(a) Schematic diagram of the multi-photon microscope. (b) Photograph of the microscope. Examples of a multiphoton images are shown in (c) the end of a mosquito needle and (d) a full cross-section of a mouse brain. Click on the image to see a larger image. A fuller description of the apparatus appears below.

Multiphoton microscopyprocedure

A basic multiphoton microscope described here can be constructed for approximately $10,000 (not including the femtosecond fiber laser which is commercially available at the cost of ∼$10,000). A research grade microscope will cost around $30k using state-of-the-art new components that are commercially available. For comparison, a basic commercial multiphoton microscope would cost over $500,000.

Multiphoton microscopywiki

We have been depositing and optimizing Anti-Reflective (AR) Coatings for laser applications. In our industry, every photon of light matters. That’s why we have designed our AR coatings to optimize transmission through optical components by minimizing front and backside reflection losses. AR Coatings are typically used on windows and lenses to reduce reflection losses to optimize the transmission through the optic. ARO can also apply AR coatings to the backside of optics including polarizers and partial reflectors to minimize back side reflection. ARO has the capability to optimize AR coatings for narrow, multiband, and broadband specifications. We have custom AR coatings available from 193nm to 3000nm with particular experience optimizing 193, 248, 308, and 1063 nm coatings.

Skills that will be acquired during this Immersion include optical design and alignment, data acquisition and processing, and familiarity with multiphoton microscopy.

Participants will learn the design and construction of a multiphoton microscope along with all the required data acquisition and signal processing steps. A special nonlinear knife-edge technique will be applied to measure the resolution of the microscope in the x, y and z directions. Different nonlinear optical effects will be investigated to create an image including SHG, THG and multiphoton excitation fluorescence. In particular, three-photon excitation fluorescence from chlorophyll in a fresh leaf will be observed. Other prepared biological samples (mouse brain section, various fixed tissues of different diseases, etc.) will be also available for experimenting. Advanced imaging techniques such as tiling/stacking will be introduced if time allows. A sensitive spectrometer will also be available to acquire the optical spectrum of the nonlinear optical emission from samples.

Two photon excitationmicroscopy

The most important application of MPM is in biological research and diseases diagnosis. Imaging technology has and will continue to influence medicine by advancing research and development (R&D) based on mechanistic insights into biomedical processes that ultimately lead to innovative pharmaceuticals, diagnostics to better plan treatment, and clinical management of diseases to improve outcomes. Immersion participants will learn about a couple of ongoing research efforts such as brain and cancer imaging. Nonlinear material and device characterization is another interesting application where MPM can play an important role. If time allows, participants will have a chance to image some interesting 2D materials such as graphene with the multiphoton microscope. The microscope can also be used for 3D writing which is an important emerging technique for creating advanced optical and medical components.

Multiphoton microscopyprinciple

AR Coating performance depends on multiple specifications. Material selection, coating deposition type, material, bandwidth, angle of incidence, and wavelength are all factors to consider when designing your optimal AR Coating. ARO can select from E-beam, magnetron sputtering, IBS, and ion-assisted deposition coating techniques based on your environmental, laser damage threshold, and wavelength requirements. Optical coatings technology can be used with a wide range of substrate material including FS, CaF2, MgF2, BK7, and others. ARO is able to reach the lowest reflectivity specifications for narrowband coatings at normal incident angles. Broadband AR coatings see a trade-off between bandwidth and reflectivity. Reach out to our sales team, so we can help you select the best custom AR coating for your application.

Multiphoton microscopyvs confocalmicroscopy

Participants are encouraged to bring a notebook. A laptop computer would be helpful to view, store and display multiphoton images. Please feel free to bring your own samples that you want to image with the multiphoton microscope.

ARO offers anti-reflective coatings from 193-3000nm that can be applied to many substrates. We have extensive experience applying anti-reflective coatings on lenses, windows, mirrors, beam splitters and more.

Multiphotonfluorescencemicroscopy

Multi-photon microscopy (MPM) is a powerful technique that allows three dimensional mapping of samples that have a measurable nonlinear optical response such as second harmonic generation (SHG), third harmonic generation (THG) or fluorescence induced by multiphoton absorption. MPM provides us with a way to see the nonlinear microscopic world with high resolution, in 3D. After about two decades of intensive development MPM has found important applications in nonlinear materials characterization, biological research and in diagnosing medical conditions. Immersion participants will learn how to build a compact multiphoton microscope using a femtosecond fiber laser as the excitation source. Different nonlinear optical contrast mechanisms will be introduced. The optical and mechanical design of the microscope will be discussed. Participants will assemble the microscope from scratch, perform alignment, and acquire multiphoton images from various samples including biological tissues.

The laser power level used is generally considered safe but laser safety glasses for laser light at 1040nm nm and 1550 nm will be available.

Our technical team will review your optical drawing or custom specifications and provide an estimate. Typically we offer: