While most microscope objectives are designed to work with air between the objective and cover glass, objectives lenses designed for higher NA and greater magnification sometimes use an alternate immersion medium. For instance, a typical oil immersion object is meant to be used with an oil with refractive index of 1.51.

DE weapons have been tested in the field, in air, on land and in the sea. Although those developments and applications are understandably classified, it is possible to find some literature describing the motivations and models. For example: “Adaptive Optics for Directed Energy: Fundamentals and Methodology” (here).

Both the objective lens and the eyepiece also contribute to the overall magnification of the system. If an objective lens magnifies the object by 10x and the eyepiece by 2x, the microscope will magnify the object by 20. If the microscope lens magnifies the object by 10x and the eyepiece by 10x, the microscope will magnify the object by 100x. This multiplicative relationship is the key to the power of microscopes, and the prime reason they perform so much better than simply magnifying glasses.

Figure 14: images of drosophila tissues acquired with the microscope described on Figure 13 at 2 different depths ( 22µm, 42µm) without (left) and with (right) adaptive optics.

By continuously measuring and correcting wavefront errors in a loop, the adaptive optics system constantly produces much sharper and clearer images than would normally be possible to acquire.

In this application note, among the many examples of adaptive optics implementation, we choose to describe only the following few examples:

A basic achromatic objective is a refractive objective that consists of just an achromatic lens and a meniscus lens, mounted within appropriate housing. The design is meant to limit the effects of chromatic and spherical aberration  as they bring two wavelengths of light to focus in the same plane. Plan Apochromat objectives can be much more complex with up to fifteen elements. They can be quite expensive, as would be expected from their complexity.

What is the purpose of theobjective lensin alightmicroscope

At Avantier we produce high quality microscope objectives lenses, ocular lenses, and other imaging systems. We are also able to provide custom designed optical lenses as needed. Chromatic focus shift, working distance, image quality, lens mount, field of view, and antireflective coatings are just a few of the parameters we can work with to create an ideal objective for your application. Contact us today to learn more about how we can help you meet your goals.

Although today’s microscopes are usually far more powerful than the microscopes used historically, they are used for much the same purpose: viewing objects that would otherwise be indiscernible to the human eye.  Here we’ll start with a basic compound microscope and go on to explore the components and function of larger more complex microscopes. We’ll also take an in-depth look at one of the key parts of a microscope, the objective lens.

Figure 15: Retinal Imaging using Rtx1 from Imagine Eyes to improve the resolution: see the difference when the AO is ON.

Adaptive optics (AO) systems consist of measuring and compensating distortions in the incoming wavefront in order to recover signal or resolution. The wavefront measurement is typically performed with a wavefront sensor such as a Shack-Hartmann wavefront sensor, whereas the compensation is carried out by a deformable mirror. A control system or software will then apply a closed loop algorithm and the ensemble will provide a corrected output wavefront which can then be processed by detectors or cameras. Adaptive optics systems have demonstrated significant resolution improvement. With the recent progress in camera technologies,  wavefront sensors, deformable mirrors and real time computers, AO systems have become very popular and are now used in many fields such as high-power lasers, free space optical telecommunications, micro and nano-manufacturing, fluorescence microscopy, optical coherence tomography and retinal imaging, just to name a few.

Unlike in astronomy, the speed of wavefront correction in UHIL is not critical and the key factors are rather the optical quality of the deformable mirror, the performance of the wavefront sensor and the ability to optimize the final spot. Figure 9 shows a focal spot before and after adaptive 0ptics correction obtained at the Laboratoire Irène Joliot-Curie, Université Paris-Saclay in Orsay, France. The adaptive optics system used was composed of an ILAO-Star deformable mirror, a HASO wavefront sensor and the Wavetune software from Imagine Optic.

A microscope is an optical device designed to magnify the image of an object, enabling details indiscernible to the human eye to be differentiated. A microscope may project the image onto the human eye or onto a camera or video device.

This page is an overview of adaptive optics and its applications from high intensity lasers to microscopy and retinal imaging. Learn how deformable mirrors are used to compensate for wavefront errors and help imaging systems go from blur to clarity.

The first AO system based on a wavefront sensor, wavefront reconstructor and deformable mirror comparable to those used today was developed in the early 70s within the scope of a DARPA agency grant aiming at imaging and tracking satellites. The RTAC (Real Time Atmospheric Compensator) developed in association with the company ITEK, MA was first demonstrated in 1974 [Hardy and al.] and the first implementation of an AO system (CIS – Compensated Imaging system) was made on a 1.6 m telescope located on Mt Haleakala on Maui Island. This system was associating a piezoelectric DM with 168 actuators, separate Tip-Tilt Correction and an intensified shearing interferometer as a wavefront sensor, all together able to perform closed loop correction up to 1000Hz.

