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.
<aside> 📌 Sample prep
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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).
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.
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.
Here are a few specific recommendations for preparing your samples for imaging.
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.
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.
<aside> 📌 Epi-fluorescence microscopy
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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.