Cell Culture Sample Prep Recommendations for the Tilt

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.

Tilt light sheet with 4 well sample holder
Photo of the Tilt light sheet illuminating a 4 well sample holder.

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.

Schematic of light approaching from right
On the Tilt, the light that creates the light sheet at your sample enters from the right. Ensure that the path the light travels is not obstructed in any way.

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.

Example of 4 well sample holder with lip
Be careful of 4 or 8 well sample holders that have a large lip around the bottom edge (red arrows). These can interfere with the 4 light beams making the light sheet on the Tilt.

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.

Screenshot of Ibidi u-slide 4 well #1.5 coverslips
Screenshot of Ibidi u-slide 4 well sample holder with selection for #1.5 coverslip highlighted in red box. When purchasing sample holders for imaging on the Tilt, ensure you are selecting the product with #1.5 coverslips. Screenshot captured here: https://ibidi.com/chambered-coverslips/37–slide-4-well.html

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.

Cartoon of high vs low plating density.
Plate your monolayers of cells at 30% or less. This will help prevent obstructions in the path of the light.

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.

Cartoon with 20% Optiprep added
For optimal imaging on the Tilt, add 20% Optiprep to your media.

Cell Culture Sample Prep: Key Take Homes

1) Use a sample holder that is flat on the side, free of imperfections and lips. Ibidi u-slide 4 well chamber slides or u-slide 8 well chamber slides work well.

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!

 

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