Compact confocal laser scanning microscope for 3D fluorescence imaging

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Technology abstract

Multifunctional microscope, which can be used both as a confocal scanning microscope (LCSM) with dual wavelength fluorescence imaging and as a differential imaging contrast (DIC) microscope. Very compact and low power consumption.

Individual DIC and LCSM microscopes are mostly used to image and analyse biological samples. Now a combined system is presented.

This company has extensive knowledge of space optics and space projects. The resulting microscope has advantages not covered in commercially available microscopes. Compactness and the merging of two microscopy methods being the main features. If this (device) is not enough, it shows the potential/expertise of the company in optical metrology.

- Sam Waes -

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Technology Description

This microscope was engineered by a Belgium company boasting a wide-ranging expertise in the field of vision and optical metrology.

The system is basically an inverted microscope using 2 parallel working imaging systems - DIC as well as LCSM - both using the same microscope objective. The total system consumes just 50W of power and is very compact with overall dimensions around 400 x 600 x 400mm. Note the compactness!

The DIC system uses a near infrared LED as its light source. This allows the light to be separated from the visible light used for the LCSM system. As a side benefit, IR light also penetrates the sample deeper.

A CMOS camera interprets the IR light and an image is created.

The LCSM system uses 2 laser excitation light sources working at 488nm and 630nm wavelength. The choice of these wavelengths was dictated by the best adaptation to current most popular fluorophore labels in cellular biology, as well as manufacturing limitations of dichroics and filters. 2 photomultiplier tubes, one for each wavelength, acquire the fluorescence signals and an image is generated. Axial scanning is also possible, so 3D images can be generated.

The sample holder is motorized in the x, y and z direction, with 0.1µm repeatability. It can move ±12.5mm lateral and ±2.5mm vertical. Removal and insertion of the samples is easy and user friendly. Sample holder and microscope objective is temperature controlled.

Software and hardware support the modes of operation: fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP) and fluorescence lifetime imaging (FLIM, time resolution 200ps).

This microscope could have potential applications in every field where DIC and/or LCSM microscopes are currently used, including:

  • 3D fluorescence imaging of biological samples
  • Analysis of planar silicon semiconductor processing
  • Study the effectiveness of pharmaceutical systems
  • Food structure analysis
  • Failure and fracture analysis

Innovations & Advantages

The main advantages of this microscope with respect to the most advanced confocal technology commercially available, are its very compact size and it having both a DIC and LCSM system on board without compromising functionality.

The DIC system can be used to easily find interesting zones and it can create images with high contrast. It also uses IR light which allows for deeper sample penetration. The LCSM is used for a more detailed analysis of the biological samples using fluorescence.

DIC and LCSM system work in parallel and use the same microscope objective, so there is no need to swap samples or move parts when switching between DIC or LCSM system. Sample holder and MO are temperature controlled.

The total power consumption of this microscope is very low.

Further Information

  • Motorized scan stage (0.1µm repeatability), ±12.5mm lateral movement, ±2.5 axial movement.
  • Total volume occupied by complete system: 53dm3

Sample holder and MO temperature controlled

Current and Potential Domains of Application

  • 3D fluorescence imaging of biological samples
  • Analysis of planar silicon semiconductor processing
  • Study the effectiveness of pharmaceutical systems
  • Food structure analysis
  • Failure and fracture analysis