Light Microscopy of Liposomes

• Plating volume
• Surface chemistry
• Settling times
• Specific examples
• Microscope and coverslip:
• Ibidi chamber slides
• Choosing an objective
• Fluorophore selection:
• Emission filters
• Wide field vs confocal
• What to report

After preparing liposomes it’s time to have a look at them. Here we’ll walk you through some considerations for preparing samples for imaging and things to consider at the microscope. Also included is a checklist of things to report when sharing your data.

Plating volume

Expect your liposome yields to vary. In general, diluting liposome suspensions by a factor 2-5x gives good density for imaging. Remember to dilute your samples in appropriate outer solution to ensure that system is osmolarity matched. The exact volume you might need will depend on your set up (see below for specific examples).

Surface chemistry

Liposomes are composed of lipids. When they come in contact with a surface there is the surface to destabilize the lipid membrane. Highly charged surface such as plasma treated glass might lead to liposome stability. Conversely, appropriate surface treatment can be used to immobilize liposomes on the surface. Future releases of Nucleus will support liposome immobilization.

Settling times

Depositing liposomes on the surface it will take some time for them to settle on the imaging surface. In most sample preps we recommend waiting approx. 30 mins to allow for liposomes to settle. Otherwise, it might be a challenge to focus on the sample for long term observation.

Specific examples

Here are a few specific recommendations for preparing your samples for imaging.

Microscope and coverslip:

The simplest method will be to use an untreated microscope slide (75mm x 25 mm) and a coverslip. We use microscope slides from VWR (catalog number 48311-703). The key will be to ensure sufficient spacing between the slide and coverslip to ensure that the samples are not compressed. This can be done using double sided tape - these can be configured to allow for spiking solutions. These ring stickers are particularly convenient. We use glass cover slips also from VWR (catalog number 16004-332, thickness 0.13-0.16 mm). Approximately 5 uL of reaction solution are sufficient to prepare a sample.

Ibidi chamber slides

For imaging multiple liposome preparations in parallel we use these 18-well coverslips from Ibidi (catalog number 50-305-825) with polymer (#1.5 grade). The open format of these slides make them more prone to evaporation and associated internal flows. 15 uL of reaction solution are sufficient to prepare a sample.

Choosing an objective

If using wide-field fluorescence microscopy we recommend that to cleanly resolve the liposome annulus and the interior cytosol requires working with a low working distance objective. Additionally the relatively low intensity signal means that objectives with high numerical aperture are preferable. If your objective has a collar correction, be sure to set it to match the thickness of the cover slip.

Fluorophore selection:

Nucleus currently supports liposomes labelled with rhodamine and cytosol labeled with eGFP. Future distributions will support a wider variety of fluorescent reporters. It is important to consider the emission spectra of your scope to ensure compatability.

Emission filters

To individually resolve reports on a given color channel it will be necessary to use appropriate emission filters.

Wide field vs confocal

Many of the wonderful images founded in publications are taken using confocal fluorescence microscopy. The use of a low WD and high NA objective approximates a confocal configuration. Future versions of Nucleus will offer guidelines for confocal microscopy.

What to report

When sharing microscopy data within the nucleus community we encourage reporting the following information:

[Expected release: ≥ v0.2.x]

Resources and References

Other Protocols

Papers

Considerations for reporting microscopy data [Nature Methods, 2021]

Resources

Great beginner microscopy tips from the Broad Institute [link]

Practical introduction to epi fluorescence microscopy [link]