Most microscopes rely on background illumination such as daylight or a lightbulb rather than a dedicated light source. In brightfield illumination (also known as Koehler illumination), two convex lenses, a collector lens and a condenser lens,  are placed so as to saturate the specimen with external light admitted into the microscope from behind. This provides a bright, even, steady light throughout the system.

Deformable mirrors are mirrors with electronically controlled surface shapes. They are commonly used to compensate aberrations. They can be based on piezoelectric transducers, mechanical or electromagnetic actuators. Spatial Light Modulators with phase modulation are high resolution arrays that can control the phase of the optical wavefront, pixel by pixel. Although they present lower spectral bandwidth and smaller damage threshold, they have much higher resolution than deformable mirrors and can especially be used for beam shaping applications. For a list and description of deformable mirrors, please click here.

-In retinal imaging: the patient eye itself creates aberrations which greatly limits the final resolution of the retina image acquired.

Microscope objective lenses are typically the most complex part of a microscope.  Most microscopes will have three or four objectives lenses, mounted on a turntable for ease of use. A scanning objective lens will provide 4x magnification,  a low power magnification lens will provide magnification of 10x, and a high power objective offers 40x magnification. For high magnification, you will need to use oil immersion objectives. These can provide up to 50x, 60x, or 100x magnification and increase the resolving power of the microscope, but they cannot be used on live specimens.

The eyepiece or ocular lens is the part of the microscope closest to your eye when you bend over to look at a specimen. An eyepiece usually consists of two lenses: a field lens and an eye lens. If a larger field of view is required, a more complex eyepiece  that increases the field of view can be used instead.

For example, a classical imaging system such as an objective lens, is typically considered “high performing” if the wavefront error (wfe) introduced by its lenses is minimal (typically wfe < Lambda/20 rms). It is also qualified as “diffraction limited,” meaning the resulting point spread function is close to the perfect Airy function. Optical designers from various industries make great efforts to calculate combinations of lenses or optics to reduce this wfe. For imaging lenses or optical components, the wavefront error is fixed and comes from the system itself, from its design on one hand, and errors of fabrication on the other hand. In other cases, wavefront distortion can come from other sources along the path, with time or space dependency. For example:

There are two major specifications for a microscope: the magnification power and the resolution. The magnification tells us how much larger the image is made to appear. The resolution tells us how far away two points must be to  be distinguishable. The smaller the resolution, the larger the resolving power of the microscope. The highest resolution you can get with a light microscope is 0.2 microns (0.2 microns), but this depends on the quality of both the objective and eyepiece.

Historically, bimorph and monomorph deformable mirrors, developed for directed energy and astronomy, were the only large deformable mirrors available until Imagine Optic introduced its new line of ILAO mechanical deformable mirrors. ILAO and ILAO Star are specific DMs developed for UHIL applications. They meet all critical requirements for UHIL: large aperture, optical quality (1o nm rms active flat), extreme stability (no drift) and quasi perfect linearity. Figure 10 shows a 400 mm diameter ILAO STAR – custom designed for petawatt femtosecond laser manufactured by Imagine Optic.

Directed-energy (DE) weapons have been in development for the past three decades. On one hand, high energy lasers (HELs) offer many advantages over conventional weapons, including the delivery of energy at light speed, a low cost per shot, unlimited magazines, and stealth. On the other hand, the performance of DE weapons is dependent on atmospheric conditions. This is why adaptive optics have a key role in those developments in order to optimize the irradiance on target, by compensating the aberrations coming from the laser (thermal effects), the optical train (beam transportation optics) and of course from the turbulence along the emitter to target optical path.

Typesof microscope objectives

Wavefront sensors provide the 3D map of the optical wavefront or phase front with speeds up to several KHz. They quantify atmospheric disturbance, or optical aberrations with a high precision of λ/100 RMS. Shack-Hartmann wavefront sensors are by far the most used sensors for adaptive optics, because they are easy-to-use, accurate, fast and robust. Other types of sensors can also be used depending on the application. For a list and description of wavefront sensors, please click here.

A basic compound microscope could consist of just two elements acting in relay, the objective and the eyepiece. The objective relays a real image to the eyepiece, while magnifying that image anywhere from 4-100x.  The eyepiece magnifies the real image received typically by another 10x, and conveys a virtual image to the sensor.

The working distance of a microscope is defined as the free distance between the objective lens and the object being studied. Low magnification objective lenses have a long working distance.

