Abstract
Wide field fluorescence microscopy is the most commonly employed fluorescence imaging modality. However, a major drawback of wide field imaging is the very limited imaging depth in scattering samples. By experimentally varying the control of illumination, we found that the optimized illumination profile can lead to large contrast improvement for imaging at a depth beyond four scattering path lengths. At such imaging depth, we found that the achieved image signal-to-noise ratio can rival that of confocal measurement. As the employed illumination control is very simple, the method can be broadly applied to a wide variety of wide field fluorescence imaging systems.
Highlights
Thanks to the development of genetic and synthetic fluorescence indicators, fluorescence imaging [1,2] has profoundly transformed life science and medical research
To explore various imaging conditions, we developed a versatile system (Fig. 1) that allows the accurate control of illumination area, numerical aperture (NA) and angle and allows a direct comparison between wide field imaging and confocal imaging
We tried to mimic the illumination with an extended incoherent spatial light source by adding a diffuser
Summary
Thanks to the development of genetic and synthetic fluorescence indicators, fluorescence imaging [1,2] has profoundly transformed life science and medical research. Compared with other imaging technologies, fluorescence imaging has its key advantages in sensitivity, resolution and functionality. Imaging resolution beyond the diffraction limit has been enabled for fluorescence imaging through either nonlinearity or photo-switchable fluorophores [4,5,6,7]. The fusion of fluorophores with molecule binding groups has enabled a wide variety of function imaging probes, which allows tracking various biological signaling within live animals [8]. These advances have positioned fluorescence imaging as the dominant optical imaging modality for both structural and function imaging of biological systems
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