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Photonics Handbook

Lasers for Microscopy: Major Trends

Marco Arrigoni, Nigel Gallaher, Darryl McCoy, Volker Pfeufer and Matthias Schulze, Coherent Inc.

Laser development for the microscopy market continues to be driven by key trends in applications, which currently include superresolution techniques, multiphoton applications in optogenetics and other areas of neuroscience, and even a shift in multiphoton imaging toward preclinical and clinical usage.
In spite of its long history, optical microscopy, and particularly laser-based microscopy, is a very dynamic field. New techniques continue to be developed, while existing techniques are being applied to new applications. In order for biologists, drug developers, clinical lab professionals and other scientists to fully exploit these new techniques and applications, parallel developments in laser technology are often required. In this article, we present a broad overview of three important trends in laser-based microscopy and examine how laser manufacturers are responding with products optimized to match the needs of these applications.

Superresolution microscopy – optical switching of fluorescent labels

In order to better understand the details of processes like signaling and the control of gene expression, biologists need to correlate molecular-level events with macroscopic structures and dynamics. This has fueled rapid growth in superresolution microscopy techniques, often referred to as nanoscopy, that go beyond the classical spatial resolution limit set by diffraction. This limit is about half the wavelength of light (i.e., for visible light, approximately 200 to 250 nm in the XY plane) and, in the case of confocal microscopy, about 500 nm in the Z direction. Most superresolution techniques use CW lasers, often with fast internal or external (on/off) modulation.

The Nobel Prize Committee recognized the importance of superresolution microscopy by awarding the Nobel Prize in chemistry 2014 jointly to Eric Betzig, Stefan W. Hell and William E. Moerner, for their pioneering work in developing superresolution microscopy techniques.

All optical superresolution or nanoscopy techniques are based on the principle of optically and reversibly preparing states of a fluorescence label that differ in their emission characteristics (e.g., a bright “on” and a dark “off” state). Based on their different mechanisms for this on/off photoswitching, optical nanoscopy methods can be loosely divided into two groups: those that directly improve microscope effective spatial resolution by deterministic photoswitching in space and time, and those that achieve the higher resolution by (random) stochastically switching single-molecule fluorescence on and off in space. Examples of the first group include simulated emission depletion (STED) microscopy, ground state depletion microscopy, reversible saturable optical fluorescence transition (RESOLFT) microscopy or parallelized RESOLFT microscopy, often denoted nonlinear or saturated structured illumination microscopy (SIM). Examples of the latter include direct stochastic optical reconstruction microscopy (d-STORM) and fluorescence photoactivation localization microscopy (f-PALM).

Principles of STED.

Figure 1.
Principles of STED. (a) Schematic drawing of the setup of a STED nanoscope with phase plate, objective lens dichroic mirror (DC), fluorescence filter (F), detector, scanning device, and excitation and STED lasers with their focal intensity distribution (right) and a representative, sub-diffraction sized observation area. (b) STED nanoscopy is based on inhibiting fluorescence emission by de-exciting the excited S1 ON state to the S0 OFF ground state via stimulated emission. Increasing the power of the STED laser drives the inhibition into saturation. (c) Combined with an intensity distribution that features at least one intensity zero, this ON-OFF fluorescence inhibition realizes sub-diffraction sized observation volumes: The volume in which fluorescence emission is still allowed (green, insets) decreases with increasing STED laser power. Courtesy of Christian Eggeling.

Figure 1 explains how STED works. A donut shaped laser beam is coincident with a transverse electromagnetic TEM00 (Gaussian profile) beam of a different wavelength. The donut beam depletes the population of excited fluorophores via stimulated emission. This beam shape serves to constrict the volume of fluorophores that are excited by the TEM00 beam. The donut-shaped depletion beam has a smooth spatial profile so that increasing the power in the STED beam increases the “bleached” area and thus confines fluorescence excitation by the TEM00 beam to a smaller area. This is equivalent to having a “knob” that controls the spatial resolution. Optimized on/off time-gating of the two lasers enables signal discrimination and high signal-to-noise images. Figure 2 is an example showing the effectiveness of STED.