Objective lens function

Numerical aperture NA denotes the light acceptance angle. Where θ is the maximum 1/2 acceptance ray angle of the objective and n is the index of refraction of the immersive medium, the NA can be denoted by

Functionofcondenserin microscope

One example of successful implementation of adaptive optics for retinal imaging was performed by the company Imagine Eyes. Figure 16 below shows  a real time image of a live retina with adaptive optics ON and OFF. The system drastically improves the resolution and provides instant visualization of  cellular details (rods and the cones) of the retina.

A reflective objective works by reflecting light rather than bending it. Primary and secondary mirror systems both magnify and relay the image of the object being studied. While reflective objectives are not as widely used as refractive objectives, they offer many benefits. They can work deeper in the UV or IR spectral regions, and they are not plagued with the same aberrations as refractive objectives. As a result, they tend to offer better resolving power.

The optical performance of an objective is dependent largely on the optical aberration correction, and these corrections are also central to image quality and measurement accuracy. Objective lenses are classified as achromat, plan achromat, plan semi apochromat, plan apochromat, and super apochromat depending on the degree of correction.

The number of facilities employing ultra high intensity lasers have been on the rise since the late 90s. Those sources typically use mode locked oscillators that are amplified to produce femtosecond type pulses with peak power varying between 10s of TW to several PW on target. This paves the way for experimental production of extreme electromagnetic conditions required for relativistic physics. Not only are those sources more compact and cheaper to build and assemble than linear accelerators and synchrotrons, they are also much easier to operate and maintain.

There are some important specifications and terminology you’ll want to be aware of when designing a microscope or ordering microscope objectives. Here is a list of key terminology.

In the early 90s, several astronomical agencies such as the National Optical Astronomy Observatory (NOAO), the European Southern Observatory (ESO), and Office National d’Etudes et Recherches Aerospatiales (ONERA) in France started their own developments aiming at astronomy applications.

Each beamline of the NIF includes an adaptive optics system, with the deformable mirrors being located at the end of the main amplifier, correcting residual thermal distortions, imperfect optical materials and amplifier distortions due to flash lamp heating. This wavefront correction allows to achieve smaller spot size and produce higher power density on the target, therefore facilitating the fusion process.

In modern microscopes, neither the eyepiece nor the microscope objective is a simple lens. Instead, a combination of carefully chosen optical components work together to create a high quality magnified image. A basic compound microscope can magnify up to about 1000x. If you need higher magnification, you may wish to use an electron microscope, which can magnify up to a million times.

Ophthalmology has immensely benefited from the use of adaptive optics. Human eyes are very imperfect optical elements: they present irregularities which introduce aberrations and distort light waves. As a consequence, retinal examinations have always been limited to a rather low level of details and early signs of diseases occurring at the level of cells, had remained invisible to eye doctors.

Typesofobjective lenses

The Adaptive Optics control software is in charge of controlling all the components of the AO loop, receiving signals from the wavefront sensor and computing the right commands to send to the deformable mirror. In a lot of cases, a closed loop approach is chosen to compensate for time dependent aberrations (such as atmospheric turbulences). However, when wavefront sensing becomes very challenging, an open loop approach could also be chosen, for example in microscopy.

Multiphoton microscopy is an alternative to laser-scanning confocal microscopy. It uses the same principle of scanning the excitation beam over the sample, but differs to the extent that it is using a multiphoton excitation beam to create the fluorescence signal. This technique is broadly used for in-vivo imaging or deep imaging because it minimizes background fluorescence signal, overcomes to some extent scattering of sample tissues and is less photo-toxic than conventional confocal techniques. However, as the imaging goes deeper in the tissue, optical aberrations quickly become significant, which destroys the quality of the focal spot and reduces the 2P signal generated drastically. Adaptive optics was proven to be a good solution to recover performance when imaging deep, with better, tighter focus, improved axial sectioning, resolution and 2P signal emitted with typical x2 to x5 gains obtained. Perhaps the most well known microscope with adaptive optics is the one developed by Betzig and Wang at Janelia farms published in 2014 (click here to access it). Two subsequent advantages of adaptive optics and the signal gain are: 1) the ability to reach much deeper layers of the sample and 2) to decrease the intensity of the excitation laser, therefore reducing photo-toxicity. Below are two examples of adaptive optics applications to 2P imaging that have been published. In the example 1 below, AO is implemented on the excitation path of the multiphoton microscope. An iterative algorithm was used to optimize the DM shape to get the most signal.

The promising results obtained at the NIF triggered a flood of investments into private companies promising to deliver fusion power in the 2030s and the use of adaptive optics for high energy lasers is therefore expected to grow.

The wavefront from a distant object under observation, typically a star, is distorted by atmospheric turbulences. A part of the beam is directed to a wavefront sensor which measures the wavefront error of the incoming wave. The control system processes this measurement and sends a command to the deformable mirror to change its shape and compensate the distortion. The resulting output beam is now corrected and sent to the science camera for imaging.

