Examples of Microscope Methods

Set up is given. You can use these as a base and switch out specific components (i.e. the lenses, channels used, DIC, etc.)

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Examples for 6D Microscope

See 6D microscope hardware specifications here for more details

Set up: Fluorescent and Brightfield imaging using a 10X lens

Fluorescent widefield images were captured on an inverted Nikon TI microscope run with NIS-Elements 5.20.21 (Nikon) and equipped with an automated stage and stage up kit, using a DS-Qi2 CMOS Camera (Nikon), a Plan Apo 10x/0.45 lens, Lambda LS lamp with filter wheel and shutter (Sutter), Lambda 10-3 controller and emission wheel (Sutter), using a 5-band dichroic FF408/504/581/667/762 (DA/FI/TR/Cy5/Cy7-5X5M-B-000, Semrock) in the upper turret, with excitation filters 387/11x, 485/20x, 560/25x, 650/13x (Semrock) in the Lambda LS lamp and emission filters 440/40m, 525/30m, 607/36m, 684/24m (Semrock) in the lower turret position for DAPI, GFP, Cy3, and Cy5, respectively. Color brightfield images were captured on the same system using a DS-Ri2 CMOS Camera (Nikon).

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Examples for CSU-W1

See CSU-W1/Highspeed hardware specifications here for more details.

Set up: CSU-W1, sCMOS Camera, 100X lens, 3 channel images — Red, Green, Blue

Confocal images were captured on an inverted Nikon Ti microscope run using Micro Manager 2.0 Gamma [CITATION BELOW], equipped with a Zyla 4.2 CMOS camera (Andor), piezo XYZ stage (ASI), CSU-W1 Spinning Disk with Borealis upgrade (Yokogawa/Andor), Spectra-X (Lumencor), CSU-W1 Penta Dichroic 405/488/561/640/755, ILE 4 line solid-state Laser Launch (405/488/561/640nm; Andor). Images were taken using a Plan Apo VC 100x/1.4 Oil (DIC N2 / 100X I; Nikon) using solid-state lasers 405, 488, and 561nm and emission filters 447/60, 525/50, 607/36 (Semrock), for DAPI, GFP, and RFP, respectively.

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Examples for CREST/C2

See CREST/C2 hardware specifications here for more details

Set up: CREST, 100X

Images were generated using a Nikon Ti2-E microscope equipped with a Crest X-Light-V2 spinning disk confocal (Crest Optics), Celeste Light Engine (Lumencor), Piezo stage (Mad City Labs), and a Prime 95B 25mm CMOS camera (Photometrics) using a Plan Apo VC 100×/1.4 Oil (Nikon). Five channel images were captured using a penta dichroic 405/488/561/640/750 (Nikon), solid-state lasers 405nm, 477nm, 546nm, 638nm and 749nm and emission filters FF01-438/24, FF01-511/20, FF01-560/25, FF01-685/40 (Semrock), Nikon Multi-band, for DAPI, Alexa 488, Alexa 568, Alexa 647, and Alexa 750 respectively. The data was captured with NIS-Elements software (v. 5.41.01 build 1709, Nikon) and processed with Fiji/ImageJ2 [CITATION FOR FIJI].

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MicroManager Citations

Arthur D Edelstein, Mark A Tsuchida, Nenad Amodaj, Henry Pinkard, Ronald D Vale, and Nico Stuurman (2014), Advanced methods of microscope control using μManager software. Journal of Biological Methods 2014 1(2):e11

Arthur Edelstein, Nenad Amodaj, Karl Hoover, Ron Vale, and Nico Stuurman (2010), Computer Control of Microscopes Using μManager. Current Protocols in Molecular Biology 14.20.1-14.20.17

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ImageJ / Fiji Citations

Please visit to double check for variants of ImageJ and Fiji: https://imagej.net/contribute/citing

ImageJ Citation

Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9(7), 671–675. doi:10.1038/nmeth.2089

Fiji Citation

Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., … Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682. doi:10.1038/nmeth.2019

ImageJ2 Citation

Rueden, C. T., Schindelin, J., Hiner, M. C., DeZonia, B. E., Walter, A. E., Arena, E. T., & Eliceiri, K. W. (2017). ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics, 18(1). doi:10.1186/s12859-017-1934-z