Lasers for Life Sciences

There is a need to conduct advanced laser imaging as part of the private and publicly funded research into diseases such as cancer, Alzheimer’s and Parkinson’s which are prevalent due to the ageing population.

Various imaging techniques are in common use by biologists and there is a growing demand for higher resolution, faster and more cost-effective cell imaging.  Imaging modalities such as laser scanning fluorescence microscopy and single plane illumination microscopy are core techniques that are used routinely in life science and medical laboratories.

To obtain these images life scientists require reliable picosecond and femtosecond lasers that provide powers that won’t damage the biological sample under test while generating a suitable non-linear excitation from the sample plane. In this instance, our range of lasers can be used to excite fluorescent dyes and obtain images of particular sub-cellular components such as membranes, mitochondria, nuclei, proteins and ions.  These everyday imaging techniques can help to indicate the presence or absence of certain cell dynamics and medical conditions.

The section below highlights just a few of the imaging techniques that have incorporated our  Chromacity Spark 1040 laser system, to help illustrate its capabilities in helping others to discover more.

Multi-photon single plane illumination microscopy

Zebra Fish

Lightsheet fluorescence microscopy (or single plane illumination microscopy) is a new paradigm in two-photon imaging. This technique is used in cell biology and for microscopy of intact, often chemically cleared, organs, embryos, and organisms.

The technique provides excellent optical sectioning capabilities and high speed (up to 1000 times faster than those offered by point-scanning methods). In contrast to epifluorescence microscopy only a thin (0.5 – 5 µm) slice of the sample is illuminated. The sample is then observed perpendicular to the illumination plane.

Chromacity has the expertise to provide its Spark 1040 as the ideal source for multi-photon light sheet fluorescence microscopy. The technique combines good z-sectioning and only illuminates the observed plane. By generating a sheet of light, the optical power is spread across the whole image. This method reduces photo-damage and stresses induced on living samples. Additionally, the excellent optical sectioning capability increases the SNR and creates images with higher contrast, when compared against confocal microscopy.

The Chromacity Spark’s excellent beam quality and high average power makes it ideal for this technique.

Contact us to discuss integrating a Spark 1040 laser in your light sheet microscopy system

Application Note - Two Photon Light Sheet Microscopy

Multi-photon optogenetics

Fluorescence Rat Brain

There is a drive in multi-photon microscopy applications in optogenetics to study increasingly larger groups of neurons, and to image deeper into live brain tissue or over wider areas. Today’s cutting-edge experiments involve simultaneous stimulation/interrogation of a few tens of neurons, but some researchers would like to push this number to as many as 1,000 neurons. Optogenetics is already used in a broad range of experiments. In microscopy, optogenetics has enabled all-optical physiology experiments where optical microscopy is used to image the morphology of a group of live neurons.

A range of multi-photon imaging techniques make use of the non-linear processes that can be generated by using ultrashort pulses. Multi-photon absorption (i.e., excitation) in a fluorophore can be generated by using a near-IR laser wavelength that excites a label, fluorescent protein or indicator with a strong single-photon absorption at about half the near-IR laser wavelength. Other nonlinear techniques used for 3D microscope imaging include three-photon excitation of fluorescence and second harmonic generation (SHG). Through these techniques the light generated by the sample only occurs at the beam waist allowing for high resolution imaging.

Using the Chromacity Spark 1040 allow for excitation microscopy with three key benefits:

  • Inherent 3D resolution allows cells or groups of cells to be imaged at typically micron resolution in the Z-axis and a few hundred nanometers in the XY plane. Using a pulse laser source also allows time resolved measurements to be taken.
  • Using the Spark 1040 allows the capture of image at greater sample depths. Light scatter is often the limiting factor for microscopy imaging of thicker samples. Mie scattering within a sample can have a significant effect on the signal to noise ratio of the detected signal, as it has a highly nonlinear dependence on wavelength. Use of a 2-times or even 3-times longer wavelength results in a 16-times or 81-times reduction in scatter as well as an increase in imaging depth.
  • Photobleaching and photothermal degradation is reduced, allowing in vivo experiments to be more readily accessible.

Historically, optogenetic experiments have made use of solid-state laser sources. When a second wavelength is needed an expensive and bulky independently tunable optical parametric oscillator (OPO) has been required. However, as researchers are keen to image larger neuron populations there is a requirement for more than 1 watt at wavelengths longer than 1 micron.

To meet this demand, Chromacity has developed a high average power ultrafast laser that can deliver up to 2.5 W at 1040 nm.

This new generation of ultrafast lasers offer the requisite power and pulse duration to perform multineuron studies while also providing reliability that allows you to focus more on the imaging and less on the laser.

Application Note - Multi-Photon Microscopy

Second Harmonic Generation Microscopy

mtmg liver 500mw

In recent years, SHG microscopy has proven its capability in the study of crystallized bio-molecules such as starch, collagen and myosin. SHG imaging can reveal the structural organization and molecular orientation within non-centrosymmetric tissue structures.

Starch, which is an important food source and a promising future energy candidate, has been shown to exhibit strong SHG response and is a relatively new tool for plant research and other applications.

The Chromacity Spark can be used to generate an SHG signal in a solution of starch molecules. The Chromacity Spark 1040 output beam originates from a single-mode fibre, making the system ideally suited for coupling into commercial laser-scanning microscopes. This saves time, when setting up samples, and allows images to be acquired sooner.

The figure above illustrates typical images acquired from the system in the forward direction. The power levels from the Spark were more than adequate for generating these fluorescence images, which were recorded with 150 mW incident on the galvo-scanning mirrors.

Application Note - SHG Imaging

Second harmonic generation (SHG) imaging of collagen fibres

The non-centrosymmetric molecular structure of collagen also makes it possible to image collagen fibres in SHG using the Chromacity Spark 1040. Images can be acquired in both the forward and backward directions.

Including the Spark 1040 femtosecond laser as part of a SHG imaging system allows users to generate exceptionally clear, high-resolution images, a result of the Spark’s excellent beam quality and high average power levels.