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Agarose pad or lectin (Concanavalin A) methods for yeast live-cell imaging

Agarose pad or Concanavalin A (lectin) methods to imobilize and image live yeast cells

1. Imaging yeast cells using an agarose pad

1.1. Prepare your thin agar pad

To get the full efficiency of our CherryTemp temperature controller and ensure the accurate control we provide, samples (cells/embryos) must be placed close to the thermalization chip (<500µm).

Following this protocole, you will prepare thin agar pads perfectly suitable with our CherryTemp chip holders and inserts.

  1. Place 3 slides next to each others and stick standard lab tape lengthwize on the two external slides (Fig. 1).
  2. Drop about 50-100µl of melted agarose on the middle slide (Fig. 2).
  3. Cover on top with a clean slide, perpendicular to the 3 others (Fig. 3); apply gentle pressure (Fig. 4).

agar-pad-slide-microscopy-temperature-control-fig1b

Figure 1: Align 3 slides and stick lab tape (red) on the 2 external ones.

Figure 2: Drop melted agarose on the middle one.

Figure 3: Top the agarose with another clean slide, perpendicularly.

Figure 4: Apply gentle pressure and allow agarose to solidify.

1.2. Mounting cells on an agarose pad
  1. Once the agarose is cooled and when ready to image, twist slides relative to each other to expose the pad (Fig. 5).
  2. Place the CherryTemp chip upside down (engravements down, cover slip and liquid microfluidic up) and transfer the pad on it.
  3. Add the cells in a 10-15µl drop medium.
  4. Top with a 170µm cover slip (or a 140µm for SR-microscopy).
  5. UPRIGHT SCOPE – Gently clamp the system into our chip holder and go to your upright microscope (see section 3.).
  6. INVERTED SCOPE – Turn the system upside-down, place it in the provided insert and gently clamp (see section 3.).

Figure 5: Gently remove the top slide.

Figure 6: Transfer the pad onto our chip. It must be centered on the thermalizing pattern.

Figure 7: Drop the embryos on the pad.

Figure 8: Top with a high-resolution cover slip.

1.3. Place the mounting system on either an upright or an inverted configuration
  1. Gently clamp the system into our chip holder and go to your upright microscope (Fig. 9)
  2. Turn the system upside-down, place it in the provided insert, gently clamp and instal on your inverted stage (Fig. 10)

Figure 9: Use a dedicated chip holder.
(from bottom to top: lower holder part, chip manifold, thermalizing pattern, closing cover slip, agarose pad, cells, top cover slip, top holder parts)

Upright configuration2

CherryTemp mounting system in an UPRIGHT configuration

Figure 10: Use customized inserts to maintain the system.
(from bottom to top: chip insert, cover slip, spacer, yeast cells, closing cover slip, thermalizing patternchip manifold)

Inverted configuration2

CherryTemp mounting system in an INVERTED configuration

2. Imaging yeast cells with a lectin (ConA) coating

2.1. Prepare your Con A functionalized cells

Figure 11: Drop Concanavalin A (Con A lectin) on a cover slip.

Figure 12: Allow the liquid to evaporate under the hood.

Figure 13: Place the functionalized cover slip in our chip insert.

Figure 14: Top with our dedicated spacer.

Figure 15: Drop your cells (10-15µl) in the spacer’s hole.
Caution: medium must not touch the sides.

Figure 16: Top with our microfluidic chip.



2.2. Place the mounting system on either an upright or an inverted configuration
  1. Gently clamp the system into our chip insert and go to your inverted microscope (Fig. 17)
  2. Turn the system upside-down, place it in the provided chip holder, and instal on your upright stage (Fig. 18)

Figure 17: Use customized inserts to maintain the system.
(from bottom to top: chip insert, cover slip, spacer,  yeast cells, closing cover slip, thermalizing patternchip manifold)

Inverted configuration2

CherryTemp mounting system in an INVERTED configuration

Figure 18: Use our dedicated upright chip holder.
(from bottom to top: lower holder part, chip manifold, thermalizing pattern, closing cover slip, spacer, yeast cells, top cover slip, top holder parts)

Upright configuration2

CherryTemp mounting system in an UPRIGHT configuration

Methods adapted from P. Tran lab (Institut Curie) and from our user’s feedback.

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