Biophotonics: A Guide to Using Lasers in Life Sciences Research

Topics Covered

Introduction
Confocal Microscopy for Life Sciences Research
Nonlinear Imaging and Multiphoton Microscopy for Life Sciences Research
Cycometry and High Content Screening for Life Sciences Research

Introduction

Modern imaging methods can be used to provide a better understanding of cell, organ and protein functions. By penetrating a cell with light a significant amount of data can be obtained via the measurement of the light that is transmitted and reflected. Light-based methods integrate deep penetration, non-destructive sampling, high spatial resolution and 3D imaging, and allow the real-time observation of biological samples.

Lasers are used in methods such as flow cytometry and microscopy and are vital components in pharmaceutical and medical instruments.

Figure 1. Advanced microscopy uses multi-color laser illumination.

In medical and life science applications, the increasing number of optical technologies that are required is linked to the growing need for advanced photonic sources, where each source is optimized to perform its particular task. Toptica Photonics addresses biophotonical applications by providing high-quality laser sources with emission of ultraviolet (UV), visible (VIS) and infrared (IR) light. Toptica provides laser solutions that are optimized for sophisticated biophotonics applications. This includes highly-reliable continuous wave (cw) diode lasers, as well as pulsed, femtosecond fiber lasers.

This article will explore the different ways in which lasers can be applied in life sciences research. We will discuss what can be achieved using optical methods, the specific advantages of each and which laser should be used to give the best outcome.

Confocal Microscopy for Life Sciences Research

Solid state lasers, fiber lasers, and diode lasers have replaced the gas lasers which were used historically for confocal imaging. In the field of optical imaging methods, it is critical to meet the unique and different requirements of customers in terms of high speed modulation of the laser source, a long lifetime and a high beam quality, as well as being able to access almost any wavelength in the NIR, VIS and UV regions.

The diode lasers supplied by Toptica provide a high power stability, a high beam quality and excellent lifetimes whilst remaining cost-effective. They deliver excellent performance to any AOM of AOTF and have direct modulation capabilities. These aspects reduce the complexities associated with optical systems and reduce investment expenditures.

Figure 2. Laser-based microscopes can be used to observe biological samples.

Figure 2 shows how advanced microscopy can employ multi-color laser light. In addition, a diffraction-limited beam with a superior wavefront is provided by diode lasers. The wavefront is the base for realizing high resolution confocal images.

Toptica has developed special features that make laser of the   iBeam smart seriesa suitable option for demanding biophotonic applications. These features include the first purely electronic Feedback Induced Noise Eraser (FINE), and SKILL, a Speckle KILLer, which cuts down laser speckle without the introduction of moving components. Simple, high quality, single-mode and polarization maintaining fiber delivery can be provided by Toptica. All of Toptica's lasers are optimized for their individual wavelength.

Even more sophisticated are Toptica’s multi-laser engines, the iChrome MLE and the iChrome SLE. They integrate up to 8 individual colors with up to 100 mW each in one box. An unified user interface to control all integrated lasers enables unique modulation capabilities and high-speed modulation up to 20 MHz. Two output fibers enable new microscopy techniques like FRAP. They also allow for operating several microscopes in parallel which is very cost-effective for many laboratories.

Nonlinear Imaging and Multiphoton Microscopy for Life Sciences Research

For studies focused at the cellular level, optical sectioning and determination of a sample’s 3D structure plays a major role in cellular biology. This is made possible by the constant development of a wide range of fluorescent markers. CFP, YFP and GFP (and its derivates) are the latest series of these markers. In the case of multiphoton microscopy techniques, usually a pulsed infrared light source is required for fluorophore excitation.

GFP markers are expressed within living cells and can be linked to proteins as required by researchers. This is not the case for artificial dyes. The light sources for laser scanning microscopy (LSM) can be tailored to work alongside fluorescent tags.

Fluorescent lifetime imaging microscopy (FLIM) is facilitated using pulsed lasers as their wavelengths can be tuned for specific fluorescent labels. Ultrafast lasers that deliver laser pulses with durations of several femtoseconds also allow non-invasive techniques that depend on multiphoton excitation techniques such as THG (Third Harmonic Generation), SHG (Second Harmonic Generation) and CARS (Coherent Anti-Stokes Raman Scattering).

Titanium-sapphire (Ti:Sa) based lasers are able to deliver pulses with femtosecond duration over a relatively broad wavelength tuning range. Nevertheless, there are several drawbacks. They have large space requirements and need water cooling. In addition, regular realignment and cleaning of optics makes Ti:Sa lasers time-consuming.

In contrast, fiber lasers provide  an alternative solution and can be used to concentrate on the particular requirements of research laboratories or industrial microscopy systems. Fiber-based laser systems are low-cost and are perfect sources of femtosecond pulses. They are fully highly-reliable, hands-off turnkey solutions with minimum space requirements and no water cooling.Femtosecond fiber lasers are available in a variety of different versions to suit bespoke applications.

