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Multi-Photon MicroscopyThe neuroscience field strongly depends on acquiring faster images at greater depths for deep tissue in vivo imaging. Scattering media not only inhibits subsurface cell-scale imaging within tissues, such as the brain, but also limits the penetration depth of current optical imaging. Multi-photon microscopy (MPM) overcomes this problem at depths up to a few scattering mean free path lengths (e.g. 500 µm in mouse brain). Adaptive optics allows optical wavelength control needed for transformative impacts on deep tissue imaging, a goal in the neuroscience field.

A major problem standard Multiphoton Microscopy faces is correcting for scattering media. Super Penetration Multi-Photon Microscopy (S-MPM), was pioneered by the Cui Lab at Howard Hughes Medical center and recently reported by Boston University on focusing light through static and dynamic strongly scattering media. Developed at Boston University and commercialized by Boston Micromachines Corporation (BMC), the enabling component is a fast MEMS spatial light modulator (Kilo-SLM), incorporated into the test bed shown in Fig. 1. With this component, images of 1 µm diameter fluorescent beads through 280 µm thick mouse skull reached image depths of about 500  µm.

The images in Fig. 2 depict these measurements.

Multi-Photon Imaging PMT image Optical Window
Camera image (Transmission, before optimization)
PMT image (Reflection) Optical Window ~20 µm
Fig. 2. Imaging fluorescent beads (1  µm diameter) through mouse skull (280  µm thick) using a glass cover slip (150 µm thick).

Additonal observations made:

  • 10x – 100x advance in signal enhancement with higher resolution and contrast.
  • SLM corrects for low order spherical aberrations as well as higher order scattering effects.
  • The optomized SLM phase improves imaging over a field of view of 10–20 µm for samples tested to date.

The Boston Micromachines DM model used for both HHMI and BU are:

Kilo-S-DM MEMS Deformable Mirror

  • Segmented Surface
  • 9.3 mm aperture
  • Gold Coating
  • Latency (First word written to last DAC updated): 22.7 µsec
  • Maximum Frame Rate: 60 kHz
  • Resolution: 12 Bit
  • Average Voltage Step Size: 14 mV

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