High numerical aperture objective lenses and optical system improved objective type total internal reflection fluorescence microscopy

Recently, the total internal reflection fluorescence microscopy (TIRFM) has gained popularity among many biologists for observing cells and single molecules. The objective lens type TIRFM is a very useful tool because the near-field fluorescence image can be observed with leaving an open space on the other side of the lens. Therefore, a manipulation on the preparation becomes possible, and simultaneous observations by scanning probe microscopy or by DIC microscopy are also possible. Currently, many investigators are using 100x, NA 1.40 objective lens for this purpose. Some scientists have little experience in aligning optical systems and tend to have difficulty because the laser needs to pass through the very perimeter of the 100x NA 1.40 objective lens. The critical angle for the 100x NA 1.40 objective lens with a water-immersed specimen (cells) is 65.63 degrees and the maximum light pass angle is 67.53 degrees. The angle available for the total internal reflection is 1.9 degrees. As a result, it is very difficult to align the laser beam to this NA 1.40 objective lens, which makes the evanescent wave illumination fail most of times. To solve this problem, we designed Apo 100x NA 1.65 and Plan Apo 60x NA 1.45 objective lenses, When we use these lenses for water-immersed specimens, the marginal angle available for the total internal reflection becomes larger compared with NA 1.40 objective lens. Therefore, the alignment of the laser beam to the objective lens becomes very easy. We also designed the TIRFM illuminator. A single mode fiber is connected between the laser and illuminator. The laser beam is conducted through the fiber and supplied to the illuminator with a pre-adjusted positioning. Almost no additional alignment of illumination Light is necessary.