Time to celebrate cell culture light sheet imagers! Cell culture sample prep is a piece of cake on the Mizar Tilt Light Sheet! Read on to learn how to prepare tissue culture cell samples for maximum light sheet success on the Tilt.
Cell Culture Sample Prep: Something to Keep in Mind
When preparing your tissue culture cells for imaging on the Tilt Light Sheet it is important to keep in mind that the excitation light is entering your sample from the right. This is different than what you are used to with other modes of imaging. With widefield, confocal and TIRF imaging on inverted stands, the excitation light enters your sample from below and then the emitted light (fluorescence) is also collected from below. This is not the case with many forms of light sheet microscopy and why you will hear people saying that excitation and detection objectives are decoupled.
On the Tilt, the light sheet illuminates your sample from the right and then is collected by the detection objective below. This means that it is important to keep the path of the light unobstructed. Objects in the path of the light that absorb or scatter light can cause artifacts in your image.
Recommended Sample Holder: Ibidi u-slide 4 or 8 Well Chamber Slides
The side of your sample holder needs to be flat (sorry, no round 35mm imaging dishes here) and free of imperfections (yes, this includes fingerprints). We recommend using Ibidi u-slide 4 well chamber slides (cat no 80426) or u-slide 8 well chamber slides (cat no 80826). Other similar sample holders can work as well, but be careful of a lip around the bottom edge. This lip can completely prevent focusing of the light beams.
Remember the Tilt has 4 separate beams of light that converge at the sample to form a long skinny light sheet. All 4 of the beams have to pass through the same amount of material. If one or more has to pass through more or less plastic, glass, air or media than the others this can affect the light sheet. Need to review how the light sheet is created on the Tilt? Check out our blog post on this here.
Use #1.5 coverslips
When companies manufacture objectives they have to make an assumption about the thickness of the coverslip the end user will be using. The coverslip is part of the optical path and must be taken into account when correcting defects in the objective. Manufacturers always assume the end user is using a #1.5 coverslip.
A #1.5 coverslip is 170 microns thick. For the best imaging results and quality always ensure that you are using a #1.5 coverslip.
Plate at Low Density
Remember that anything in the path of the light beams entering the sample to form a light sheet can result in stripe artifacts. This includes fingerprints on your sample holder, debris and other cells in your sample. If your cell type allows for it, plate at 30% confluency or less.
Use 20% Optiprep
Optiprep or Iodixanol is a chemical that helps bring the refractive index of the media closer to what is found in your cells. Check out this paper from Boothe et al., 2017. We love this stuff. It helps focus the light sheet on your sample, helps minimize lensing artifacts and is safe for live cell imaging.
Optiprep can be purchased from Stemcell Technologies (catalog #07820) and comes as a 60% solution diluted in water.
Cell Culture Sample Prep: Key Take Homes
2) Always use #1.5 coverslips.
3) Plate at 30% confluency or less.
4) Add 20% Optiprep to your media.
Want an infographic of this to take with you on the microscope? Download here: Mizar Tilt sample prep tissue culture cells!