Methods Examples

Brightfield Imaging with Large Imaging

Brightfield images were captured on an inverted Nikon TiE microscope (Nikon) run using NIS-Elements 5.21.03 (b1489_64bit) that was equipped with a fully automated Nikon stage, the Nikon Perfect Focus System, halogen illumination housing, and a 2.5X C-mount adaptor in front of the DS-Ri2 Color Camera (Nikon). Images were captured under Kohler illumination, using a Plan Apo 40x/0.95 NA Corr Objective (Nikon), pixel size 220nm, 30ms exposure, software white balance (red 1.07, Blue 3.12), Gain at 1.4x, a captured shading correction applied during imaging, and tile scanned using HCA/jobs to set coordinates with a 15% overlap, then stitched with NIS-elements.

---

CSU-W1 (before laser change in Feb 2024)

Excerpt from:

Calia, Giuliana P., Xinyue Chen, Binyamin Zuckerman, and Leor S. Weinberger. “Comparative Analysis between Single-Cell RNA-Seq and Single-Molecule RNA FISH Indicates That the Pyrimidine Nucleobase Idoxuridine (IdU) Globally Amplifies Transcriptional Noise.” bioRxiv, March 15, 2023, 2023.03.14.532632. https://doi.org/10.1101/2023.03.14.532632.

Images were taken on an inverted Nikon TiE microscope (Nikon) run using Micromanager 2.0 [NEEDS TO BE CITED] equipped with a CSU-W1 Spinning Disk with Borealis Upgrade (Yokogawa, Andor), ILE Laser launch with 4 laser lines (450/488/561/646nm, Andor), quad-band dichroic ZT405/488/561/647 (Chroma), emission filters for DAPI (ET447/60), GFP (ET525/50), RFP (ET607/36), and Cy5 (ET685/40) (Chroma), piezo XYZ stage (ASI), and Zyla 4.2 CMOS camera (Andor), using a Plan Apo VC 60x/1.4 Oil objective (Nikon). Approximately 10 XY locations were randomly selected for each condition. For each image, XY pixel size was 108nm/px, and a Z-step size of 250nm was used with over 60 image planes to fully cover the tissue.

---

CSU-W1 (before laser change in Feb 2024)

Excerpt from:

Touhara, Kouki K., Nathan D. Rossen, Fei Deng, Tifany Chu, Andrea M. Harrington, Sonia Garcia Caraballo, Mariana Brizuela, et al. “Crypt and Villus Enterochromaffin Cells Are Distinct Stress Sensors in the Gut.” bioRxiv, April 25, 2024. https://doi.org/10.1101/2024.02.06.579180.

Confocal images were captured on an inverted Nikon Ti microscope run using Micro Manager 2.0 Gamma, 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), ILE 4 line Laser Launch (405/488/561/640 nm; Andor). Images were taken using a Plan Apo λ 20x/0.75 using lasers 405, 488, and 561 nm and emission filters 447/60, 525/50, 607/36, for DAPI, GFP, and RFP, respectively. Maximum intensity projections were generated in Fiji v2.14.

---

Crest-V2 Spinning Disk

Excerpt from:

Zhong, Xiaofang, Qiongyu Li, Benjamin J Polacco, Trupti Patil, Aaron Marley, Helene Foussard, Prachi Khare, et al. “A Proximity Proteomics Pipeline with Improved Reproducibility and Throughput.” Molecular Systems Biology 20, no. 8 (August 2, 2024): 952–71. https://doi.org/10.1038/s44320-024-00049-2.

Images were generated using a Nikon Ti2-E microscope equipped with a Crest X-Light-V2 spinning disk confocal (Crest Optics), emission filters 438/24 (DAPI), 511/20 (GFP), 560/25 (RFP), and 685/40 (Cy5), and Celeste Light Engine excitation lasers 405/477/546/638 nm used respectively (Lumencor), Piezo stage (Mad City Labs), and a Prime 95B 25mm CMOS camera (Photometrics) using a Plan Apo VC 100x/1.4 Oil (Nikon). The data was captured with NIS-Elements software (v. 5.41.01 build 1709, Nikon) and processed in Fiji/ImageJ2 (Schindelin et al, 2012).

---

Crest-V2 Spinning Disk with FRAP

Adapted after a preprint.

Condensates were allowed to settle in the well for at least 1 hour before imaging with a Plan Apo VC 100x/1.4NA (Nikon) oil immersion objective on a Nikon Ti2 microscope equipped with a CREST-V2 LFOV Spinning Disk (Crest Optics), Celesta Light Engine (Lumencor), with excitation wavelengths 546nm or 647nm and emission filters FF01-595/31 or FF02-685/40 (Semrock) to image Alexa Fluor 555 or 647 respectively, a 405/488/561/640/750 dichroic (Crest), a Prime 95B 25mm sCMOS camera, run using NIS Elements 5.41.01 build 1709 (Nikon). Fluorescence recovery after photobleaching was achieved with an Opti-Microscan FRAP unit with a 405nm Reflect/430-800nm Trans Dual TIRF (FRAP) dichroic and 405nm laser for photobleaching. 20% laser power was used to bleach Alexa Fluor 555-labeled H1 and 15% laser power was used to bleach Alexa Fluor 647-labeled chromatin arrays.

---

CVRI Spinning Disk

Adapted after preprint.

Confocal images were taken using an inverted Nikon Ti microscope equipped with a CSU-X1 (Yokogawa) spinning disk confocal using a Plan Apo VC 60 × 1.4 NA (Nikon) oil objective, 4 line Vortan laser launch (405/488/561/647nm), with respective emission filters ET460/50m, ET525/50m, FF01-609/57, ET630/75m (Chroma, Semrock), an ASI XY stage with Z piezo, and a Prime 95 sCMOS camera (Photometrics).

---

CSU-22

Written for fixed slides.

A fully automated Nikon Ti equipped with a CSU-22 spinning disk confocal (Yokogawa), piezo stage (Mad City Labs), 4-line Coherent OBIS laser launch 405/488/561/640nm (Solamere), a quad dichroic 405/491/561/640 (Yokogawa), and corresponding emission filters ET460/50m, ET525/50m, ET610/60m, ET700/75m in a filter wheel controlled by a Lambda 10-3B (Sutter) for channels DAPI/GFP/RFP/Cy5 respectively. Images were captured using a Plan Apo VC 60x/1.4 Oil (Nikon) and a 1.7x relay lens in front of the Photometrics Evolve Delta EMCCD Camera (154nm/pixel).

---

OMX

Here was text used by Rose for methods: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0190530#sec003

Microscopy Acquisition and Analysis

SIM data was acquired on a DeltaVision OMX SR microscope (GE Healthcare) using a 60x/1.42 NA oil immersion PSF objective and three sCMOS cameras. Immersion oil with a refractive index of 1.514 was used to match with the refractive index of the sample. Z stacks of 9μm thickness were collected with 0.125μm step-sizes using 5 phases and 3 angles per image. Reconstructions were performed using SoftWorx 6.5.2 (GE Healthcare) with a 0.003 Wiener filter for the Alexa Fluor 488 channel and 0.002 Wiener filter for Alexa Fluor 555 as suggested by analysis using the ImageJ plugin SIMcheck22. Channel specific optical transfer functions were used for reconstructions. Alignment, averaging and calculation of the radial distribution were performed using custom python scripts previously described5. Monomeric CM2 data was fit with an additional central Gaussian to distinguish the central intensity from the ring distribution.

---

MicroManager Citations

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. doi:10.14440/jbm.2014.36

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

Page on citing Micromanager: https://micro-manager.org/Citing_Micro-Manager