Microscopy Methods Reporting

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Here is a visual guide that will help you track your microscopy method reporting. We also provide a general template followed by two specific examples of widefield and confocal imaging. You will find instrument-specific information on our web pages but we also suggest contacting our staff to assist with your method writing.

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Please also remember to acknowledge our core facility, HiLIFE, **and** Biocenter Finland.

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**Acknowledgements example:** Imaging was performed at the Biomedicum Imaging Unit, University of Helsinki, with the support of the Helsinki Institute of Life Science (HiLIFE) and Biocenter Finland

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[[image:attach:Microscope_Methods_Layout_Figure_20210303_v1.png]]

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== General Methods Template ==

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Cells were **[__SAMPLE INFORMATION__]**. Imaging was performed using a [**__1. Microscope MAKE & MODEL__]**. The objective used was a **[__2. OBJECTIVE]__**. Live imaging was performed at **[LIVE IMAGING]**. Samples were imaged with [**__3. LIGHT SOURCE__**] with [**__4. FLUORESCENCE FILTERS__]**. Images were acquired [**__5. STAGE and 3D Capture__]** using a [**__6. CAMERA__**]

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== (% style="color: rgb(51,51,153);" %)Widefield example – Nikon Eclipse Ti2-E(%%) ==

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Cells were grown (fluorophores?) on No. 1.5 coverslips, fixed with 4% PFA and mounted on slides using Prolong Gold. Imaging was performed using a Nikon Eclipse Ti2-E widefield inverted microscope using Nikon NIS Elements 4.11 acquisition software. The objective used was a Nikon 63x Plan Apochromat 1.4 NA with Nikon oil immersion 1.518. Live imaging was performed with Okolab bold line heating system at 37°C, 20% O2 and 5% CO2 with images captured every 10 minutes for 24 hours. Samples were imaged with 475/28nm and 575/25nm excitation filters and 525/50nm and 600/30nm emission filters (Chroma). Images were acquired by taking a z stack of 30 slices with 300nm spacing using a Hamamatsu ORCA-Flash 4.0 v3 sCMOS camera with a pixel size of 6.5μm. Images were 16bit with pixel dimensions 2048×2048.

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== (% style="color: rgb(51,51,153);" %)Confocal example – Zeiss LSM880 with Airyscan(%%) ==

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Cells expressing GFP and RFP were grown on 35 mm Mattek dishes. Imaging was performed using a Zeiss LSM880 confocal with a Axio Observer.Z1 microscope using ZEN 2.3 SP1 black edition acquisition software. The objective used was a Zeiss C Plan-Apochromat 63x/1.4 Oil DIC M27. GFP was imaged with a 488 nm Argon laser and emission window at 500-550 nm while RFP was imaged with a 543 nm HeNe laser with emission window at 580-630nm. The pinhole was set to Airy 1 with bidirectional imaging and a pixel dwell time of 1.92µs. A GaAsp detector was used to record the images and no averaging was performed. Live imaging was performed at 37°C and 5% CO2 with image channels captured sequentially in frame mode every 10 minutes for 2 hours. 3D Images were acquired by taking a z stack of 30 slices with 300nm spacing with a pixel size of 100nm.

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Wiki source code of Microscopy Methods Reporting

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