Holographic laser scanning microscopy

We realize a new optical microscopy technique (Holographic Laser Scanning Microscopy, H-LSM) based on holographic laser scanning illumination of the sample. In each scanning step, a multispot illumination pattern is generated by phase modulating a laser beam via Computer Generated Holography (CGH). A CCD acquires an image of the light signal backscattered from the sample in each scanning step, and the elaboration software (Henriques R, et al. Nat Methods 7:339-340, 2010) reconstructs the image of the sample by localizing the centroid of each recorded spot (Bobroff N, Rev Sci Instrum 57:1152-1157, 1986). The elaboration software operates a spatial filtering on the recorded spots, accepting and processing only those satisfying the minimum SNR and the maximum FWHM controls. The centroid of the valid signals is displayed in the elaborated image (H-LSM image) as a single bright pixel. We implement two completely different methods in illumination schemes. A first illumination condition is realized by focusing an array of light spots onto the sample plane. This intensity pattern is obtained in objective focal plane imposing the calculated phase map (kinoform) on the laser wavefront via a Spatial Light Modulator (SLM). The array of light spots can be made scanning the sample by imposing a grating-like function to the original kinoform (Leach J, et al. Appl Opt 45:897-903, 2006). The second illumination scheme is realized by dynamically changing the random Speckle pattern originating from the random mutual interference of the laser beam wavefronts. Such Speckle pattern is easily generated by imposing a random phase profile on the laser beam using the CGH apparatus, while the mapping of the sample is realized by changing the phase profiles in time.We tested the H-LSM method on some samples presenting structures whose lateral dimensions are below the resolution limit of the optical setup. The H-LSM image of a region of a sample illuminated with an array of light spots, realizing the ordered illumination scheme, is shown in Fig. 64.1a. For comparison, in Fig. 64.1c the image obtained summing up the CCD acquired stack of the scanning procedure, is also shown. Fig. 64.1b,c,d display respectively theH-LSM and the summed image of the sample obtained with the random illumination scheme. Despite a standard resolution limit, the H-LSM images show improved contrast and sharpness respect to the summed images. Moreover, the filtering on the contributing light signals operated by the elaboration software makes the method comparable to software confocal technique, with very small depth of field for the ordered illumination scheme and large depth of field in random illumination scheme compared to brightfield microscopy. The absence of moving mechanical parts provided by the holographic nature of illumination makes the proposed method useful for vibration-free measurements, with a possible miniaturized setup and 3D imaging.