The effectiveness of STED

Figure 2.
The effectiveness of STED is clear in this pair of images of fluorescently tagged microtubuli in mammalian cells using a 577 nm Coherent Genesis laser to drive the STED effect: conventional confocal (left) and gated STED image (right). Courtesy of Dr. Giuseppe Vicidomini and Prof. Alberto Diaspro, Instituto Italiano di Tecnologia.

Stochastic-based techniques such asf-PALM and d-STORM also use two lasers (Figure 3). Here one laser is used to drive most of the fluorophores between the “on” and “off” states, so that only a very small, random subset of well-separated molecules are in their “on” state and available to be excited by a second laser. The fluorescence spread from each of these camera-imaged point sources is then analyzed to find the centroid (i.e., the point source or spatial localization, based on the point spread function (PSF) of the microscope). Repetitive on-off switching brings up different random subsets in subsequent camera images for single-molecule localization. After many cycles, the microscope computer assembles all the idealized point sources into a subdiffraction image, since the localization of a single molecule can be performed with high, subdiffraction spatial precision.

Principles of STORM microscopy.

Figure 3.
Principles of STORM microscopy. (a) A wide-field fluorescence image (blue area) is observed from overlapped fluorophores in a small target structure (grey lines), (b) No or little fluorescence are left after pre-acquisition bleaching with an appropriate laser, (c) Individual fluorophores are sparsely activated by an activation laser, and then emit fluorescence (blue spots) after applying an excitation laser (usually the same one for pre-acquisition bleaching). (d) The excitation laser is again used to bleach the fluorophores. The center positions (red crosses) of the fluorophores are found using an appropriate molecule localization algorithm. The uncertainty of the center positions (red spots) is significantly smaller than the diameter of the single molecule fluorescence from the same fluorophores (the large blue spot in the left corner). (e) Repeating the activation, excitation, localization and photobleaching process for more than 1,000 times allows reconstruction (via overlaying the center positions of the fluorophores) of a superresolution image of the target structure (red lines). Courtesy of Professor Zhen-li Huang at Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology.

Trends in CW lasers to support superresolution techniques

Laser manufacturers are supporting the rapid growth in superresolution microscopy with new products that provide higher power and smoothly adjustable power, new wavelengths, and simplified fiber coupling and fiber combining.

Higher laser power is necessary because researchers want output in the 1 to 3 W range for STED, and typically a few hundred milliwatts for PALM and STORM. And in the case of SIM, microscope manufacturers are looking to move up to 300 mW lasers to decrease image acquisition times, whereas early users of this method worked with as little as 100 mW. Both deterministic and stochastic switching of fluorescence also need adjustable power output in the “bleaching” laser, in order to optimize these techniques.

Lasers based on optically pumped semiconductor laser (OPSL) technology are proving ideal to meet these higher power/adjustable power needs. That’s because OPSL technology is readily scalable, simply by increasing the pump diode power. And the OPSL output can be smoothly adjusted from a few percent to maximum power with no effect on beam quality or beam pointing, because OPSL technology does not suffer from thermal lensing, which prevents wide power adjustment in diode pumped solid-state (DPSS) and other solid state lasers.

Another key trend is the growing demand for “new” laser wavelengths, and particularly longer wavelengths, to optimally excite many of the new fluorophores. These include the mFruit series of fluorescent proteins and a host of new green, yellow and orange excited dyes originally developed for multichannel flow cytometry applications. And, as with wide-field microscopy, new nanoscopy methods often use combinations of multiple fluorophores to permit simultaneous mapping or probing for more than one type of structure or biochemical.

Fortunately, wavelength scalability is another key advantage of OPSL technology; lasers can be fabricated for any arbitrary output wavelength across much of the visible and near-UV spectrum, merely by tweaking the design of the gain semiconductor chip. For example, new lasers at 588 nm and 594 nm were recently introduced for exciting orange fluorophores. Other models at 550 nm also have been developed as this wavelength can be used to simultaneously excite green dyes and several new yellow fluorophores with a single laser. Another new OPSL wavelength is the legacy (krypton ion) wavelength of 568 nm.

