Cryo Micro Station 3.1

Low-temperature measuring cell, fluorescence microscope expansion module

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Fig. 1: CMS concept outline

Functionality

The Cryo Micro Station (CMS) 3.1 provides fluorescence microscopy professionals with an extraordinary measuring cell that enables samples for analysis to be cooled in a vacuum to temperatures as low as 20K, so that the fluorescence yield of ‘molecules of interest’ is increased. The measuring cell can hold a Chamber Slide with up to 8 samples. The cooled samples can be examined in reproducible manner through a quartz glass inspection window (diameter 10 mm / thickness 0.8 mm). The XY drive is mounted inside the cooled measuring cell and can be used for scanning a sample area of 50 x 25 mm (standard slide work area). It can be operated optionally with a joystick or by operator input.

The internal memory function of the µm-drive units allows memory function for 10 different measurement points, which can be revisited as necessary for further measurements. In order to guarantee confocal recordings, the measuring cell is slaved vertically to the z-lift of the microscope stage.

The CMS 3.1 has been constructed so that it can be coupled to the most widely used standard microscope models from Leica, Zeiss etc..

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Fig. 2: CMS pictorial schematic

The sample for investigation is spread on a Chamberslide (CS) (25 mm x 76 mm) with up to 8 chambers, and the slide is then place in the measuring cell. The cover, with a circular quartz glass inspection port, is sealed. After the sample has been positioned in the measuring cell (MC), the CMS 3.1 is advanced by a roller mechanism along the centreline with the glass inspection port in the cover until it is beneath the microscope beam path. The vacuum chamber (VC) is evacuated to by a vacuum pumping station until an insulation vacuum (high vacuum) is created. A closed helium circuit, consisting of a compressor, flexlines and cold head generates cold, which is transported to the sample holder via a cold finger (CF) which extends into the measuring cell. With this system, a temperature of 25 K at the sample is reached within 100 minutes. A positioning system can be used to travel to all positions (x-/y direction) on the Chamberslide in the cooled state. And the desired position can be input at the control computer via the user interface, or approached directly with a joystick. Focussing on the sample in the z-direction is carried out by adjusting the microscope stage (MS). A distance sensor adjusts the position of the measuring cell according to the movement of the stage.

Applications in marker-free analysis

Fluorescence microscopy analyses are widely used to detect molecules in cells or tissue. For this, the cell components or molecules for analysis are usually market with fluorescing dyes. In order to do this, certain modifications have to made to the cells, such as fixing and permeabilisation or microinjection, or changes must be made to the molecules, such as direct coupling of dyes or expression of fluorescing fusion proteins. However, such changes to the sample are not acceptable for all scientific investigations, so in recent years there has been a pronounce trend towards analytical procedures that do not use markers.

The Nanoscopix Cryo Micro Station (CMS) offers a way to perform analyses either entirely without using markers or with significantly lower quantities of markers. Simply cooling the microscopic sample reduces the influence of a number of different fluorescence quenchers, and so enhances the expression of specific auto-fluorescences of the molecules.

Thus it becomes possible to trace the path of a substance inside the cell, for example (e.g. the path of medication to the site of action). Usually, these molecules must be suitably marked. But such marking also alters many important chemical and physical parameters of the substance or cell. The natural environment of the molecule and/or its original path is modified (e.g., marked medication can no longer penetrate a cell). Therefore, a great deal of time must be spent selecting a suitable marker, and sometime this proves to be impossible. With the aid of the CMS, we were able to analyse a target molecule with the aid of its specific auto-fluorescence, so we could dispense with the marking. (Tondera et al., 2013). //Tondera, C., Laube, M., Wimmer, C., Kniess, T., Mosch, B., Großmann, K., Pietzsch, J. (2013) Visualization of cyclooxygenase-2 using a 2,3-diarylsubstituted indole-based inhibitor and confocal laser induced cryofluorescence microscopy at 20 K in melanoma cells in vitro. Biochemical and Biophysical Research Communications 430, 301–306//

General advantages for applications in cellular biology

Amplification of specific fluorescence

One the most frequently encountered problems in fluorescence microscopy is that the fluorescence signal is not strong enough.

