• get training from George on how to do the steps below (I am deliberately not including pictures here - these steps are the brightfield microscope equivalent of 'learning to walk', or more mechanically, learning to ride a bicycle - should become automatic for you.

1. turn on Zeiss.A1 microscope power, light path to eyes, transmitted light shutter open, condenser turret to "H" (German for Hole? ... not Ph# or DIC), filter cube turret to empty position, turn up the lamp voltage to be comfortable to your eyes (and white light, not orange). I recommend start with the 10x objective lens.
2. Focus your specimen (i.e. H&E slide) with the microscope focus (left and right side of base).
3. Shrink the condenser light path field aperture diaphragm all the way. If this is an in focus octagon, great ... if not, use the condenser focus knob to bring it to focus.
4. Adjust the condenser numerical aperture (the 'dial' on the left side of the condenser) to that the illuminated area (conter of the octagon) is (almost) full brightness, while the aperture diaphragm is black (or nearly black).
5. Open the field aperture diaphragm enough to get it out of the way ... I like to park it just inside the eyepiece field of view (which is way outside the camera field of view).
6. In Olympus CellSens software, use manual exposure time (ex. 40 milliseconds), to get to 'reasonable brightness', while not saturating the empty (bright) areas. 
7. If you change objective lenses: ideally re-do for each objective lens. Typically one objective lens images will be most critical for your experiment, so simplest to optimize the Numerical Aperture Diahragm for that lens, and not mess with it during your session (the other lenses will be not-quite-perfect, but the premise here is one lens is the priority).
8. Color balancing: CellSens software has a control for this (icon).
9. I note there is plenty of free (or almost free) software to stitch. For example, Microsoft ICE (Image Composite Editor). Fiji ImageJ and Adobe Photoshop can also stich.

• if already acquired, see my 2005 article, color balancing with Adobe Photoshop, http://www.home.earthlink.net/~geomcnamara/McNamara2005JoH28n2pp81-88.pdf

TBP 400 + 495 + 570

20200629Mon: widefield brightfield microscopy vs widefield fluorescence microscopy vs confocal fluorescence microscope resulution

Ideally, in 2020, no one should need to sit down at a brightfield transmitted light microscope and acquire histology or immunohistochemistry images. The JHU pathology core that has a high resolution transmitted light digital slide scanner, aka whole slide imager (DSI, WSI),  to image your slides as a service without training (and with reasonable pricing and turn-around time). For  one example, see https://tmalab.jhmi.edu/scanning.html (I do not have an exhaustive list of core facilities or scanners on campus). If you would like to donate a working good quality slide scanner, ideally brightfield and fluorescence (ex. Olympus VS-200 or Leica Biosystems/Aperio scanner), we would love to have it (require approval of image core Director Bin Wu and NIDDK P30 Center Director Mark Donowitz). Vendors: we are happy to host demos, currently (6/2020) no fundign to uy a scanner. 

Our core does not have any microscopes set up for “high resolution” transmitted light (brightfield) image acquisition. We might be able to get you “good enough” image quality – will need to wait until phase 2 re-opening (I could acquire a few images for you so you can see what we can do with our current equipment). Please note: “high resolution” implies working at or near the optical limit of the wavelength of light and high quality optics. In practice, this usually not needed for immunohistochemistry (ESR1 detected by DAB “brown” IHC using HRP). You have access to the Leica SP8 confocal microscope, whose fluorescence images can operate at “full resolution”. I also note that tyramide signal amplification (TSA) with, say, Alexa Fluor 488 tyramide, can be used as a direct replacement for DAB as HRP substrates (co-substrate being H2O2).

The usual microscope for this is our Zeiss AxioImager A1 inverted microscope with DP80 color & monochrome camera (dual CCD).

http://confocal.jhu.edu/current-equipment/zeiss-invert-scope-w-olympus-camera  (this web page, so did not enable link).

It has a long working distance condenser, whose numerical aperture (NA) is 0.35, so will not produce “very high resolution” images”. This is because for brightfield (aka “transmitted”) light, the resolution (dxy) is based on this equation (I’m using “green light 500 nm and 1.3NA of the oil immersion objective lens on our microscope):

                       1.22 * wavelength                      1.22 * 500nm

Dxy =     -------------------------------------- =          -------------------  = 370 nm = 0.370 um

               NAcondenser + NAobjective               0.35 + 1.3

--> the main reason for long working distance (LWD) condenseris to enable this and similar microscopes to be used for SBS plates, T25 and T75 flasks, and petri dishes. We have confocal micoscopes (and FISHscope) for high resolution imaging, usually by fluorescence. If someone needs high resolution, I recommend microscope slide or 35 mm imaging dish (ex. mattek.com, https://www.mattek.com/store-category/35mm-dishes    (the glass bottom is the coverglass, no need to seal the specimen ... which enables iterative rounds of label-image-"de-label", if desired). As noted above, our FISHscope has a 0.55NA long working distance condenser, though practical users are unlikely to see benefits of FISHscope for transmitted light. 

For comparison, same objective lens in epi-illumination fluorescence:

                       1.22 * wavelength                     1.22 * 500nm

Dxy =     -------------------------------------- =         -------------------  = 235 nm = 0.235 um

               NAcondenser + NAobjective               1.3  + 1.3

Our confocal microscopes (Leica SP8 and Olympus FV3000RS) have similar NA objective lenses, for Leica SP8 (plan apo 60x / 1.4NA, and replacing the 1.22 (which is 0.61+0.61) in numerator with confocal pinhole 1.0 Airy Units value of 0.51, and simplifying denominator since same lens in and out:

                                                        0.51 * wavelength               0.51 * 500nm

Dxy (confocal 1.0 Airy unit) =     -------------------------------- =      -------------------  = 182 nm = 0.182 um

                                                                    NA                                1.4

Jeff Reece (NIH) wrote (10/2019 confocal listserv, see below) wrote  “When there is plenty of signal (most fixed specimens), I recommend reducing pinhole for better resolution, with a practical limit of roughly 0.5AU.  The sweet spot is usually 0.6-0.7AU.” … I am not a fan of ranges, so I simplify this to the “confocal sweetest spot” 0.666 Airy Unit, which can result in ~10% improvement in resolution (0.46 Airy Unit), and note that Brilliant Violet 421 (BV421) emission maximum is 421 nm (in practice, would use 415-455 nm so will use 420nm below),

                                                              0.46 * wavelength               0.46 * 420nm

Dxy (confocal 0.666 Airy unit) =     --------------------------------- =      -------------------  = 138 nm = 0.138 um

                                                                         NA                                 1.4

I note that all the dxy are resolution distances, and that pixel size needs to be smaller to enable Nyquist sampling (see Wikipedia for who Nyquist is and why sample); for a sine wave, one needs to sample 2.2 data points per wave; I recommend 3x sampling for biological images, that is,

 pixel size = Dxy / 3.

For point scanning confocal microscopes like our Leica SP8 and Olympus FV3000RS, pixel size can be optimized by image dimensions (ex. 4096 x 4096 pixels) and zoom, for any given objective lens, and the wavelength of fluorescence emission. Slightly higher resolution can be obtained using reflected light (405 nm laser, 405 nm detection), for example, Cox-Golgi ‘stained’ neurons (see Sivaguru 2019 J Microscopy, for example, with small confocal pinhole).