Fiber coupling/fiber combining

Many end users and microscopy OEMs – and not just in superresolution – are increasingly utilizing fiber delivery with CW lasers. To maintain high beam quality, this necessitates using polarization-preserving, single-mode fiber which has a core diameter around 3.5 µm. At first, users would couple the laser into the single-mode fiber using mounts with up to six degrees of freedom that had to be individually optimized and then locked. This typically required many hours (per laser) from a skilled technician, and the fiber often lasted only 100s of hours because of degradation of the small output facet. This problem was eliminated with the advent of bio-instrumentation lasers with permanent fiber-coupled outputs.

The past few years have seen a similar paradigm shift in combining multiple lasers in response to the needs of applications to use multiple wavelengths. This formerly required multiple optics (beamsplitters, waveplates, etc.) for each laser wavelength, and demanded hours of painstaking adjustment. But there are new plug-and-play combiners on the market that now simplify this process. These passive devices can integrate up to eight different laser wavelengths using a combination of a minimum number of refractive optical components, together with a novel fiber having constant NA over the entire 405 – 640 nm range. This reduces the task to simple plug and play with snap-in (FC/UFC) fiber connectors.

Neuroscience dominates growth in multiphoton imaging

In multiphoton excitation (MPE) microscopy using ultrafast lasers, the single biggest driver in techniques and laser development is neuroscience. Billions of dollars in public and private funding are enabling dramatic advances in understanding how the brain processes information, from the synaptic to the network level. Moreover, increased life expectancy brings an emphasis on improved quality of life in old age, and other neuroscience goals include a better understanding of how to reduce and treat age-related mental impairments such as Alzheimer’s and Parkinson’s. MPE is playing a key role in much of this research because it enables deep (1 - 1.5 mm) in vivo imaging with full 3D resolution and low photodamage. The classic mammalian setup involves a mouse with a thin glass cover replacing a small part of the skull to provide visual access to the cortex at layers 1 through 6. The rest of the MPE market is a combination of various fields including cancer research, stem cell studies and other aspects of immunology. While many of these applications involve laboratory research, there is growing interest in the elusive goal of moving toward clinical (i.e., human) applications.

The overarching trends in neuroscience rely on faster and deeper in-vivo imaging. At the same time, novel applications are being enabled by new fluorescent tools and methods, many of which use gene expression in laboratory animals, from insects to mice. Advances in these genetically expressed probes include the development of more efficient red-shifted fluorescent probes for deeper imaging with less scatter, as well as new types of so-called functional probes. These functional probes include proteins whose fluorescent characteristics are modified by specific local physiological conditions that take place, and nonfluorescent proteins that manipulate cell membrane conditions when illuminated, as in the relatively new field of optogenetics, pioneered by Dr. Karl Deisseroth at Stanford University and Dr. Ed Boyden at MIT. Deisseroth and Boyden were among the recipients of the 2016 Breakthrough Prize in Life Sciences for their work.

Optogenetics uses a class of proteins called opsins that reside in the cell membrane and act as single-element ion gates that can be controlled by pulses of light. When irradiated at the appropriate wavelength, they enable Ca2+ or other ions to flow across the membrane, leading to a spike in the membrane potential that mimics the normal action potential used for neuron conduction and dendritic signaling processes in neural cell networks. Optogenetics has thus enabled researchers to progress beyond studying the structure of neural networks to provoking (or inhibiting) their activity in real time. What about monitoring the actual signals as they propagate in the neural network, typically in terms of Ca2+ spatiotemporal concentration? This also can be done using another family of genetically expressed probes that fluoresce strongly only in presence of this ion – the so-called GECI or genetically encoded calcium indicators.

Early optogenetics research used LED light sources for wide-field photoactivation, somewhat of a “bazooka” approach. Thanks to the exquisite spatial selectivity and penetration of multiphoton excitation (MPE), an appropriate ultrafast laser can be used for highly specific optogenetics activation of particular sites, possibly in conjunction with a spatial light modulator. As shown in Figure 4, a second ultrafast wavelength can be deployed for imaging the Ca2+ activity using a GECI excitable at a convenient wavelength.

