and 4X objectives). In particular for adult morphological structures of Drosophila (wings and legs). This system has plan apochromatic objectives (to maximize resolution and flatness of field).
  2. Getting high quality images of the developing structures (imaginal discs) for wings, legs, etc. We also have the plan apochromatic objectives for these. At higher magnification parallaxes increases (less flatness of field).

  • As such, this scope has the highest quality 2X, 4X, 10X, 20X and 40X objectives (PLAN- apochromatic, see here and here for more details. In particular these objectives had the highest numerical aperature I could afford, which (in combination with a good camera) should provide excellent detail of the images, including for counting wing hairs (cells) on the wing. These are also corrected for flatness of image (PLAN). However we still have to empirically test how well this flatness correction works (parallax distortions near edge).

  • This microscope does not do Nomarksi (Differential Interference Contrast), or phase contrast. If you need this for your project, let me know as there are several other excellent microscopes in the building with this capability.

  • The current filters for fluorescent microscopy are for DAPI, Texas Red (possibly including Alexa594, DyLight594?), Fluorescien/FITC (Alexa 488, DyLight488), TRITC, GFP and mCherry. More information on each of the filter sets is below.

  • As is commonly the case, the higher magnification lenses (10X, 20X and 40X) need to be used with the condensor in, so you will need to set up the microscope under Kohler Illumination. This takes less than 1 minute, and is worth checking each time when you are using the scope.

Some specifics about the filters we have:

  • DAPI filter from Semrock (OSF-D50LPBX3, DAPI-50LP-A-OFF; 377/50X, BS409, 409M).

    • Highly compatible with: Alexa Fluor 350, Alexa Fluor 405, Fluoro-Gold, LysoSensor Blue, Marina Blue, Pacific Blue).
    • Compatible with: DyLight 350,DyLight 405, Hoechst 33258, Hoechst 33342 (works very well), Hoechst 34580).
    • See compatability with other fluorophores here.

  • FITC filter from Semrock (OSF3-FITC-A 1 FITC-A-BASIC-OFF;BAND PASS FILTER SET FITC, BODIPY, ALEXA488).

    • Highly compatible with: Alexa Fluor 488, Fluo-4, FITC (Fluorescein).
    • Compatible with: Calcein, GFP, Rhodamine 110, Rhodamine Green, super glow GFP, syber GOLD, syber green I, turbo GFP, wild type GFP
    • Compatability with other fluorophores is here.

  • Texas Red from Semrock (OSF3-TXRED-A 1 TXRED-A-BASIC-OFF;BAND PASS FILTER SET TXRED, ALEXA 568).

    • Highly compatible with:5-ROX,Alexa Fluor 568, mCherry, mRFP1 (but not mRFP).
    • Compatabile with:DY-590, Propidium iodide
    • Other compatible fluorophores here.

  • TRITC from Semrock (OSF3-TRITC-A 1 TRITC-A-BASIC-OFF;BAND PASS FILTER SET TRITC, ALEXA 546, DII)

    • Highly Compatabile with dTomato
    • Compatabile with Alexa Fluor® 546, mRuby, Propidium Iodide (PI), TRITC (Tetramethylrhodamine) - "reddish" appearance

  • mCherry from Semrock (OSF-MCHLEDBX3)

    • Highly compatible with: Alexa Fluor® 594, DyLight 594, Alexa Fluor® 568, 5-ROX (carboxy-X-rhodamine), ATTO 590, BOBO™-3, BODIPY TR-X phallacidin, Calcium Crimson, CellTracker Red CMTPX, FluoSpheres Red fluorescent microspheres, HCS LipidTOX Red neutral lipid stain, HCS LipidTOX Red phospholipidosis, HiLyte Fluor™ 594, KFP-Red, mCherry, MitoTracker™ Red, mPlum, mRFP1, ReAsH-CCPGCC, Red 580, Texas Red dextran, Texas Red DHPE, Texas Red-X®, TurboFP635(Katushka), X-Rhod-1 Indicator, SNARF (carboxy) 514 Excitation pH9, SNARF-1 488nm (ph 9.0), SNARF-1 514nm (ph 9.0)
    • Other compatable fluorophores here.

  • GFP from Semrock (OSF3-GFP-3035D)

    • Highly comptabile with: GFP (EGFP), Cy2™, DiO, sgGFP™ (super glow GFP), wtGFP (wild type GFP, non-UV excitation), YO-PRO-1, YOYO-1
    • Other compatible fluorophores here

Using the microscope for capturing wing images just for splining

Using the microscope for capturing wing images for splining and/or wing hair counts

Using the microscope for brightfield

Other notes