In tirf microscopy the evanescent wave is limited to 200 nm from the glass surface so where deeper sample penetration is required hilo becomes a very desirable mode. Other optical sectioning schemes like total internal reflection microscopy tirf or highly inclined laminated optical sheet microscopy hilo already became very successful in this field but because of their limited penetration depth they are not suitable for single molecule tracking in extended 3d samples. Highly inclined and laminated optical sheet hilo microscopy significantly improves the signal to background ratio by increasing the image intensity and decreasing the background intensity.
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