With the CMS, the specific fluorescence of the target molecule can be supported, meaning that lower marker concentrations can be detected (see Fig. 3)

Fig. 3: Murine melanoma cells (B16 F10), stained with 5-(hexadecanoylamino)fluorescein (membrane marker λexc 488 nm) and DAPI (DNA marker λexc 351 nm) at a sample temperature of 20 °C (RT) and 22 K with the addition of various concentrations of marker dyes: top) 1,5 µM 5-(hexadecanoylamino)fluorescein / 30 nM Dapi and bottom) 150 nM 5-(hexadecanoylamino)fluorescein – 3 nM Dapi. All settings on the microscope except the temperature conditions and the digital zoom factor were kept constant.

Fig. 3: Murine melanoma cells (B16 F10), stained with 5-(hexadecanoylamino)fluorescein (membrane marker λexc 488 nm) and DAPI (DNA marker λexc 351 nm) at a sample temperature of 20 °C (RT) and 22 K with the addition of various concentrations of marker dyes: top) 1,5 µM 5-(hexadecanoylamino)fluorescein / 30 nM Dapi and bottom) 150 nM 5-(hexadecanoylamino)fluorescein – 3 nM Dapi. All settings on the microscope except the temperature conditions and the digital zoom factor were kept constant.

Reduction of photobleaching

After prolonged exposure to light, most dyes are bleached quite significantly at room temperature. As a result, it often happens that the fluorescence light given off by the sample is not strong enough to enable the entire sample to be reproduced in three dimensions. When CMS is used, the molecule under examination is stabilised against bleaching by light, which means that measuring time can be extended considerably, depending on the molecule under consideration (see Fig. 4).

Fig. 4: Murine melanoma cells (B16 F10), stained with 5-(hexadecanoylamino)fluorescein (membrane marker λexc 488 nm) at a sample temperature of 20 °C (RT) and 22 K. The laser light with wavelength of 488 nm was focused on the sample for 24 minutes, and bleaching of the sample was captured in images. All settings on the microscope except the temperature conditions and the digital zoom factor were kept constant.

Fig. 4: Murine melanoma cells (B16 F10), stained with 5-(hexadecanoylamino)fluorescein (membrane marker λexc 488 nm) at a sample temperature of 20 °C (RT) and 22 K. The laser light with wavelength of 488 nm was focused on the sample for 24 minutes, and bleaching of the sample was captured in images. All settings on the microscope except the temperature conditions and the digital zoom factor were kept constant.

 

Measurements with excitation in the UV range

Measurements with excitation in the intense UV range can only be taken at room temperature to a limited degree, because the stability of biological samples in particular is compromised by high-energy light. At the same time, many interesting and viable biomarkers reach their optimum excitation precisely in the UV range. The use of CMS stabilises the molecule under investigation against the more energy-rich UV radiation, which in turn results in significantly longer measuring times.

Further fields of application

  • Material research – investigations into the temperature behaviour of materials (alloys / inclusions etc.)
  • Electrical engineering – temperature-dependent behaviour of superconductor materials
  • Material research /electronics– organic electronics, breakdown of nanostructures at low temperatures
  • Coating technology– temperature behaviour of OLED coatings
  • Environmental chemistry/ Environmental analysis – Propagation behaviour of heavy metals of concern for environmental analysis (actinides – lanthanides) in Nature

Technical specifications

Adaptation to commercial microscopes Coupling in a few seconds using a sliding system:
The measuring cell can be docked with and undocked from the microscope stage easily during the warm-up and cool-down periods.
Lenses Up to 63x magnification:
Maximum numeric aperture of lenses: NA = 0.95 (air lens)
Sample positioning,
horizontal (x, y)
in the measuring cell (stepping motors)

  • Approach via direct and indirect input
  • Step size 0.2 µm to 3 mm
  • Max. travel speed 3 mm/s
  • Maximum travel path 50 x 25 mm
  • Storage capacity and recovery for up to 10 measurement points
Sample positioning,
vertical (z-axis)
The complete measuring cell is slaved to the microscope stage

  • Movement: by direct and indirect input
  • Step size depending on programmed step size of the microscope stage
  • Maximum travel path +/- 10 mm
  • Storage capability and recovery for up to 5 measuring points
Temperature management
  • Range: 300 K – 20 K
  • Temperature increments: 1 K
Pressure control Pre-vacuum 10-3 bar / High vacuum 10-7 bar
Cooling time 1.5 h
Warming time 0.5 h
Automation PLC-supported automation of all parameters with the aid of standardized software (PC
Vibration suppression Passive system