Correct option is C
(3) B and C only
Sol. In Laser Scanning Confocal Microscopy (LSCM), certain statements are correct while others are not, and it is essential to differentiate between them for accurate understanding.
A. LSCM is a wide field technique with Köhler illumination system: This statement is incorrect. LSCM is not a wide-field technique; it is a point-scanning technique where the illumination focuses on a specific point in the sample. Köhler illumination is a feature of wide-field microscopes, not LSCM.
B. Spatial resolution higher than that achieved in wide field imaging could be obtained if only the central portion of an Airy Disk is used to form an image: This statement is correct. By using the central portion of the Airy disk, confocal microscopy can achieve higher resolution compared to conventional wide-field microscopy, effectively eliminating out-of-focus light and improving optical sectioning.
C. Scanning mirrors sweep the excitation beam over the sample point-by-point to build the image: This is a correct statement. In LSCM, the scanning mirrors direct a laser beam across the sample, point by point, to form the image. This method enables the collection of detailed images with high optical sectioning and resolution.
D. An altered pinhole size does not make any impact on the resolution of the image: This statement is incorrect. The pinhole size plays a significant role in controlling the resolution in confocal microscopy. A smaller pinhole improves optical sectioning and enhances resolution, while a larger pinhole reduces resolution and increases light intensity but may allow more out-of-focus light.
E. A photomultiplier tube (PMT) in LSCM helps in generating real color of fluorophores: This statement is not entirely correct. The PMT in LSCM is used to detect fluorescence emitted by the sample, but the color of the fluorophore is determined by the emission wavelength, not the PMT. The PMT amplifies the signal, but the color corresponds to the specific fluorophore being excited, not to the PMT.
Information Booster
The correct combination is B and C only, as they describe fundamental principles of Laser Scanning Confocal Microscopy:
Spatial Resolution: In LSCM, spatial resolution is improved by focusing on the central part of the Airy disk, effectively reducing out-of-focus light and increasing resolution. This technique is a key advantage of confocal microscopy over wide-field techniques, allowing for detailed imaging at a high resolution.
Point-by-point Scanning: The point-by-point scanning of the laser beam, as described in statement C, is a hallmark of LSCM. This allows the microscope to construct an image by scanning each pixel in succession, as opposed to illuminating the entire field at once as in wide-field microscopy.