An advanced optogenetics experimental scenario involves MPE imaging to map the neurons

Figure 4. An advanced optogenetics experimental scenario involves MPE imaging to map the neurons, followed by activation of one or more neurons using multiphoton excitation at wavelength 1, and probing of Ca2+ activity in remote neurons by multiphoton excitation at wavelength 2. Wavelength 1 (or sometimes wavelength 2) is also used for the initial MPE mapping.

Higher laser power is needed to enable stimulation of more neurons and also for faster imaging speed to increase the number of neurons that are probed. At present a typical multiphoton experiment with individual neuron resolution might involve up to 10 neurons. But ultimately researchers would like to simultaneously survey as many as 10,000 neurons, i.e., a column of cortex measuring 250 × 250 µm with a depth up to 1 mm. This will require very fast scanning with higher laser power.

Optogenetics is not the only area of neuroscience where multiple laser wavelengths are needed simultaneously. For example, an interesting technique for mapping different neuron types is “brainbow” imaging. Here, subject animals are transfected with genes for three or more different fluorescent proteins, whose emission peaks at red, green and blue wavelengths. The various cell types in the brain express these proteins in unique stochastic ratios, each characterized by a different ratio of the fluorophores and, therefore, a unique emission spectrum and color signal. Brainbow imaging needs two or more independently tunable ultrafast source. CARS and SRS imaging similarly rely on two simultaneous ultrafast wavelengths.

Trends in ultrafast lasers driven by neuroscience needs

To better support these new imaging methods in neuroscience, laser manufacturers are focused on providing ultrafast sources with higher power at longer wavelengths, as well as maintenance-free lasers that are ever simpler to use. Until 2012, the only proven gain material for ultrafast lasers was Titanium:sapphire (Ti:S). Wavelengths at 1050 nm and longer were then generated by using part or all of the Ti:S output to pump a tunable optical parametric oscillator (OPO). Moreover, the optional use of fan-poled crystal technology provided a revolutionary way to generate two independently tunable wavelengths – one from the Ti:S laser and one from an OPO pumped by this laser, all under push-button integrated computer control. Emmanuel Beaurepaire et al. showed that independent tunability of two synchronous pulses can excite all the brainbow expressions by using two photons from the Ti:S laser, two from the OPO and one photon each from the OPO and the Ti:S (Figure 5).

Trichromatic two-photon imaging of brainbow mouse cortical tissue using wavelength mixing.

Figure 5.
Trichromatic two-photon imaging of brainbow mouse cortical tissue using wavelength mixing. Courtesy of E. Beaurepaire, P. Mahou, K. Loulier, J. Livet, Ecole Polytechnique, Palaiseau/ Vision Institute, Paris.

Several key advances have recently offered improved laser performance – in terms of higher power and longer wavelengths – benefiting both single and dual-wavelengths MPE applications, including optogenetics. Some Ti:S lasers now provide brighter images through a combination of shorter pulsewidths and higher average power than all earlier OPOs.

Even more exciting has been the development of ultrafast lasers using ytterbium doped gain media – the first successful solid state alternative to Ti:S for short pulsewidths (< 100 fs). These lasers are direct diode-pumped and are complementary to Ti:S based lasers. But in many instances they represent a superior option, particularly in the 1030 to 1100 nm window that is specifically important for optogenetics. This window is important since it enables optimum two-photon excitation of new fluorescent proteins like mCherry, long wavelength photoactivation proteins like C1V1 and red-shifted calcium indicators like RCaMP. Moreover, this is also a useful wavelength window for label-free MPE techniques like SHG imaging which are being investigated in transitional research studies with possible future use as clinical tools.

The first of this new generation were fixed wavelength lasers based on ytterbium-doped fiber. With an output wavelength of 1055 nm, these were the first ytterbium-based commercial lasers to successfully combine multi-watt (> 2 W) output power with a pulsewidth of only 70 fs. More importantly, these rugged, streamlined and sealed lasers are standout examples of the new trend in ultrafast lasers that relies on HALT/HASS* engineering development and rigorous testing protocols in order to combine best in class performance with an industrial revolution in 24/7 reliability and ease of use. (HALT refers to highly accelerated life testing and HASS refers to highly accelerated stress screening. These are rigorous practices long-used outside the photonics industry, including protocols often colloquially referred to as “shake and bake.”)

