Confocal Microscope

Overview

A confocal microscope builds an image one point at a time, and in doing so produces something a conventional microscope cannot: a clean optical section. By scanning a focused laser across the sample and rejecting any light that did not come from the focal plane, it images a thin slice through a thick fluorescent specimen, free of the blur that out-of-focus material would otherwise add. Stack those slices and the result is a three-dimensional view of cells, tissue or whole small organisms.

The instrument is the standard tool of modern fluorescence biology. Multiple labels are imaged in separate colour channels, time-lapse follows living cells, and Z-stacks reconstruct structure in depth. Everything depends on the trick at its heart: a pinhole that only lets the in-focus point through.

How it works

The optics live in the Laser Scan Head. Several lasers are combined and steered by the X Scan Galvanometer and Y Scan Galvanometer, a pair of galvanometer mirrors that raster the beam across the field, one sweeping fast along each line, the other stepping between lines. Dichroics separate the returning fluorescence from the excitation, and the Confocal Pinhole at the conjugate focus blocks everything except light from the focal point, which is what gives the image its sectioning.

The beam enters the sample through the Microscope Body & Turret, whose motorized turret swaps high-NA objectives onto the axis, while the Motorized XY Stage positions the specimen for tiling and multi-point work. Emission photons returning through the pinhole land on the PMT Detector Array, photomultipliers that count them for each pixel, split into colour channels so several labels are captured at once. A separate Widefield Camera on a side port grabs widefield and brightfield views for navigation.

Depth comes from the Z Focus Drive, a stepper-and-piezo column that steps the objective through the sample to collect a Z-stack. The whole acquisition is run by the Scan Controller & Workstation, which synchronizes galvos, lasers and detectors pixel by pixel and streams data to its workstation. Because the image is diffraction-limited, the microscope sits on the Vibration Isolation Table table, floated on air legs so floor vibration never blurs the result.