Below are the images of drosophila tissues acquired with that same microscope at three different depth (2µm, 22µm, 42µm) without (left) and with (right) adaptive optics (Court. of Wei Zheng, Yicong Wu, Peter Winter & Hari Shroff, NIH, 2017 ).

Objective lensmicroscopemagnification

An microscope objective  may be either reflective or refractive. It may also be either finite conjugate or infinite conjugate.

While a magnifying glass consists of just one lens element and can magnify any element placed within its focal length, a compound lens, by definition, contains multiple lens elements. A relay lens system is used to convey the image of the object to the eye or, in some cases, to camera and video sensors.

Inertial Confinement Fusion (ICF) is a process that uses lasers to heat a small micron size target in order to produce energy through the fusion process. This approach was validated in the USA with the OMEGA and NOVA lasers in the late 70s and 80s but the first real milestone was achieved on December 5, 2022 at the NIF facility at the Lawrence Livermore laboratory where 192 beamlines combined delivered 2.05MJ of UV (351nm), over a few ns, which resulted in producing 3.15 MJ of energy – more energy than the target had received.

In this second example of AO implementation in a multiphoton set up, the deformable mirror is on the excitation and emission path. The wavefront measurement is carried out on “descanned” guide star. A flip mirror directs the light onto a wavefront sensor or the imaging camera (Court. of Wei Zheng, Yicong Wu, Peter Winter & Hari Shroff, NIH, 2017).

Nuclear fusion is expected to become one of the next big technological breakthroughs in the coming decades. The utilization of high energy lasers for nuclear fusion is well on its way, and will hopefully allow us, one day, to produce low cost “clean energy” in the near future.

-In fluorescence microscopy, depth dependent index changes of biological tissue induces aberrations that makes the imaging or fluorescence excitation inefficient and distorted at a certain depth.

Refractive objectives are so-called because the elements bend or refract light as it passes through the system. They are well suited to machine vision applications, as they can provide high resolution imaging of very small objects or ultra fine details. Each element within a refractive element is typically coated with an anti-reflective coating.

The idea behind adaptive optics is to compensate for these distortions and recover the system resolution. Originating from astronomy, the idea of adaptive optics was first introduced by the American astronomer Horace W. Babbock as early as 1953 for the correction of atmospheric turbulences.

Adaptive Optics brought major improvements to UHIL facilities, allowing the lasers to deliver quasi theoretical maximum intensity on target.

What isobjective lensin microscope

Figure 12 is an image of a drosophila larva acquired with this microscope (Court. of Drs. Beaurepaire, Débarre & Olivier, Ecole Polytechnique, France, 2012). We can see a 3x improvement of the SNR thanks to AO.

Adaptive Optics is commonly used in several microscopy techniques such as non linear two photon microscopy, confocal microscopy, light sheet microscopy and superresolution microscopy (PALM/STORM). One of the biggest challenges in applying adaptive optics to microscopy is measuring the disturbed wavefront at the desired location. For example if the goal is to get a perfect beam focusing at a certain depth of the sample, how could we measure the wavefront inside the sample? Scientists have come up with different tricks to perform this measurement, in some cases, guide stars are used or generated  inside the sample to run a closed loop. In other cases, open loop algorithms were implemented. This sections provides examples of different AO implementation schemes.

-In high intensity or high energy lasers, the high power of the beam passing through optics causes time and space dependent index refraction changes that highly distorts the focal spot.

Historically microscopes were simple devices composed of two elements. Like a magnifying glass today, they produced a larger image of an object placed within the field of view. Today, microscopes are usually complex assemblies that include an array of lenses, filters, polarizers, and beamsplitters. Illumination is arranged to provide enough light for a clear image, and sensors are used to ‘see’ the object.

The field of view (FOV) of a microscope is simply the area of the object that can be imaged at any given time. For an infinity-corrected objective, this will be determined by the objective magnification and focal length of the tube lens. Where a camera is used the FOV  also depends on sensor size.

What is thefunctionof thestage ona microscope

The parfocal length of a microscope is defined as the distance between the object being studied and the objective mounting plane.

Figure 7 below shows the wavefront correction of the NIF baseline design. The aberrated wavefront of the uncorrected beam (a) is compensated by DM shape (b). The residual (c) shows a 6-fold decrease of the WFE amplitude and 18x increase of the strehl ratio.

Stay tuned for regular updates as we keep pace with the ever-evolving landscape of Adaptive Optics technology. This page is dedicated to serving as a comprehensive resource for applications in adaptive optics, ensuring you stay informed of advancements. Check back frequently for the latest updates and insights.

-In astronomy, (see fig 1 below) because of atmospheric turbulences, a perfectly designed telescope, (i.e. with minimal aberrations) will still generate blurry images.