Multi-Photon Imaging

MBF Bioscience > Multi-Photon Imaging


Multiphoton microscopy has become an important tool for probing in vivo neuronal activity of individual neurons and circuits in modern neuroscience research. It has also proven to be very valuable in skin cancer research.


Multiphoton microscopy refers to both two-photon excitation fluorescence microscopy (2PEF) and three-photon excitation fluorescence microscopy (3PEF).  2PEF was developed first and is most commonly used in research laboratories, while 3PEF is a more recent invention gaining in use.


Both 2PEF and 3PEF share some important characteristics in relation to one photon microscopy, e.g. widefield fluorescence and confocal.  The first is that is their smaller point spread functions intrinsically eliminates most of the out of plane fluorescence and provides very thin optical sectioning. This is because the objective lens’ focal point is the only space with a high enough photon density to ensure simultaneous presentation of two photons to the fluorophore. Effectively, this means there is no out of focus emission light and all light at the emission wavelength must have come from that single spot


The second common characteristic is they use much longer wavelengths of light to stimulate or excite the fluorescent molecules. This yields less scattering in tissues like the brain, which that means you can image a lot deeper in the in the brain than you could with a one photon type of experiment. Therefore, multiphoton microscopy provides optical access deeper into tissue compared to single-photon fluorescence microscopy.


2PEF can image up to approximately 1 mm in living tissues. In comparison, single photon confocal microscopy can only penetrate to about 200 µm. 3PEF imaging is ideal for imaging deeper in scattering tissue, or when imaging through thin scattering layers. Due to the light having a longer wavelength, it is able to penetrate deeper into tissue. The light scatters less, enabling clearer images of structures deep in scattering tissue to be obtained. Fluorophores deeper in tissue can be excited, and, as with other optical sectioning techniques, structures can be visualized in 3D.

Multiphoton microscopy allows for optical sectioning while imaging in scattering tissue. When it is used in combination with rapid axial-scanning, this allows for the acquisition of three-dimensional (3D) representations of neuronal structure and multiple focal plane imaging of neuronal activity. As optical proteins, such as genetically-encoded fluorescence Ca2+ sensors, have continued to improve, 2PEF microscopy has been a dominant tool for in vivo imaging in neuroscience research.


To perform multiphoton imaging, you need a pretty complex microscope. This starts with a fast laser, something that’s usually firing at  80 megahertz with pulse widths of about 150 seconds, about 10 nanoseconds between each pulse. Then you’re going to scan that laser across the sample in a raster pattern using galvanometers and/or resonant scanners. Then the light will hit the sample and fluorescence emission is going to happen. Then you’re going to collect photons not with a camera but with a photomultiplier tube. So, what you’re doing is recording point-based fluorescence from the sample which requires you to then reconstruct that image with a computer as you’re acquiring the data. So, obviously to be able to do that you have to have a piece of software that can not only control the microscope but reconstruct the image on the fly in real-time. ScanImage is the software that makes this happen.



Further developments in multiphoton imaging include:

    • Photostimulation
    • Holography
    • Online Analysis
    • Time demultiplexing


Very recent advancements in multiphoton imaging include the following microscopes:

    • SLAP2 – Scanned line angular projection microscopy
    • Light Beads microscopy

What is ScanImage software?


ScanImage is a state-of-the-art software product for controlling laser scanning microscopes. The application was first released in 2003 and the original version (r3.0) is described in [1].


ScanImage uses the powerful vDAQ hardware (described below) for microscope control. For legacy systems, National Instruments hardware is also supported. ScanImage runs on custom built microscopes and on commercial microscopes from Scientifica, Sutter and Thorlabs.


In an article titled “Biological imaging software tools” published in the journal, Nature Methods, ScanImage was described as follows [2]: “ScanImage provides a software framework to control laser-scanning microscopes and is used extensively for two-photon excitation microscopy. It implements most standard modes of image acquisition and basic automation, and supports continuous image acquisition synchronized to behavioral or physiological data, which is particularly useful for imaging in intact animals. The software framework is object-oriented and event-driven to promote extensibility, online analysis and plug-in development. ScanImage can control laser-scanning microscopes, such as inhouse-built confocal systems, and allows for complex recordings where high signal-to-noise ratio is needed, such as tracking axon signaling in neuron cultures.”



[1]     Pologruto TA, Sabatini BL, Svoboda K. ScanImage: flexible software for operating laser scanning microscopes. Biomed Eng Online 2003;2:13. doi: 10.1186/1475-925X-2-13.

[2]     Eliceiri KW, Berthold MR, Goldberg IG, Ibáñez L, Manjunath BS, Martone ME, Murphy RF, Peng H, Plant AL, Roysam B, Stuurman N, Swedlow JR, Tomancak P, Carpenter AE. Biological imaging software tools. Nat Methods 2012;9(7):697-710. doi: 10.1038/nmeth.2084.



What are the RMR scanner and vDAQ Data Acquisition Card?


The Rapid Multi Region (RMR) Scanner combines the flexibility of galvo mirrors with the speed of resonant scanning. Its novel design combines two galvo and one resonant mirror into one compact device. The scanner is fully compatible with ScanImage’s powerful scanning modes. The flexible mounting options allow installation on a Thorlabs BScope, a Sutter MOM and “do it yourself” microscopes.


The vDAQ is an all-in-one data acquisition card for microscope control with ScanImage. It controls Galvos, resonant scanners, Pockels cells, Piezo objective positioners, shutters and much more. It greatly simplifies the wiring complexity of microscopes by eliminating the need for additional 3rd party data acquisition hardware. 

Fields Of Study

  • In vivo brain imaging
  • Calcium imaging
  • Imaging synaptic activity
  • Cancer research


ScanImage is a software package for controlling multiphoton and laser scanning microscopes. It enables advanced techniques such as time multiplexed acquisition and single photon counting.


The RMR is a resonant-galvo galvo module for 2P microscopes. It was developed as a novel solution for neuroscientists seeking to scan multiple regions of interest with high time resolution.


The vDAQ is an all-in-one card and breakout box that’s engineered to provide the most advanced and dependable microscope control and data acquisition.

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