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Using Mounting Frames for DLS Sample Preparation

Sample Preparation for Light Sheet Microscopy

Sample handling is usually an important topic in the context of Light Sheet Microscopy. The TCS SP8 DLS integrates Light Sheet technology into an inverted confocal platform and can hence make use of general principles concerning sample mounting and xy-stage functionality. Here, we describe a set of mounting frames that allow preparation of a higher number of samples, also in the context of potentially harmful mounting media like BABB (Benzyl Alcohol Benzyl Benzoate).

Image: 3D glomeruli in a portion of an ECi-cleared kidney scanned by Light Sheet Microscopy. Courtesy of Prof. Norbert Gretz, Medical Faculty Mannheim, University of Heidelberg [1].

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Sample Holders

The set of sample holders described here has been designed for Digital LightSheet (DLS) imaging, but is suitable for imaging with conventional upright and inverted microscopes as well.

Introduction

For imaging with the SP8 DLS module, the samples are placed between the TwinFlect mirrors.
The travel range in the x-direction is limited by the distance of the used TwinFlect mirror (2.5 mm, 5 mm, or 7.8 mm). The maximum travel range is approximately half of the distance between the mirrors.

In the y-direction, the shape and size of the sample holder, for example a petri dish, determines the travel range.

With standard 35 mm petri dishes (20 mm diameter of the glass bottom cover slip), only a small movement is possible in y-direction without contacting the edges of the dish:

Especially with the sample holder for rectangular 24 x 50 mm cover slips, the y-movement is increased enabling the use of larger/longer samples.  Also, for multi-position experiments, the number of samples imaged in one experiment can be scaled up.

Materials and Methods

Mounting Frame Set (#158007063) consists of one sample holder for 24 x 50 mm glass cover slip and a second sample holder for round cover slips with a diameter of 30 mm. Please note that the glass cover slips are not included.

The sample holder for 24 x 50 mm cover slips is only compatible with the following z-Galvo stage inserts:

  • 158004118 Rotatable Insert Inverse and
  • 158004119 Insert Universal.

Sealing the Sample Holder/Attaching a Glass Cover Slip

For imaging in aqueous solutions, a glass cover slip can be attached using silicone (e.g. Elastosil® 43, Wacker) or the two-component twinsil®.

These holders are also suited for imaging in more aggressive solutions like benzyl alcohol/ benzyl benzoate (BABB). Because BABB can dissolve many materials, it is necessary to use a special type of silicone for sealing the vessels, e.g., Elastosil® E41 (Wacker) (personal communication with Jürgen Pinnecker, Rudolf Virchow Zentrum University of Würzburg, Germany).

  1. Apply silicone/Twinsil to the inner edge of the recess at the bottom of the sample holder.
  2. Add a glass cover slip and press it lightly to ensure that it is sitting evenly for a good seal at all points.
  3. To check the chamber seal, add some distilled water to the sample holder and see if leaks occur.

You can reuse the sample holders with the glass cover slips attached. It is not necessary to replace the cover slips after each session. Cleaning with alcohol is possible.

Mounting Your Samples

Once the mounting frame with respective glass cover slip is assembled, you can proceed with different sample mounting options:

Mounting based on agarose

  1. Fill the surface of the cover slip with a thin layer of agarose ( 0.5 – 1 mm) and wait for it to polymerize, so that it creates a spacer for your samples
  2. Add your samples, suspended in liquid agarose (low melting agarose for living samples), on top of this layer
  3. Align your samples (ideally in a line) for imaging
  4. Remove excessive agarose from the sides to create space for the mirrors.

U-shaped glass capillaries

Follow the protocol as described in the Science Lab article:

DLS Sample Preparation: Using U-Shaped Glass Capillaries for Sample Mounting.

Super Glue for Mounting of cleared tissue

Larger (clarified) samples, when glued directly onto the glass cover slip, can be used with the sample holders.

Customer Application DLS Preparation

Visualization of 3D glomeruli in a portion of an ECi-cleared kidney scanned by Light Sheet Microscopy. Courtesy of Prof. Norbert Gretz, Medical Faculty Mannheim, University of Heidelberg