From a practical viewpoint, it is also important to note that the broader spectral bandwidth of a 70 fs pulse means that group velocity dispersion (GVD) in the beam delivery optics will stretch the initial laser pulsewidth. For this reason, these lasers incorporate a software-controlled precompensator to enable the user or system builder to offset downstream GVD and thus minimize the pulsewidth at the sample.

However, numerous optogenetics researchers were soon reporting they need even higher power in this wavelength window, in excess of 10 W. To meet this need and future trends, models were already available with power as high as 18 W. While this may seem excessive for live tissue imaging, it’s important to remember that in multiphoton excitation, only a small portion of the total power is absorbed. Indeed, as far back as 1997, Schoenle and Hell showed that irradiation with 100 mW of power at 800 nm on a diffraction limited spot for 1 s resulted in only a 0.2 °C temperature rise, well compatible with cell functionality. Furthermore, techniques such as holographic “scanless” imaging are power hungry because they involve reaching the focused intensity for two-photon excitation over many areas of the sample simultaneously.

Another series of ytterbium-based lasers now produce even higher power by combining a mode-locked oscillator with a pulse picker and a regenerative amplifier, some of which generate 40 W of average power (40 µJ pulse energy at a repetition rate of 1 MHz), at pulsewidths of under 400 fs. This laser technology actually was originally developed to meet the needs of some precision materials processing applications. But the insatiable appetite for higher power prompted its packaging in a format suitable for multiphoton imaging and other research applications.

At the same time ytterbium-based lasers have also been developed to meet the need for a fully featured ultrafast source with wide tunability and dual wavelength output. This tunability is needed in several MPE applications, for instance where different wavelengths are required in quick succession or where the laser is used a shared resource for different experiments. And the dual wavelength output enables sophisticated optogenetics experiments as well as brainbow type work. The type of laser is continuously tunable from 680 to 1300 nm, with short pulsewidth (100 fs) and power up to 1.4 W. And as with some of the fixed wavelength lasers, an internal and fully automated GVD precompensator enables the shortest possible pulsewidth at the sample. Just as important, the secondary output simultaneously provides 1.5 W of output in that all important 1030 to 1100 nm window – specifically at 1040 nm. This combination supports optogenetic experiments based on established schemes using longer wavelength opsins like C1V1, as well as alternative wavelength pairings based on red-shifted calcium indicators of the GCaMP family currently under development. And the availability of dual wavelength pulses that are phase-correlated also makes this new type of laser a perfect source for other multiphoton microscopy techniques like CARS and SRS.

Summary

Laser-based microscopy continues to be one of the most valuable tools in the life sciences. As researchers develop and refine new techniques and methods, laser sources need to be adapted and re-optimized in order to fully exploit the potential of these techniques. By closely dialoging with key researchers and institutions, laser manufacturers are responding in a timely manner with new products designed specifically for the changing needs of these applications.

Acknowledgments

The authors wish to acknowledge the assistance of Dr. Christian Eggeling (Principal Investigator Human Immunology Unit and Scientific Director Wolfson Imaging Centre, Weatherall Institute of Molecular Medicine, University of Oxford, U.K.) in the form of personal communications about optical nanoscopy techniques and specifically STED technology, including supplying Figure 1. They also wish to acknowledge the assistance of Professor Zhen-li Huang at Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, for important insights in STORM imaging and for providing Figure 3.

GLOSSARY
optogenetics
A discipline that combines optics and genetics to enable the use of light to stimulate and control cells in living tissue, typically neurons, which have been genetically modified to respond to light. Only the cells that have been modified to include light-sensitive proteins will be under control of the light. The ability to selectively target cells gives researchers precise control. Using light to control the excitation, inhibition and signaling pathways of specific cells or groups of cells...
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