In recent development of fluorescence microscopy, the extent of out-of-focus fluorescence background is vital in determining the penetration depth achievable in visualization of biological tissues [1
]. Even conventional Confocal Microscopy (CM) with a detection pinhole to reject out-of-focus fluorescence is limited to use near the tissue surface (tens of microns) as multiple scattering dominates at large depths which could obscure the in-focus details and diminish the appreciable imaging contrast [3
]. In multi-photon microscopy (MPM), nonlinear light-matter interaction is being utilized to concentrate the fluorescence excitation in a submicron focal volume that greatly suppressed the out-of-focus excitation and thus could increase the imaging depth up to 1mm [4
]. However, MPM uses expensive pulse laser. Furthermore, the concern of nonlinear photon-damage/photobleaching [8
] and availability of fluorescence probes with large “two-photon absorption cross-section” make single-photon excitation sometimes favored. Another rapidly developed technique termed photo-acoustic tomography (PAT) [9
] based on ultrasonic detection of pressure waves generated by the absorption of pulse light in elastic media also enable high-resolution visualization of absorbers a few millimeters deep within highly light-scattering living organisms. However, the in-plane spatial resolution of the photo-acoustic images achievable is limited by the effective bandwidth of the ultrasonic detector; leading to few tenths microns diffraction limited spatial resolution and even worse for axial resolution [10
]. Dynamic speckle illumination microscopy (DSI) [11
] presents a simple yet robust technique to obtain optical sectioning and out-of-focus background rejection with a widefield microscope coupled with speckle illumination and spatial wavelet prefiltering. However, there are no demonstrations of DSI imaging with penetration depth larger than 100um up to date. Structured illumination microscopy (SI) [12
] is another widefield microscopy technique that could improve the spatial resolutions up to 2 folds of that of CM by exploiting the interference patterns in the images captured at different angles corresponding to the illumination diffraction gratings. However, SI is limited in acquisition times due to requirement of capturing multiple images, and no penetration depths larger than 100um have been demonstrated as well. Other super-resolution optical imaging techniques such as stimulated emission depletion (STED) microscopy [13
] which uses superlocalized depletion of the excited state by stimulated emission with few tens nanometers spatial resolution being demonstrated are severely limited in penetration depth to few tenths microns and limited for deep imaging applications.
Focal Modulation Microscopy [14
] is an emerging fluorescence microscopy technique that can provide sub-micron spatial resolution at large penetration depths in highly scattering media such as biological tissues by preserving the signal-to-background ratio. In FMM, intensity modulation at focal point is being induced by interference of two periodically phase modulated (or frequency-shifted) excitation beams, which are spatially separated except when brought to the focal point by the objective lens. Ballistic photons contribute mainly to the oscillatory excitation confined exclusively at the focal point as they have well defined phase and polarization compared to scattered photons, though both of them could reach the focal point. The fluorescence emission from the sample is modulated at the same frequency as excitation. Subsequently, by implementing a lock-in technique to demodulate the fluorescence signal collected by the pinhole detector, we can effectively remove fluorescence signal excited by diffusive photons. Experimental [14
] and theoretical studies [16
] of FMM have shown its immense potential in noninvasive imaging of thick biological samples. Previously, we reported our FMM system implemented using tilting plate phase modulator [15
] and modulation frequency up to a few kHz has been achieved. However, compared to most commercial laser scanning microscopes with microsecond pixel dwell time, that modulation frequency presents a fundamental limit on the imaging speed, temporal resolution and imaging throughput we can attain. In this letter we would like to describe a novel implementation of FMM with AOMs onto a commercial Confocal Microscope and demonstrate the improved image acquisition speed and quality.