Novel calibration device and calibration method for total internal reflection fluorescence microscope penetration depth

Abstract

The invention discloses a calibration device for the total internal reflection fluorescence microscope penetration depth. The device comprises a glass slide, square cover glass and a glass sheet, wherein the square cover glass covers the upper surface of the glass slide; the right sides of the square cover glass and the glass slide are aligned; the glass sheet is arranged on the glass slide; the left side edges of the glass sheet and the glass slide are in contact and are aligned; the lower surface of the glass sheet is supported by the direction cover glass; the lower surface of the glass sheet is uniformly coated with fluorescent dye. The invention also discloses a method for calibrating the total internal reflection fluorescence microscope penetration depth by using the calibration device. The device has the advantages that the structure is simple; the cost is low; the operation is convenient and flexible.

Description

technical field [0001] The invention belongs to the field of fluorescence microscope imaging, and in particular relates to a novel calibration device and calibration method for the incident depth of a total internal reflection fluorescence microscope. Background technique [0002] Total internal reflection fluorescence microscopy (TIRFM) is an emerging optical imaging technology in recent years. Very thin regions of the interface. Due to the characteristic of the evanescent wave field that the energy decays exponentially in the axial direction, the dynamic range of the observable area is usually below 200nm, which effectively controls the excitation volume and greatly reduces the interference of background light noise, so it has other optical imaging technologies. Unmatched high signal-to-noise ratio and contrast. Therefore, this technology has been widely used in the dynamic observation of substances on the surface of cell membranes. [0003] According to the characteris...

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Problems solved by technology

[0009] Although these two methods can obtain better calibration results, they require special fluorescent beads or experimental tools, and complex image processing and data fitting are required in the later stage, which is not very convenient to use.

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