Fiber lasers from Toptica are highly reliable and can be handled easily. A fiber coupled laser, FemtoFiber pro TVIS has been developed specifically for confocal microscopy, and spans the full visible range between 488 and 640 nm. Its 40 MHz repetition rate makes it ideal for FLIM. Unlike pulsed diode lasers, fiber coupled lasers do not show any after-ringing effects which can impact measurement. With the FemtoFiber pro range, non-linear microscopy and multi-photon methods can be achieved easily. For instance, femtosecond pulses spanning wavelengths between 780 and 1400 nm allow access to novel red markers used in two-photon excitation fluorescence (TPEF) or FLIM measurements using Alexa 594 (Figure 3).

Figure 3. SLIM measurement using Alexa 594.

 

TOPTICA’s customers have successfully demonstrated THG and SHG methods. Tunable and synchronized outputs deliver picosecond pulses, which are suitable for SRS (Stimulated Raman Scattering) or CARS (coherent anti-Stokes Raman scattering).

Erbium fiber-lasers can be used in the place of Ti:Sa lasers in applications where high output powers and tunability are not nessecary. This holds true for several multiphoton applications for two reasons. First, the 2-photon absorption spectra are relatively broader and also the peak of 2-photon absorptions are often blue-shifted. This means the majority of dyes can be easily 2-photon excited by using a laser at 780 nm. Secondly, 2-photon excitation is often used for live-cell imaging as it causes minimal photodamage in contrast to 1-photon excitation.

Using a nonlinear procedure ensures that photodamage is restricted to the focal region only; however, multiphoton microscopy can still result in some phototoxicity and photodamage when carried out at higher power levels. With commercial fiber lasers of lower output powers, users still need to select the right imaging-parameters to avoid photodamage.

Figure 4 shows a multiphoton image of nerve cells stained with YFP dyes.

Figure 4. Multiphoton image of nerve cells stained with YFP.

 

2-Photon fluorescence microscopy is an incoherent nonlinear imaging method that is often applied in cell biology studies to image thick samples with excellent resolution. Coherent nonlinear methods, like SHG and THG (Second and Third Harmonic Generation) also provide similar resolutions depending on the degree of photon scattering. SHG processes involve a non-linear interaction between a pair of equivalent photons which results in the production of a new photon which has twice the energy, and therefore half the wavelength, of the incident photons.

This phenomenon was first observed on crystalline quartz, and the process was later applied to study biological samples. Recently second harmonic imaging microscopy (SHIM) has became a popular technique for viewing the structure of intact tissues and cells in order to determine their functions. Figure 5 shows an SHG-image of collagen fibers.

Figure 5. SHG-image of collagen fibers

Appropriate methods for long-term imaging of living model organisms, cells and tissues are needed in life sciences research. For these types of applications, THG microscopy provides a suitable solution because it offers a high resolution and  does not require any sample staining.

The advanced fiber laser, FemtoFiber pro, makes it easy to combine non-linear microscopy methods in biological studies. It is a rugged, small and low-cost system, which requires a minimum ammount of maintenance. The FemtoFiber pro IR emits at 1560 nm, and creates a THG signal in the visible range. This means the laser can be used with conventional microscope systems and can be easily identified using standard PMTs (PhotoMultiplier Tubes).

Figure 6 shows a THG image of cell division in C. elegans embryo.

Figure 6. THG image of cell division in C. elegans embryo.

Cycometry and High Content Screening for Life Sciences Research

Advanced flow cytometry techniques facilitate the rapid detection and isolation of cells. Biologists can use flow cycometry to acquire detailed data about specimens by recording fluorescence signals and scatter data at the same time. A cell’s size, shape and type are automatically recorded. Because of this flow cycometry and it's related methods have become the preferred methods for life science research. Figure 7 shows the use of lasers in flow cytometry.

Figure 7. Lasers are the state of the art tool in flow cytometry.

 

TOPTICA provides a number of sources, which are optimized for flow cytometric applications. These sources have:

  • Low noise
  • Perfect power stability
  • Superior beam pointing stability
  • High output power
  • High reliability and life time
  • Fast direct modulation capabilities

Industrial iBeam smart lasers supplied by TOPTICA meet and surpass applicable field specifications from 375 to 830 nm. These lasers have special features such as high power SM-Fiber delivery, FINE and SKILL which allow customers in the cytometric field to work easily and productively.

In pharmaceutical research, drug discovery and modern cell biology, high content screening (HCS) has become a popular method. With the aid of this technique, scientists can easily and rapidly run tests on many different drugs or compounds or run genome-wide RNAi experiments.

The diode laser iBeam smart by TOPTICA is a one-box laser solution that has low noise, an excellent power stability and provides reproducible and consistent results over time. It can be used in pulsed and cw mode. High output powers ensure high acquisition speeds and high throughputs can be realized using confocal HCS systems. The small footprint of the diode laser also makes it easy to integrate into HCS systems. If several individual colors are required for biophotonics applications, the iChrome MLE and iChrome SLE multi-laser engines are ideal solutions.

This information has been sourced, reviewed and adapted from materials provided by Toptica Photonics.

For more information on this source, please visit Toptica Photonics.

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