Applications

Intrinsic Fluorescence Imaging

Application 1: Protein intrinsic Fluorescence for Crystal Detection or distinguishing Protein Crystals from Salt Crystals

Example 1: Protein crystals among ammonium sulfate crystals

Ammonium sulfate crystals illuminated with white light (left) and UV-light (right). The crystal structure of ammonium sulfate is orthorhombic and therefore it shows a distinct birefringence when illuminated with polarized white light. Whereas, when those crystals are illuminated with UV-light they become invisible, they completely merged with the background.

Example 2: Pure salt crystals:

 
Crystals of ammonium sulfate

Example 3: Crystal screening results:

A: Protein crystals (Diameter ~0.2 mm)
 
 
B: Protein crystals (Diameter ~0.1 mm)
 
 
C: Protein crystal in oily phase (Diameter ~0.3 mm)
 

 

Application 2: RNA and Aptamer Identidication applying the Nucleic Acid specific Dye "SYBR-GOLD":

Example 1: Distinguishing RNA from Salt:

In this example, an RNA-hexamer was crystallized with sodium cacodylate as precipitant. Later, under white light illumination two crystals (A, B) became visible in the drop (left). After addition of SYBER-GOLD to the drop and illumination with UV-light, crystal "A" could be identified as a salt crystal (sodium cacodylate) and crystal "B" as crystals of the RNA-hexamer. 

Example 2: Aptamer-RNA Complex Identification:

Identification of included RNA inside of an Apdamer: White light illumination showed a crystal after screening. In UV-light, the crystal showed intrinsic blue fluorescence and therefore it could be identified as proteinogenic. After addition of SYBR-GOLD to the drop and illumination again with UV-light, the crystal showed green fluorescence, indicating incorporated RNA in the crystal.

 

Trace Fluorescence Imaging

Trace Labeling Procedure

Example: Sample in green light

A10C-1

A11B-1

In situ Dynamic Light Scattering

In Situ DLS Applications

Dynamic Light Scattering

A versatile DLS-System for Cuvettes and NMR-Tubes

Examples for DLS-Analysis of Samples prior to NMR Measurements

Cryo EM Applications

Workflow for Cryo-EM Sample Selection

A) Condition Screening: After purification, a sample might not be in the desired aggregation state or a biological relevant complex of molecules has not formed yet. Some buffer conditions might support complex formation apart from its natural environment but often such conditions are unknown. One strategy to obtain biological complexes is the application of various conditions (e.g. sparse matrices) systematically to a sample by adding corresponding buffers. However, a manual pipetting procedure is a tedious task and errors are likely. In order to even the workflow, automated dispensing systems (like the Oryx8) are available enabling a time, material and manpower efficient way to apply such buffer matrices. Usually, standard microbatch plates and samples sealed under inert paraffin oil are used.

B) Sample Qualification: However such an approach results in many conditions (usually 96) exceeding grid holding capacities of most grid carriers by far. Furthermore, most of the applied conditions have a negative effect on the sample, leading to a very inefficient usage of a cryo-EM when all samples would be transferred to grids and investigated by cryo-EM. A key technology to select samples for subsequent cryo-EM analysis is in situ DLS (performed with the SpectroLight 600). It allows to identify suitable buffer conditions by particle size determination, non-invasively and directly in wells of a microbatch plate. Size and dispersity determination enables identification of the few conditions that stabilizes the desired macromolecular complex. The few good conditions can be selected for subsequently investigation for cryo-EM, resulting in a significant increase of positive hits on the grids.