Blog 4 Tips to Achieve High Quality Imaging in Microscopy Applications

4 Tips to Achieve High Quality Imaging in Microscopy Applications

Julien Clavereau

Julien Clavereau | September 18, 2019
Sales Manager for Western Europe, developing relationship with customers and striving to provide solutions

Contributing Author: John Hsu, Applications Engineer

Microscopy is an important technique across many disciplines both in research and commercially. There are many techniques used in microscopy including, spanning confocal, lattice light sheet and multi photon—and for all, image resolution is a key factor. Resolution plays a big role in microscopy’s success as it refers to the microscope’s ability to select detail in the specimen. Every day advances in these techniques help further push the boundaries of image resolution while avoiding sample damage. Today, we are going to go through some tips on how to achieve high quality imaging using supporting equipment like lasers and laser scanning technology.

High power, range of wavelengths
In order to achieve the highest resolution, the first step is to find a suitable laser source. In fluorescence microscopy, dyes including Cyanine, Atto and Azo are used to identify the presence, position and concentration of specific molecules. Each dye has a peak excitation wavelength, where fluorescence is at its highest for any given excitation power. Using a laser as closely matched as possible to this peak wavelength improves the efficiency, reducing sample heating and increasing the achievable imaging speeds in high throughput applications, or image brightness in low light applications.

In Stimulated Emission Depletion (STED) microscopy, super-resolution images, i.e. images exceeding the diffraction limited resolution, are generated by employing a second laser to selectively deactivate fluorophores close to the focal point of the excitation laser, minimizing the illuminated area to well below the spot size of the excitation laser and thereby increasing the achievable resolution.

The laser power level required for microscopy is dependent on many factors, including the size of the sample area, the specimen type, the required imaging speed and the microscopy technique employed. Finding the right balance of image brightness vs sample lifetime can be challenging, so having access to a wide range of maximum powers and the ability to continuously vary the power below that maximum is a vital tool for optimizing your setup.

High-speed imaging
Other key factors to consider beyond the type of laser and wavelengths is related to the scanning solution. It’s important to choose a scanning solution that enables the frame rate and field of view through its scan speed and angle of the beam steering components. Which is why selecting the right beam steering components is critical for developing each microscopy system.

Cambridge Technology offers beam steering components such as galvanometers and resonant scanners that are highly regarded in the microscopy field for both performance and long-term reliability. Galvanometers have been the popular choice due to its precise control of speed and position. This provides the flexibility to adjust scan frequency and scan patterns according to the microscopy technique employed. With the advancement in life sciences and the need to observe fast movements and morphologies within cells and tissues, even higher scan rates are desirable for in-vivo imaging. This is enabled with Cambridge Technology’s CRS resonant scanner. The CRS oscillates at a fixed, resonant frequency more than 10x faster than standard galvanometers.  Though less flexible in its variety of scan types, the CRS optimization for high speed scanning allows microscopy systems to reach over hundreds of frames per second.

Tightly Focused Focal Spot
Another consideration to improve resolution is the focused spot size of the laser. A tightly focused TEM00 laser with an M2 close to unity can be focused to a spot size approaching the diffraction limit of a few hundred nanometers (wavelength and numerical aperture dependent), with an easily modeled Gaussian intensity profile, allow the highest possible image resolution and simple interpretation of results.

A tight focal spot also enables high accuracy point-to-point scanning of the image, again leading to improved resolution.

Similarly, in Two Photon Microscopy, sample excitation relies on the intensity dependent two-photon absorption process. Two-photon absorption is highly localised in the focal region of the excitation beam, so achieving a tight focal area through selection of an excitation laser with an M2 close to unity enables generation of the highest resolution images.

Easy integration due to long operational lifetimes and compact size
As with any system, easy integration and long operational lifetime is a must. In microscopy, due to the industry’s small footprint, having a compact-size solution is also a must-have. A laser and laser scanning solution that can meet all these requirements will help the overall system performance, including achieving high quality images over a long period of time. For example, Laser Quantum’s lasers have long meantime to failures (MTTF) which averages >400,000 hours of usage. Cambridge Technology’s laser scanning technology also offer reliability with extremely long lifetimes for a variety of environments. Add compact-sized technology with adaptability and users can achieve an easy integration into OEM equipment meeting that important demand of compatibility and ease of use.