Fluorescence sectioning with dynamic speckle illumination microscopy

We present a novel fluorescence microscopy technique that provides depth sectioning in thick tissue. The technique relies on dynamic speckle illumination, and depth sectioning is obtained from an a priori knowledge of speckle statistics. We demonstrate nearconfocal imaging in a mouse brain labeled with green fluorescent protein. Confocal microscopy [1] is a popular technique in the bioimaging community, mainly because it provides optical sectioning. However, its standard implementation requires 3-dimensional scanning of focused illumination throughout the sample. Efficient non-scanning alternatives have been implemented, among which the simple and well-established incoherent structured illumination microscopy [2, 3]. We recently proposed a technique similar to structured illumination microscopy wherein the incoherent grid illumination pattern is replaced with a speckle illumination pattern from a laser, and we have demonstrated that this new technique confers optical sectioning in fluorescent biological tissue [4]. The technique relies on the illumination of a sample with a sequence of dynamic speckle patterns and an image processing algorithm based only on an a priori knowledge of speckle statistics. Our initial demonstration of dynamic speckle illumination (DSI) microscopy yielded a depth sectioning strength that was quasi-confocal. We recently made significant improvements to our technique, and now obtain depth-sectioning strengths that are very near confocal. Two different strategies led to this increased sectioning strength. One strategy utilizes a sequence of laterally translated speckle patterns and a differential-intensity variance algorithm to derive a final sectioned image[5]. A second strategy utilizes a sequence of independent random speckle patterns and a novel post-processing algorithm. Both algorithms yield the same sectioning strength. Our experimental setup consists of a standard widefield microscope with an argon laser source (λ = 488nm). A simple diffuser plate in the laser beam path is imaged onto the back aperture of the objective and rotated with a stepper motor to produce dynamic speckle illumination (DSI). The images are recorded using a CCD Camera. We demonstrate DSI fluorescence sectioning by imaging GFP-labeled sensory neurons in an excised (non-fixed) mouse olfactory bulb (see Figure). We find that a good image quality is maintained throughout a depth of at least ~100 μm. Figure: Simultaneous conventional widefield (left) and DSI (right) images of GFP-labeled axon fibers in a mouse olfactory bulb