Soft Bioelectronics Lab

Total internal reflection fluorescence microscopy (TIRF) is an essential imaging tool that facilitates to image a very thin layer (~< 200 nm depth) of near-surface and interfacial processes at the sample and glass slide interface without background noise. TIRF microscope finds usefulness in numerous scientific studies such as molecular genetics [1] DNA biophysics [2], biosensor design [3], and basic separation theory [4], etc. Moreover, the TIRF microscope is also an essential tool to study various molecular activity at the cell plasma membrane which includes kinetic rates of binding proteins to cell surface receptors [5], membrane trafficking [6], cytoskeleton remodeling [7], cell adhesion [8], cargo transport by molecular motor [9], single-molecule imaging [10] and nanoparticle-membrane interaction events for targeted drug delivery [11] etc. Despite these many important applications of the microscope, the use of this microscope sometimes is limited by its expensiveness and some inherent drawbacks.

The conventional TIRF microscope uses either prism or high numerical aperture objective lens to generate evanescent waves at the interface. In a prism-based TIRF microscope, the evanescent field is generated at the coverslip/buffer interface through a prism which directs the laser light towards the coverslip. Although prism-based TIRF generates a thin and clean evanescent field without undesirable disturbance due to reflected light, its not free from remarkable shortcomings such as it is bulky, complex to set up, and usually expensive. Moreover, the prism-based TIRF microscope is associated with direct coupling of the sample with the prism which limits the sample movement and often requires unconventional sample mounting systems. On the other hand, In an objective-based TIRF microscope, an objective lens with a high numerical aperture (N.A) ( >1.4)  is used for the purpose of both delivery of light as well as the collection of fluorescence light from the sample. Even though a high objective-based TIRF microscope is easy to use, however, typically too expensive and generates background noise due to specular reflection from the incident light as the same objective is used to deliver and collect the light. In addition, a conventional TIRF microscope uses laser light to produce enough intensity of evanescence wave to excite the fluorescent molecule. This typically expensive laser source adds an extra expense to the conventional TIRF microscope.

Therefore, in recent times, researchers have noticed those limitations of the conventional TIRF microscope and come up with some innovative idea of introducing low-cost LED light source and waveguide-based evanescence field generation to resolve the problems confronted by conventional TIRF microscope. However, despite the introduction of LED and waveguide-based smart configuration of these recent reports, LED light may not produce enough evanescence field intensity compared to a laser light source, which remains insufficient for a single objective TIRF microscope to collect maximum possible photons emitted by the fluorescence molecule. Therefore, LED as a light source may not give the expected result when a single objective TIRF microscope is used to collect the fluorescence light.

Therefore, we have come up with a novel configuration to overcome the possibility of insufficient evanescence field strength due to the use of a LED light and enhance the light collection capability of the TIRF microscope from the fluorescence molecule. The problem is overcome by introducing dual aspherical objective lenses facing each other for collecting light from both sides of the sample and, also proposed a smart design of waveguide to sufficiently enhance the evanescence field due to a LED light source. The dual objective lens configuration can not only give more information about the specimen but also each objective lens can focus on a different 2D plane to capture movements of the interfacial events at different focal planes. The proposed waveguide is modified by introducing a reflecting coating on the oppositely facing wall of the waveguide to reduce the loss of light coming out from the waveguide. Moreover, we have proposed to use custom-made cost-effective aspherical objective lenses and other necessary optical components to make the device cost-effective and compact (Earlier work )

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